This research was funded by the Central Public-interest Scientific Institution Basal Research Fund of State Key Laboratory of Tree Genetics and Breeding (grant number CAFYBB2020ZY001-2), Enterprise research projects(202404016-4320), and National Natural Science Foundation of China (NSFC 32271896).

1. Collection of <img alt="Equation 2" src="/files/ftp_upload/67350/67350eq02.jpg" style="margin: auto;" /> B. xylophilus
<ol>
	<li>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.
	<ol>
		<li>Collect round logs (5 cm thick) from the upper, middle, and lower sections of the main trunk of the felled trees.</li>
		<li>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.</li>
		<li>Place the small wood strips into sealed bags and store them in a laboratory for further use.</li>
	</ol>
	</li>
	<li>Extraction of <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus
	<ol>
		<li>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.</li>
		<li>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. 
		&#8203;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.</li>
		<li>Release the pinchcock at the bottom of the funnel slowly, and let 2000-5000 &#181;L of the mixture leak into a Petri dish (5 cm in diameter) for microscopic examination and morphological identification.</li>
		<li>Identify <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus by observing features such as stylets, median esophageal bulbs, lipid droplets, and &#34;finger-like&#34; tail shapes11.</li>
		<li>Under a microscope, transfer individual <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus onto a slide by aspirating 10 &#181;L of liquid along with one nematode using a 10 &#181;L pipette each time. Repeat this process 30 times to collect approximately 300 &#181;L of the mixture containing nematodes.</li>
		<li>After extraction of <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus, sterilize the remaining wood strips and infected wood materials to prevent further contamination.</li>
	</ol>
	</li>
</ol>
2. Osmotic regulation process control
<ol>
	<li>Preparation of osmotic regulation mixture
	<ol>
		<li>Prepare an 8% KCl aqueous solution.</li>
		<li>Using a pipette, add 10 &#181;L of the 8% KCl aqueous solution to the slide containing the mixture of <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus, yielding approximately 310 &#181;L of a mixture of the nematodes and KCl solution.</li>
	</ol>
	</li>
	<li>Regulation of increased osmotic pressure and dehydration of <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus&#8203;
	<ol>
		<li>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 &#181;L of the mixture to evaporate naturally until the KCL crystals precipitate.</li>
		<li>Observe the dehydration process (e.g., body curling, aggregation, etc.) of <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus under the microscope. When the water fully evaporates and KCl crystals precipitate, <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus stops moving and enters cryptobiosis.</li>
	</ol>
	</li>
	<li>Low-temperature stress treatment of cryptobiotic <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus
	<ol>
		<li>Place the cryptobiotic <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus in a constant temperature environment of -20 &#176;C for 24 h.</li>
	</ol>
	</li>
	<li>Regulation of decreased osmotic pressure and rehydration of <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus
	<ol>
		<li>Add approximately 300 &#181;L 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.</li>
		<li>Observe the rehydration process of <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus (e.g., body stretching, resumption of movement, etc.) under the microscope. Calculate the survival rate after 24 h.</li>
	</ol>
	</li>
</ol>

