Najie Zhu and Liqun Bai contributed equally to this work. This work was financially supported by the Special Fund for Forestry Scientific Research in the Public Welfare (201204501) and the National Natural Science Foundation of China (31170604, 31270688, and 31570638). We thank Dr. Holighaus Gerrit and Dr. Danielle Hickford for their helpful suggestions on English writing.

1. Acquisition of Virgin Adult Nematodes
<ol>
	<li>Pinewood nematode isolate and culture conditions 
	NOTE: The B. xylophilus isolate Nxy61 was originally extracted from a diseased Pinus massoniana in the Ningbo area of Zhejiang province in China.
	<ol>
		<li>Prepare Potato Dextrose Agar (PDA, See Table of Materials) plates for growing Botrytis cinerea. </li>
		<li>For inoculation, punch a piece of fungal mat with diameter of 10 mm from a B. cinerea mat and put it in the middle of a new PDA plate. Culture the B. cinerea for 5 d at 25 &#176;C in the dark.</li>
		<li>Culture the pinewood nematodes in the prepared petri dish containing B. cinerea at 25 &#176;C in the dark.</li>
	</ol>
	</li>
	<li>Acquisition of B. xylophilus eggs
	<ol>
		<li>Place a clamped rubber tube below a funnel and hold the prepared funnel in a rack to assemble the Baermann funnel apparatus. Place two layers of filter paper instead of gauze in the mouth of the funnel.</li>
		<li>Transfer the fungal cultures to the funnel setup and add water until the fungal mat is immersed. Wait 2 h, and nematodes will come out from the fungal culture, cross the filter paper, and settle down in the lowest part of the Baermann funnel.</li>
		<li>Gather the first couple of drops of water from the bottom of the tube into a 15&#160;mL tube and collect nematodes by slowly releasing the clamp on the tube.</li>
		<li>Centrifuge the above collections at 3,000 x g for 2 min at RT.</li>
		<li>Remove the upper layer of water with a pipette and resuspend it with 1 mL of sterile double distilled (dd)&#160;H2O.</li>
		<li>With a pipette, transfer the above resuspension to a new glass Petri dish (6&#160;cm diameter). Add 3 mL of sterile dd H2O to the Petri dish to ensure that the nematodes are able to swim freely.</li>
		<li>Keep the nematodes in the glass Petri dish for 10 min at 25 &#176;C in the dark. Eggs will be laid by pregnant females. Because of their glycoprotein surface, the eggs will stick to the bottom of the glass Petri dish.</li>
		<li>Remove the water and worms carefully, without disturbing the eggs. Add 3 mL of sterile dd H2O to the glass Petri dish immediately to prevent the eggs from drying out.</li>
		<li>Repeat step 1.2.8 3x to remove all the larvae and adults, and obtain the pure eggs. Add an extra 2 mL (5 mL in total) of sterile dd H2O to the Petri dish.</li>
	</ol>
	</li>
	<li>Acquisition of the fourth-stage juveniles (J4 larvae)
	<ol>
		<li>Hatch the collected eggs for 24 h in the dark at 25 &#176;C to obtain J2 larvae.</li>
		<li>Transfer the J2 larvae with a pipette into a 15&#160;mL tube, centrifuge&#160;at 3,000 x g for 2 min, and remove the upper layer of water.</li>
		<li>Resuspend the J2 larvae with 200 &#181;L of sterile dd H2O, and then transfer them to the PDA plate containing B. cinerea.</li>
		<li>Culture the J2 larvae for 52 h at 25 &#176;C in the dark. At this time point, most J2 larvae will have developed into the early stage of J4 larvae.</li>
	</ol>
	</li>
	<li>Acquisition of virgin adult nematodes
	<ol>
		<li>Prepare a needle for picking up nematodes by drawing a glass capillary after heating it over an alcohol burner. 
		NOTE: The tip of this capillary needle usually needs to be 50 &#181;m in diameter, roughly the body width of an adult nematode, so that it can be used as a tool for picking up a single nematode.</li>
		<li>Assemble a Baermann funnel and extract nematodes from the fungal cultures for 2 h, as described in steps 1.2.1 - 1.2.3.</li>
		<li>Transfer the J4 with water into a clean Petri dish.</li>
		<li>Observe the nematodes under a stereomicroscope and distinguish between males and females by genital morphology, as described by Mamiya and Kiyohara8 (Figure 1). Pick up a nematode of the desired sex with the prepared capillary needle and transfer it to the appropriate fungal-culture plate for males or females. 