Cryptobiosis of Bursaphelenchus xylophilus Juveniles

<ol>
	<li>Li, Y., Zhang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+on+the+trend+of+invasion+and+expansion+of+Bursaphelenchus+xylophilus.">Analysis on the trend of invasion and expansion of Bursaphelenchus xylophilus.</a> Forest Pest and Disease. 37 (5), 1-4 (2018).</li><li>Futai, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pine+wood+nematode,+Bursaphelenchus+xylophilus.">Pine wood nematode, Bursaphelenchus xylophilus.</a> Annu Rev Phytopathol. 51 (51), ?61 (2013).</li><li>Crowe, J. H., Cooper, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryptobiosis.">Cryptobiosis.</a> Sci Am. 225, 30-36 (1971).</li><li>Wharton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anhydrobiosis.">Anhydrobiosis.</a> Curr Biol. 25, 1107-1125 (2015).</li><li>Browne, J., Tunnacliffe, A., Burnell, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anhydrobiosis:+plant+desiccation+gene+found+in+a+nematode.">Anhydrobiosis: plant desiccation gene found in a nematode.</a> Nature. 416 (6876), 38 (2002).</li><li>Shatilovich, A. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viable+nematodes+from+late+Pleistocene+permafrost+of+the+Kolyma+river+lowland.">Viable nematodes from late Pleistocene permafrost of the Kolyma river lowland.</a> Dokl Biol Sci. 480 (1), 100-102 (2018).</li><li>Schottenfeld-Roames, J., Ghabrial, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osmotic+regulation+of+seamless+tube+growth.">Osmotic regulation of seamless tube growth.</a> Nat Cell Biol. 15 (2), 137-139 (2013).</li><li>Erkut, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trehalose+renders+the+dauer+larva+of+Caenorhabditis+elegans+resistant+to+extreme+desiccation.">Trehalose renders the dauer larva of Caenorhabditis elegans resistant to extreme desiccation.</a> Curr Biol. 21 (1), 1331-1336 (2012).</li><li>Pan, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Third-stage+dispersal+juveniles+of+Bursaphelenchus+xylophilus+can+resist+low-temperature+stress+by+entering+cryptobiosis.">Third-stage dispersal juveniles of Bursaphelenchus xylophilus can resist low-temperature stress by entering cryptobiosis.</a> Biology. 10 (8), 785 (2021).</li><li>Mao, X. F., Li, H. X., Chen, X. Y., Hu, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+efficiency+of+soil+nematodes+by+different+methods.">Extraction efficiency of soil nematodes by different methods.</a> Chinese J Ecol. 23 (3), 149-151 (2004).</li><li>Pan, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+identification+of+pine+wood+nematode+in+Pinus+koraiensis+in+Fengcheng,+Liaoning+province.">Isolation and identification of pine wood nematode in Pinus koraiensis in Fengcheng, Liaoning province.</a> Forest Pest and Disease. 38 (1), 4 (2019).</li><li>Pan, L., Cui, R., Li, Y., Feng, Y., Zhang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+pinewood+nematodes+in+Pinus+tabuliformis+Carr.+under+low-temperature+conditions+in+Fushun,+China.">Investigation of pinewood nematodes in Pinus tabuliformis Carr. under low-temperature conditions in Fushun, China.</a> Forests. 11 (9), 993 (2020).</li><li>Madin, K. A. C., Crowe, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anhydrobiosis+in+nematodes:+Carbohydrate+and+lipid+metabolism+during+dehydration.">Anhydrobiosis in nematodes: Carbohydrate and lipid metabolism during dehydration.</a> J Exp Zool. 3, 193 (2010).</li><li>Wharton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anhydrobiosis:+The+model+worm+as+a+model.">Anhydrobiosis: The model worm as a model.</a> Curr Biol. 21 (15), 578-579 (2011).</li><li>Li, Y., Zhang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+risk+of+invasion+and+expansion+of+pine+wood+nematode+in+the+middle+temperate+zone+of+China.">High risk of invasion and expansion of pine wood nematode in the middle temperate zone of China.</a> J Temperate Res. 1 (1), 4 (2018).</li></ol>

The authors have nothing to disclose.

In this study, <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus was collected from dead P. massoniana in Ningbo, Zhejiang Province. It should be noted that <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> 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 <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus. During winter, when the daily minimum temperature in Liaoning Province is around -20 &#176;C, a large number of single-instar <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> 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 <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> 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 <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> 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 <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> 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 <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> 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 <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus and the fourth-stage dispersal juveniles (<img alt="Equation 3" src="/files/ftp_upload/67350/67350eq03.jpg" style="margin: auto;" />)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. <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> 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 <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> 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.

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 (<img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" />). 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. <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> 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 <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus through osmotic regulation. This method is simple, efficient, and highly reliable, providing technical support for researching the stress resistance mechanisms of <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus.

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

inducing the entry of third stage dispersal juveniles of bursaphelenchus xylophilus into cryptobiosis through osmotic regulation

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, <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" />) 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 <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> 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, <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus entered a cryptobiotic state after gradual dehydration due to increased external osmotic pressure by natural water evaporation. <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus could resist low-temperature stress at -20 &#176;C. <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus could revive upon rehydration, with a survival rate of 92.1%. This process can regulate the entry of <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> 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.

In the 8% KCl solution, as the water evaporated, <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus initially exhibited a noticeable &#34;curling&#34; 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 <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus.
A dehydration experiment demonstrated that in an initial 8% KCl solution, water evaporation caused <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus to lose water. After dehydration, <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus were subjected to low-temperature stress at -20 &#176;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 <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus under low-temperature conditions.
During the rehydration phase following the cryptobiosis of <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus, we observed that 100% of the nematodes exhibited in vivo &#34;bubbles&#34; when being transferred from 40% KCl solution to distilled water (Figure 2).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67350/67350fig01.jpg" /> 
Figure 1: The dehydration-cryptobiosis-rehydration process of <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus. This image illustrates the cryptobiosis process in <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus, including the (A) dehydration stage, (B) cryptobiosis stage, (C) rehydration stage, (D) KCL crystallization stage, and (E) <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67350/67350fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67350/67350fig02.jpg" /> 
Figure 2: Accumulation of fat granules in unrevived <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus. This image shows the state of <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus after rehydration, characterized by the presence of in vivo bubble-shaped fat granules. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67350/67350fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

The present protocol describes a method for inducing the dispersal of third-stage juveniles (pre-dauer juveniles, J&#8546;) 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.

Cryptobiosis is a state where organisms lose nearly all their internal water and enter anhydrobiosis under extreme environmental stress. The dispersal ...

inducing-entry-third-stage-dispersal-juveniles-bursaphelenchus

Nanyang Technological University

Research

JoVE Journal

Biology

291 Views.  Chinese Academy of Forestry. Ons onderzoek richt zich voornamelijk op de koude-ontvankelijke mechanismen van de dispersie van juvenielen in de derde fase van dennenaaltjes, met name op hoe het dennenaaltje bestand is tegen lage temperatuurstress bij ongeveer min 20. De huidige experimentele uitdaging is de droogtijd, die van cruciaal belang is bij het bepalen van het overlevingspercentage van verspreidingsjuvenielen van de derde fase van Bursaphelenchus xylophilus. We adviseren dat hoe l...

Video: Induceren van de inducatie van verspreidingsjuvenielen van  Bursaphelenchus xylophilus  in de derde fase in cryptobiose door osmotische regulatie

Resurrection of Dormant Daphnia magna: Protocol and Applications

Long-term studies enable the identification of eco-evolutionary processes that occur over extended time periods. In addition, they provide key empirical data that may be used in predictive modelling to forecast evolutionary responses of natural ecosystems to future environmental changes. However, excluding a few exceptional cases, long-term studies are scarce because of logistic difficulties associated with accessing temporal samples. Temporal dynamics are frequently studied in the laboratory or in controlled mesocosm experiments with exceptional studies that reconstruct the evolution of natural populations in the wild.
Here, a standard operating procedure (SOP) is provided to revive or resurrect dormant Daphnia magna, a widespread zooplankton keystone species in aquatic ecosystems, to dramatically advance the state-of-the-art longitudinal data collection in natural systems. The field of Resurrection Ecology was defined in 1999 by Kerfoot and co-workers, even though the first attempts at hatching diapausing zooplankton eggs date back to the late 1980s. Since Kerfoot&#39;s seminal paper, the methodology of resurrecting zooplankton species has been increasingly frequently applied, though propagated among laboratories only via direct knowledge transfer. Here, an SOP is described that provides a step-by-step protocol on the practice of resurrecting dormant Daphnia magna eggs.
Two key studies are provided in which the fitness response of resurrected Daphnia magna populations to warming is measured, capitalizing on the ability to study historical and modern populations in the same settings. Finally, the application of next generation sequencing technologies to revived or still dormant stages is discussed. These technologies provide unprecedented power in dissecting the processes and mechanisms of evolution if applied to populations that have experienced changes in selection pressure over time.