		NOTE: There are more females than males, with the percentage of females at 60 - 70%. The female vulva is obviously different from the male spicule. Compared to the adult, the spicule or vulva in early-stage J4 is smaller, but they can still be easily seen and distinguished. The nematode is always moving in the water, which sometimes increases the difficulty in distinguishing males and females.</li>
		<li>Culture the J4 larvae for another 24 h at 25 &#176;C in the dark. 
		NOTE: All the nematodes should develop into adults, but since they are unmated, they are regarded as virgin adults.</li>
		<li>Assemble two Baermann funnels and extract the virgin adults from the fungal cultures for 2 h, as described in steps 1.2.1 - 1.2.3, and transfer the virgin adult males and females into two separate, clean Petri dishes.</li>
	</ol>
	</li>
</ol>
2. Observation and Video Recording of Mating Behavior
<ol>
	<li>Prepare several concave slides and add 200 &#181;L of sterile dd H2O to each of their wells.</li>
	<li>Pick up the prepared virgin adult male or and female with a capillary needle and put only one nematode into the water of each concave slide.</li>
	<li>Keep the virgin adults inside the well for 1 h to let them adapt to the new aqueous environment.</li>
	<li>Pair a virgin male and female by transferring one of them from one well to another using a pipette.</li>
	<li>Observe the mating behavior of the 1&#9792; + 1&#9794; combination in the concave slide under a stereomicroscope.</li>
	<li>To analyze the mating efficiency of virgin adults, calculate their mating success rate (R) and compare it with that of randomly selected adult pairs. Calculate R as the percentage of replicates in which copulation was observed compared to the total number of replicates studied.</li>
	<li>To investigate mating choice and intrasexual competition, group and observe virgin nematodes of two different combinations: 1&#9792; + 3&#9794; and 3&#9792; + 1&#9794;.</li>
	<li>In order to collect data for qualitative and quantitative analysis, film the mating behavior using video microscopy. Normally, the whole mating process, from the time when the nematodes are placed in the well of the concave slide to the moment when they move away after copulation, is completed within 2 h. Therefore, record the mating process continuously for at least 2 h as one replicate. Complete at least 30 replicates in total for each combination. 
	NOTE: Add more sterile dd H2O (usually 50 &#181;L) every 0.5 h to the well to compensate for evaporation during the recording process.</li>
</ol>
3. Data Collection
<ol>
	<li>View all videos carefully on a computer screen and characterize the sub-behaviors qualitatively.</li>
	<li>Measure the duration of mating using the frame-by-frame function of the video recorder. Manually subtract the starting time from the end point.</li>
	<li>For the 1&#9792; + 1&#9794; combination, analyze each video recording and measure the duration of the following events:
	<ol>
		<li>Measure the duration of searching (i.e., the time from when the female and male are introduced into the well of the concave slide to the moment they encounter and contact each other).</li>
		<li>Measure the duration of contacting (i.e., the time from the first contact to the moment when the male is precisely positioned at the female&#39;s vulva for the first copulation).</li>
		<li>Measure the duration of copulating (i.e., the time from when the male is precisely positioned at the female&#39;s vulva until he slides down from the female&#39;s body after ejaculation). Note: Some nematodes can copulate several times in 2 h. In such cases, measure the duration of copulation from the first copulation to the end of the last one, including the time in between.</li>
		<li>Measure the duration of lingering (i.e., the period after copulation until the male and female swim away from each other and are separated by a distance of more than the nematodes&#39; body length (usually 1 mm)).</li>
	</ol>
	</li>
	<li>Count the copulation frequency and duration of every copulation and the average duration of all copulations in 2 h for each of the different combinations (i.e., 1&#9792; + 1&#9794;, 3&#9792; + 1&#9794;, and 1&#9792; + 3&#9794;).</li>
	<li>Calculate the mating success rate for the first contact (P) for the different combinations as the success rate of mating after the first contact, which is conducted immediately and without any break. In the contacting phase, several contacts might be observed before actual copulation.
	<ol>
		<li>When the distance between two individuals is more than the body length, consider one contact to be over. For one replicate, record P as 100% when copulation is observed immediately after the first contact, without break, or 0.00% if no copulation was observed or if there was copulation but after two or more contacts. 
		NOTE: There were 30 replicates for each combination described here.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Tokushige, Y., Kiyohara, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bursaphelenchus+sp.+in+the+wood+of+dead+pine+trees.">Bursaphelenchus sp. in the wood of dead pine trees.</a> J. Jap. For. Soc. 51, 193-195 (1969).</li><li>Sun, Y. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bursaphelenchus+xylophilus+was+discovered+in+Sun+Yet-sen's+mausoleum+in+Nanjing.">Bursaphelenchus xylophilus was discovered in Sun Yet-sen's mausoleum in Nanjing.</a> J. Jiangsu Fore. Sci. Tech. (in Chinese). 4, 47 (1982).</li><li>Mota, M. M., 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=First+report+of+Bursaphelenchus+xylophilus+in+Portugal+and+in+Europe.">First report of Bursaphelenchus xylophilus in Portugal and in Europe.</a> Nematology. 1, 727-734 (1999).</li><li>Robertson, 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=Incidence+of+the+pine+wood+nematode+Bursaphelenchus+xylophlius+Steiner+&amp;+Buhrer,+1934+(Nickle,+1970)+in+Spain.">Incidence of the pine wood nematode Bursaphelenchus xylophlius Steiner &amp; Buhrer, 1934 (Nickle, 1970) in Spain.</a> Nematology. 13, 755-757 (2011).</li><li>Vicente, C., Espada, M., Vieira, P., Mota, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pine+wilt+disease:+a+threat+to+European+forestry.">Pine wilt disease: a threat to European forestry.</a> Euro. J. Plant Path. 133, 89-99 (2012).</li><li>Jones, J. T., Moens, M., Mota, M., Li, H., Kikuchi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bursaphelenchus+xylophilus:+opportunities+in+comparative+genomics+and+molecular+host-parasite+interactions.">Bursaphelenchus xylophilus: opportunities in comparative genomics and molecular host-parasite interactions.</a> Mol. Plant. Path. 9, 357-368 (2008).</li><li>Mamiya, Y., Furukawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fecundity+and+reproduction+of+Bursaphelenchus+lignicolus.">Fecundity and reproduction of Bursaphelenchus lignicolus.</a> Jap. J. Nemat. 7, 6-9 (1977).</li><li>Mamiya, Y., Kiyohara, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Description+of+Bursaphelenchus+lignicolus.+n.+sp.+(Nematoda:+Aphelenchoididae)+from+pine+wood+and+histophathology+of+nematode-infested+trees.">Description of Bursaphelenchus lignicolus. n. sp. (Nematoda: Aphelenchoididae) from pine wood and histophathology of nematode-infested trees.</a> Nematologica. 18, 120-124 (1972).</li><li>Liu, B. J., 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=Behavioural+features+of+Bursaphelenchus+xylophilus+in+the+mating+process.">Behavioural features of Bursaphelenchus xylophilus in the mating process.</a> Nematology. 16, 895-902 (2014).</li><li>Simon, J. M., Sternberg, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+a+mate+finding+cue+in+the+hermaphrodite+nematode+Caenorhabditis+elegans.">Evidence of a mate finding cue in the hermaphrodite nematode Caenorhabditis elegans.</a> Proc. Natl. Acad. Sci. USA. 99, 1598-1603 (2002).</li><li>Barr, M. M., Garcia, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Male+mating+behaviour.">Male mating behaviour.</a> The C. elegans Research Community Wormbook. , (2006).</li><li>Shinya, R., Chen, A., Sternberg, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+attraction+and+mating+in+Bursaphelenchus+okinawaensis+and+B.+xylophilus.">Sex attraction and mating in Bursaphelenchus okinawaensis and B. xylophilus.</a> J. Nematol. 47 (3), 176-183 (2015).</li></ol>