Resurrection of Dormant Daphnia magna: Protocol and Applications

Long-term studies are essential to understanding the process of evolution and the mechanisms of adaptation. Generally, these studies require commitments beyond the life-time of researchers. Here, a powerful method is described that dramatically advances state-of-the-art data collection to generate longitudinal data in natural systems.

Long-term studies enable the identification of eco-evolutionary processes that occur over extended time periods. In addition, they provide key empirical ...

Environment

Long-term Behavioral and Reproductive Consequences of Embryonic Exposure to Low-dose Toxicants

Bisphenols, such as bisphenol A (BPA) and bisphenol S (BPS) are polymerizing agents widely used in the production of plastics and numerous everyday-use products. Based on their chemical structure and estradiol-like biological properties, they have been classified as endocrine disrupting compounds (EDC). Long-term exposure to EDCs, even at low doses, has been linked to various health defects including cancer, behavioral disorders and infertility, with greater vulnerability indicated during early developmental periods. Cellular and molecular studies with the genetically tractable nematode model Caenorhabditis elegans have demonstrated that exposure to BPA causes apoptosis, embryonic lethality and disruption in the DNA repair mechanisms. We have previously reported that exposure of C. elegans embryos to low doses of different bisphenols decreases fecundity. In addition, we have shown that the effects of exposure during the very early stages of development persist into adulthood as assayed by quantifying habituation behavior, a form of non-associative learning. Here, we provide detailed protocols for embryonic exposure to low-dose EDCs as well as the associated fecundity and anterior touch habituation assays, along with representative results.

Exposure to environmental toxicants can acutely impact development with long-term effects. Detailed protocols are provided to illustrate a strategy using an effective lab model to study the effect of early embryonic exposure to bisphenol A. We provide fecundity and behavioral assays to monitor the effectiveness of our toxicant exposure bio-assays.

Bisphenols, such as bisphenol A (BPA) and bisphenol S (BPS) are polymerizing agents widely used in the production of plastics and numerous everyday-use ...

Developmental Biology

Reduced Itraconazole Concentration and Durations Are Successful in Treating Batrachochytrium dendrobatidis Infection in Amphibians

Amphibians are experiencing the greatest decline of any vertebrate class and a leading cause of these declines is a fungal pathogen, Batrachochytrium dendrobatidis (Bd), which causes the disease chytridiomycosis. Captive assurance colonies are important worldwide for threatened amphibian species and may be the only lifeline for those in critical threat of extinction. Maintaining disease free colonies is a priority of captive managers, yet safe and effective treatments for all species and across life stages have not been identified. The most widely used chemotherapeutic treatment is itraconazole, although the dosage commonly used can be harmful to some individuals and species. We performed a clinical treatment trial to assess whether a lower and safer but effective dose of itraconazole could be found to cure Bd infections. We found that by reducing the treatment concentration from 0.01-0.0025% and reducing the treatment duration from 11-6 days of 5 min baths, frogs could be cured of Bd infection with fewer side effects and less treatment-associated mortality.

Reduced Itraconazole Concentration and Durations Are Successful in Treating Batrachochytrium dendrobatidis Infection in Amphibians

Batrachochytrium dendrobatidis infection in amphibians can be eliminated using a reduced concentration (0.0025%) and shorter duration (five&#160;minute baths for six days) of itraconazole than is generally used. Less mortality and fewer negative side effects were observed using this modified treatment protocol.

Amphibians are experiencing the greatest decline of any vertebrate class and a leading cause of these declines is a fungal pathogen, Batrachochytrium ...