The authors have nothing to disclose.

Behavioral tests are a fundamental aspect of chemical ecology, neurobiology, molecular biology, and genetics. Nematodes, and particularly C. elegans, are extensively used for studies in these areas. The mating behavior of C. elegans has been investigated previously10-11. However, B. xylophilus is gonochoristic and different from the hermaphroditic C. elegans, and its mating process has different behavioral features9. Recently, the mating behavior of Bursaphelenchus okinawaensis, a hermaphroditic sister species of B. xylophilus, was reported12. Though different experimental methods were used, this hermaphroditic species showed similar sex attraction to B. xylophilus, and inter-species attraction between them was observed. This hermaphroditic sister species might be used as a model to conduct further investigation on the biology of Bursaphelenchus.
This protocol provides a guideline on how to observe and investigate the behavioral features of B. xylophilus during mating. Our findings showed that using virgin adult nematodes is critical for increasing the chance of successfully observing the mating process (Figure&#160;1). Obtaining J4 larvae and precisely distinguishing their sexes are two important fundamental steps for the acquisition of virgin adults. Under our experimental conditions (i.e., at 25 &#176;C in the dark), the period of 52 h is the proper time to obtain J4 from J2 larvae. However, distinguishing sex at the early J4 stage mostly depends on personal experience. To ensure accuracy, it is necessary for different people to double-check the sex allocation.
The behavioral features of B. xylophilus during the mating process can be described qualitatively and analyzed quantitatively by recording the durations of the four different phases, the percentage of mating success after first contact, the copulation frequency, and the copulation duration of different combinations. Interestingly, for the first time, we observed mating choice by females and strong intra-sexual competition in both males and females. No promiscuity was observed; however, this could be due to the limited number of nematodes used in our investigation. This protocol can not only be used for investigating the mating behavior of B. xylophilus, but it can also be useful and valuable as a reference for other nematodes.

The pinewood nematode, Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle, is one of the most destructive invasive species, causing the wilting and eventual death of pine trees. This pathogenic nematode is believed to be native to the USA, but it was transported to several countries, including China, Japan, South Korea, and Portugal. Recently, it has also been reported in Spain, killing millions of pine trees at huge economic losses and threatening forest production and ecological stability1-5.
Once a host pine tree is infected by pinewood nematodes, thousands of millions of offspring are propagated rapidly within the trunk. This causes xylem dysfunction, resulting in wilting and eventually in the death of the host tree6. However, there is currently no efficient way to control this disease. Mating behavior might play an important role in the high fecundity of this nematode7. We therefore investigated the mating behavior of B. xylophilus in the laboratory, trying to find an effective way of disturbing its mating and decreasing its fecundity. 
This protocol is intended to introduce the detailed methods of how to obtain the virgin adults of B. xylophilus and how to observe and analyze the mating behavior using a video recorder and a stereomicroscope. This protocol can also be used as a reference for behavioral studies of other nematodes.