Immunology and Infection

Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

Plants can resist herbivore damage through three broad mechanisms: antixenosis, antibiosis and tolerance1. Antixenosis is the degree to which the plant is avoided when the herbivore is able to select other plants2. Antibiosis is the degree to which the plant affects the fitness of the herbivore feeding on it1.Tolerance is the degree to which the plant can withstand or repair damage caused by the herbivore, without compromising the herbivore's growth and reproduction1. The durability of herbivore resistance in an agricultural setting depends to a great extent on the resistance mechanism favored during crop breeding efforts3.
We demonstrate a no-choice experiment designed to estimate the relative contributions of antibiosis and tolerance to spittlebug resistance in Brachiaria spp. Several species of African grasses of the genus Brachiaria are valuable forage and pasture plants in the Neotropics, but they can be severely challenged by several native species of spittlebugs (Hemiptera: Cercopidae)4.To assess their resistance to spittlebugs, plants are vegetatively-propagated by stem cuttings and allowed to grow for approximately one month, allowing the growth of superficial roots on which spittlebugs can feed. At that point, each test plant is individually challenged with six spittlebug eggs near hatching. Infestations are allowed to progress for one month before evaluating plant damage and insect survival. Scoring plant damage provides an estimate of tolerance while scoring insect survival provides an estimate of antibiosis. This protocol has facilitated our plant breeding objective to enhance spittlebug resistance in commercial brachiariagrases5.

Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

This video explains mechanisms of host plant resistance to herbivory and demonstrates a no-choice test that estimates the relative contributions of antibiosis and tolerance to spittlebug resistance in Brachiaria spp.

Plants can resist herbivore damage through three broad mechanisms: antixenosis, antibiosis and tolerance1. Antixenosis is the degree to which the plant is ...

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ease of growth and small size. This small size, though, has disallowed a number of technical approaches found in other model systems. For example, blood transfusions in mammalian systems and grafting techniques in plants enable asking questions of circulatory system composition and signaling. The circulatory system of the worm, the pseudocoelom, has until recently been impossible to assay directly. To answer questions of intercellular signaling and circulatory system composition C. elegans researchers have traditionally turned to genetic analysis, cell/tissue specific rescue, and mosaic analysis. These techniques provide a means to infer what is happening between cells, but are not universally applicable in identification and characterization of extracellular molecules. Here we present a newly developed technique to directly assay the pseudocoelomic fluid of C. elegans. The technique begins with either genetic or physical manipulation to increase the volume of extracellular fluid. Afterward the animals are subjected to a vampiric reverse microinjection technique using a microinjection rig that allows fine balance pressure control. After isolation of extracellular fluid, the collected fluid can be assayed by transfer into other animals or by molecular means. To demonstrate the effectiveness of this technique we present a detailed approach to assay a specific example of extracellular signaling molecules, long dsRNA during a systemic RNAi response. Although characterization of systemic RNAi is a proof of principle example, we see this technique as being adaptable to answer a variety of questions of circulatory system composition and signaling.

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The model organism C. elegans uses pseudocoelomic fluid as a passive circulatory system. Direct assay of this fluid has not been previously possible. Here we present a novel technique to directly assay the extracellular space, and use systemic silencing signals during an RNAi response as a proof of principle example.

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ...