<table border="0" cellpadding="0" cellspacing="0" width="944">
	<tbody>
		<tr height="18">
			<td height="18" style="height:18px;width:233px;">B. xylophilus isolate Nxy61</td>
			<td style="width:123px;"></td>
			<td style="width:125px;"></td>
			<td style="width:463px;">Extracted from Pinus massoniana in Ningbo area of China</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Grey mold strain</td>
			<td></td>
			<td></td>
			<td>Obtained from Forestry Academy of China</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Baermann funnels</td>
			<td>Sengong</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">falcon tubes</td>
			<td>Sengong</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">centrifuge</td>
			<td>Sengong</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pipette</td>
			<td>Sengong</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">sterile dd H2O</td>
			<td></td>
			<td></td>
			<td>Sterilized by high pressure of steam at 121&#176;C for 30 min.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri dishes</td>
			<td>Sengong</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PDA medium</td>
			<td>Huankai Bio.</td>
			<td>021050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">incubator</td>
			<td>Sengong</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Laminar flow</td>
			<td>Sengong</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">glass capillary</td>
			<td>Sengong</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stereoscope</td>
			<td>Leica</td>
			<td>LED5000 RL</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">inverted stereo microscope</td>
			<td>Zeiss</td>
			<td>A1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">concave slides</td>
			<td>Sengong</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The mating success rate (R) of virgin adults was 86.67% on average, which was significantly higher than that of the randomly selected adults: 13.33&#160;&#177; 4.65% (F = 1301.71, df = 1, P = 0.0001) (Figure&#160;2). This finding suggests that it is easier to observe the mating behavior of B. xylophilus with virgin adults than with randomly selected adults.
When one male was paired with one female, the entire mating process lasted 82.8 min on average. The whole process could be described by four different phases: searching, contacting, copulating, and lingering. The duration for these four phases were 21.8 &#177; 2.0 min, 28.0 &#177; 1.9 min, 25.8 &#177; 0.7 min, and 7.2 &#177; 0.5 min, respectively (Figure&#160;3). In a recent publication, we described the 11 sub-behaviors as cruising, approaching, encountering, touching, hooping, locating, attaching, ejaculating, separating, quiescence, and roaming9.
When one male met two or more females simultaneously, the male seemed to mate randomly with one of them, without any selection. However, obvious mating choice was observed for female B. xylophilus. When a female contacted a male, the female tended to touch the male&#39;s head, body, tail, and genital region gently with her head and appeared to be evaluating her potential partner. If the female did not accept the male, the female left him quickly and swam to another male for the next evaluation. One of the males was then selected by the female for mating. Mating success rates after the first contact (P) of the 1&#9792; + 1&#9794;, 3&#9792; + 1&#9794;, and 1&#9792; + 3&#9794; combinations were 46.7&#160;&#177; 9.3%, 80.0&#160;&#177; 7.4%, and 23.4&#160;&#177; 7.4%, respectively, and the differences between them were significant (F = 8.21, df = 2, P = 0.0124) (Figure&#160;4).
Intrasexual competition was observed in both males and females. When one virgin female was mixed with 3 virgin males, the males tried to approach and contact the female as much as possible to get the chance of mating with her. When the vulva was occupied by one male, the other males tried to disturb the mating male by using violent body swings or dragging to separate them. Female intrasexual competition was seen to be more or less similar to male intrasexual competition.
Mating success mainly depended on the female. When a female accepted a male, the male moved more slowly and curled or even formed a hoop, and the female moved forward actively to make it easier for the male to locate her vulva and to avoid interference from other males. If a female did not accept a male, the female tried to shake him off, even if the male had mounted and positioned himself by her vulva. Because of the intrasexual competition, copulation frequencies of the 1&#9792; + 1&#9794;, 3&#9792; + 1&#9794;, and 1&#9792; + 3&#9794; combinations were 1.43 &#177; 0.091, 1.17 &#177; 0.074 and 1.83 &#177; 0.074, respectively, and the differences between them were significant (F = 14.50, df = 2, P = 0.0001) (Figure&#160;5).
In our investigation, the copulation duration varied greatly, with a minimum of 20 s and a maximum of 42 min. According to our data, the duration of copulation could be obviously differentiated into two groups: short-term copulations, which lasted less than 5 min, and long-term copulations, which lasted longer than 10 min. Obvious differences were observed between the different groups for the average duration of short-term copulation (F = 3.81, df = 2, P = 0.0296), long-term copulation (F = 179.52, df = 2, P = 0.0001), and all copulations (F = 2.93, df = 2, P = 0.0367) (Figure&#160;6).
<img alt="Figure 1" src="/files/ftp_upload/54842/54842fig1.jpg" /> 
Figure 1: Genitalia of Male and Female B. Xylophilus. (a) spicule of male (blue arrow). (b) vulva of female (red arrow). <a href="http://cloudflare.jove.com/files/ftp_upload/54842/54842fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54842/54842fig2.jpg" /> 
Figure 2: Mating Success Rate of Virgin Adult Couples and Randomly Selected Adult Couples of B. xylophilus. The calculated mean values are from 30 replicates. The vertical bar shows the standard error. The different lowercase letters indicate significant differences at P &#60;0.05 when compared by Tukey&#39;s test. <a href="http://cloudflare.jove.com/files/ftp_upload/54842/54842fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54842/54842fig3.jpg" /> 
Figure 3: Duration of the 4 Phases in the Mating Process of B. xylophilus. The calculated mean values are from 30 replicates. The vertical bar shows the standard error. The different lowercase letters indicate significant differences at P &#60;0.05 when compared by Tukey&#39;s test. The average duration of the copulating phase is 25.80 &#177; 0.71 min, which is 31.17% of the whole mating process. <a href="http://cloudflare.jove.com/files/ftp_upload/54842/54842fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54842/54842fig4.jpg" /> 
Figure 4: Mating Success for the First Contact of B. xylophilus. The calculated mean values are from 30 replicates &#177;SE. Means with the same lowercase letter within each column are not significantly different at P &#60;0.05 when compared by Tukey&#39;s Honest Significant Difference test. <a href="http://cloudflare.jove.com/files/ftp_upload/54842/54842fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54842/54842fig5.jpg" /> 
Figure 5: Copulation Frequency of B. xylophilus in Different Groups. The calculated mean values are from 30 replicates &#177;SE. Means with the same lowercase letter are not significantly different at P &#60;0.05 when compared by Tukey&#39;s Honest Significant Difference test. <a href="http://cloudflare.jove.com/files/ftp_upload/54842/54842fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/54842/54842fig6.jpg" /> 
Figure 6: Copulation Duration of B. xylophilus. Means with the same lowercase letter within each set of columns are not significantly different at P &#60;0.05 when compared by Tukey&#39;s Honest Significant Difference test. <a href="http://cloudflare.jove.com/files/ftp_upload/54842/54842fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus at JoVE.com

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.

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. ...

observation-quantification-mating-behavior-pinewood-nematode

Nanyang Technological University

Research

JoVE Journal

Biology

9.3K Views.  Zhejiang Agricultural & Forestry University. 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.

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

Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis

The unicellular green alga Chlamydomonas reinhardtii (Chlamydomonas) has become a popular organism for research in diverse areas of cell biology and genetics because of its simple life cycle, ease of growth and manipulation for genetic analysis, genomic resources, and transformability of the nucleus and both organelles. Mating strains is a common practice when genetic approaches are used in Chlamydomonass, to create vegetative diploids for analysis of dominance, or following tetrad dissection to ascertain nuclear vs. organellar inheritance, to test allelism, to analyze epistasy, or to generate populations for the purpose of map-based cloning. Additionally, genetic crosses are routinely used to combine organellar genotypes with particular nuclear genotypes. Here we demonstrate standard methods for gametogenesis, mating, zygote germination and tetrad separation. This protocol consists of an easy-to-follow series of steps that will make genetic approaches amenable to scientists who are less familiar with Chlamydomonas. Key parameters and trouble spots are explained. Finally, resources for further information and alternative methods are provided.

Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis

Mating and tetrad separation are required for genetic analysis in Chlamydomonas reinhardtii. Here we demonstrate standard methods for gametogenesis, mating, zygote germination and tetrad dissection. This protocol consists of an easy-to-follow series of steps that will make genetic approaches amenable to scientists who are less familiar with Chlamydomonas.