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, normal development or changes in environmental conditions2-5. Understanding the cellular signaling pathways that are involved in the response to hypoxia could provide new insight into treatment strategies for diverse human pathologies, from stroke to cancer. This goal has been impeded, at least in part, by technical difficulties associated with controlled hypoxic exposure in genetically amenable model organisms.
The nematode Caenorhabditis elegans is ideally suited as a model organism for the study of hypoxic response, as it is easy to culture and genetically manipulate. Moreover, it is possible to study cellular responses to specific hypoxic O2 concentrations without confounding effects since C. elegans obtain O2 (and other gasses) by diffusion, as opposed to a facilitated respiratory system6. Factors known to be involved in the response to hypoxia are conserved in C. elegans. The actual response to hypoxia depends on the specific concentration of O2 that is available. In C. elegans, exposure to moderate hypoxia elicits a transcriptional response mediated largely by hif-1, the highly-conserved hypoxia-inducible transcription factor6-9. C .elegans embryos require hif-1 to survive in 5,000-20,000 ppm O27,10. Hypoxia is a general term for "less than normal O2". Normoxia (normal O2) can also be difficult to define. We generally consider room air, which is 210,000 ppm O2 to be normoxia. However, it has been shown that C. elegans has a behavioral preference for O2 concentrations from 5-12% (50,000-120,000 ppm O2)11. In larvae and adults, hif-1 acts to prevent hypoxia-induced diapause in 5,000 ppm O212. However, hif-1 does not play a role in the response to lower concentrations of O2 (anoxia, operational definition &lt;10 ppm O2)13. In anoxia, C. elegans enters into a reversible state of suspended animation in which all microscopically observable activity ceases10. The fact that different physiological responses occur in different conditions highlights the importance of having experimental control over the hypoxic concentration of O2.
Here, we present a method for the construction and implementation of environmental chambers that produce reliable and reproducible hypoxic conditions with defined concentrations of O2. The continual flow method ensures rapid equilibration of the chamber and increases the stability of the system. Additionally, the transparency and accessibility of the chambers allow for direct visualization of animals being exposed to hypoxia. We further demonstrate an effective method of harvesting C. elegans samples rapidly after exposure to hypoxia, which is necessary to observe many of the rapidly-reversed changes that occur in hypoxia10,14. This method provides a basic foundation that can be easily modified for individual laboratory needs, including different model systems and a variety of gasses.

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

This paper details how to use continuous-flow hypoxia chambers to generate atmospheres with defined concentrations of O2 to understand biological responses to decreased O2. This system is easy to setup and maintain, and flexible enough to suit a wide range of O2 concentrations and model systems

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, ...

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, most notably developmental arrest and reduced metabolism. Global gene expression profiling via RNA-Seq can provide important insights into the transcriptional mechanisms of photoperiodic diapause. The Asian tiger mosquito, Aedes albopictus, is an outstanding organism for studying the transcriptional bases of diapause due to its ease of rearing, easily induced diapause, and the genomic resources available. This manuscript presents a general experimental workflow for identifying diapause-induced transcriptional differences in A. albopictus. Rearing techniques, conditions necessary to induce diapause and non-diapause development, methods to estimate percent diapause in a population, and RNA extraction and integrity assessment for mosquitoes are documented. A workflow to process RNA-Seq data from Illumina sequencers culminates in a list of differentially expressed genes. The representative results demonstrate that this protocol can be used to effectively identify genes differentially regulated at the transcriptional level in A. albopictus due to photoperiodic differences. With modest adjustments, this workflow can be readily adapted to study the transcriptional bases of diapause or other important life history traits in other mosquitoes.

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

RNA-Seq analyses are becoming increasingly important for identifying the molecular underpinnings of adaptive traits in non-model organisms. Here, a protocol to identify differentially expressed genes between diapause and non-diapause Aedes albopictus mosquitoes is described, from mosquito rearing, to RNA sequencing and bioinformatics analyses of RNA-Seq data.

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, ...

Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus

A method for observing and quantifying the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, was established under a stereomicroscope. To improve the mating efficiency of B. Xylophilus and to increase the chances of mating observation, virgin adults were cultured and used for the investigation. Eggs were obtained by keeping the nematodes in water and allowing the females to lay eggs for 10 min. The second-stage juveniles (J2) were synchronized by incubating the eggs for 24 h at 25 &#176;C in the dark, and the early J4 were obtained by culturing the J2 with grey mold, Botrytis cinerea, for another 52 h. At this time point, most J4 nematodes could be clearly distinguished as being male or female using their genital morphology. The male and female J4 were collected and cultured separately in two different Petri dishes for 24 h to get virgin adult nematodes. A virgin male and a virgin female were paired in a drop of water in the well of a concave slide. The mating behavior was filmed with a video recorder under a stereomicroscope. The whole period of the mating process was 82.8 &#177;3.91 min (mean &#177;SE) and could be divided into 4 different phases: searching, contacting, copulating, and lingering. The mean minutes of duration were 21.8 &#177; 2.0, 28.0 &#177; 1.9, 25.8 &#177; 0.7 and 7.2 &#177; 0.5, respectively. Eleven sub-behaviors were described: cruising, approaching, encountering, touching, hooping, locating, attaching, ejaculating, separating, quiescence, and roaming. Interestingly, obvious intra-sexual competition was observed when one female was grouped with 3 males or one male with 3 females. This protocol is useful and valuable, not only in investigating the mating behavior of B. xylophilus, but also in acting as a reference for ethological studies of other nematodes.

Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus

A protocol for investigating the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, is presented. Behavioral features of B. xylophilus are described in the mating process.

A method for observing and quantifying the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, was established under a stereomicroscope. ...

Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions

The nematode Caenorhabditis elegans is a model system for host-microbe and host-microbiome interactions. Many studies to date use batch digests rather than individual worm samples to quantify bacterial load in this organism. Here it is argued that the large inter-individual variability seen in bacterial colonization of the C. elegans intestine is informative, and that batch digest methods discard information that is important for accurate comparison across conditions. As describing the variation inherent to these samples requires large numbers of individuals, a convenient 96-well plate protocol for disruption and colony plating of individual worms is established.

Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions

This protocol describes a 96-well disruption of individual bacterially colonized Caenorhabditis elegans following cold paralysis and surface bleaching&#160;to remove external bacteria. The resulting suspension is plated on agar plates to allow accurate, medium-throughput quantification of bacterial load in large numbers of individual worms.

The nematode Caenorhabditis elegans is a model system for host-microbe and host-microbiome interactions. Many studies to date use batch digests rather ...

Bradysia coprophila Lab Protocols and Chromosomal Research

Laboratory Maintenance of the Lower Dipteran Fly Bradysia (Sciara) coprophila: A New/Old Emerging Model Organism

Laboratory stocks of the lower dipteran fly, Bradysia (Sciara) coprophila, have been maintained for over a century. Protocols for laboratory upkeep of B. coprophila are presented here. These protocols will be useful for the rapidly increasing number of laboratories studying B. coprophila to take advantage of its unique biological features, which include (1) a monopolar spindle in male meiosis I; (2) non-disjunction of the X dyad in male meiosis II; (3) chromosome imprinting to distinguish maternal from paternal homologs; (4) germ line-limited (L) chromosomes; (5) chromosome elimination (paternal chromosomes in male meiosis I; one to two X chromosomes in early embryos; L chromosomes from the soma in early embryos); (6) sex determination by the mother (there is no Y chromosome); and (7) developmentally regulated DNA amplification at the DNA puff loci in larval salivary gland polytene chromosomes. 
It is now possible to explore these many unique features of chromosome mechanics by using the recent advances in sequencing and assembly of the B. coprophila genome and the development of transformation methodology for genomic engineering. The growing scientific community that uses B. coprophila for research will benefit from the protocols described here for mating the flies (phenotypic markers for mothers that will have only sons or only daughters; details of mass mating for biochemical experiments), checking embryo hatch, feeding larvae, and other comments on its rearing.

Author Spotlight: Exploring Bradysia coprophila's Unique Biology &#8211; A Guide to Laboratory Maintenance

This paper outlines the laboratory maintenance (including mating and feeding) of the lower dipteran fly Bradysia (Sciara) coprophila.

Laboratory stocks of the lower dipteran fly, Bradysia (Sciara) coprophila, have been maintained for over a century. Protocols for laboratory upkeep of B. ...

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

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