The unicellular green alga Chlamydomonas reinhardtii (Chlamydomonas) has become a popular organism for research in diverse areas of cell biology and ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression analyses by RNA interference and stable transgenesis remain limited in many species due to the particular anatomy and molecular cellular biology of the organism. For example, outside of the crown group Caenorhabditis that includes Caenorhabditis elegans3, stably transmitted transgenic lines in non-Caenorhabditis species have not been reported in this specious phylum (Nematoda), with the exception of Strongyloides stercoralis4 and Pristionchus pacificus5. To facilitate the expanding role of P. pacificus in the study of development, evolution, and behavior6-7, we describe here the current methods to use microinjection for making transgenic animals and gene knock down by RNAi. Like the gonads of C. elegans and most other nematodes, the gonads of P. pacificus is syncitial and capable of incorporating DNA and RNA into the oocytes when delivered by direct microinjection. Unlike C. elegans however, stable transgene inheritance and somatic expression in P. pacificus requires the addition of self genomic DNA digested with endonucleases complementary to the ends of target transgenes and coinjection markers5. The addition of carrier genomic DNA is similar to the requirement for transgene expression in Strongyloides stercoralis4 and in the germ cells of C. elegans. However, it is not clear if the specific requirement for the animals' own genomic DNA is because P. pacificus soma is very efficient at silencing non-complex multi-copy genes or that extrachromosomal arrays in P. pacificus require genomic sequences for proper kinetochore assembly during mitosis. The ventral migration of the two-armed (didelphic) gonads in hermaphrodites further complicates the ability to inject both gonads in individual worms8. We also demonstrate the use of microinjection to knockdown a dominant mutant (roller,tu92) by injecting double-stranded RNA (dsRNA) into the gonads to obtain non-rolling F1 progeny. Unlike C. elegans, but like most other nematodes, P. pacificus PS312 is not receptive to systemic RNAi via feeding and soaking and therefore dsRNA must be administered by microinjection into the syncitial gonads. In this current study, we hope to describe the microinjection process needed to transform a Ppa-egl-4 promoter::GFP fusion reporter and knockdown a dominant roller prl-1 (tu92) mutant in a visually informative protocol.

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

In model organisms, transgenesis can manipulate gene functions while RNAi can knockdown specific mRNA transcripts 1-2. This protocol aims to illustrate the techniques needed to introduce stably transmitted DNA and transient double stranded RNA into the necromenic nematode Pristionchus pacificus for studies in evolutionary, developmental, and behavioral biology.

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression ...

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo4,5. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- &beta; - like ligand DBL-1, so this assay is appropriate for identification of TGF-&beta; signaling components7. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-&beta; signaling.

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a novel Drosophila circadian output, called the temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night 3. Surprisingly, the TPR and locomotor activity are controlled through distinct circadian neurons3. Drosophila locomotor activity is a well&#160;known circadian behavioral output and has provided strong contributions to the discovery of many conserved mammalian circadian clock genes and mechanisms4. Therefore, understanding TPR will lead to the identification of hitherto unknown molecular and cellular circadian mechanisms. Here, we describe how to perform and analyze the TPR assay. This technique not only allows for dissecting the molecular and neural mechanisms of TPR, but also provides new insights into the fundamental mechanisms of the brain functions that integrate different environmental signals and regulate animal behaviors. Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR3. Drosophila are ectotherms, in which the body temperature is typically behaviorally regulated. Therefore, TPR is a strategy used to generate a rhythmic body temperature in these flies5-8. We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals.

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

We recently identified a novel Drosophila circadian output, temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night. TPR is regulated independently from another circadian output, locomotor activity. Here&#160;we describe the design and analysis of TPR in Drosophila.

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a ...

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization (FISH) with PNA Probes in Caenorhabditis elegans

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase or an alternative pathway, known as alternative lengthening of telomeres (ALT)1. Recently, C. elegans has emerged as a multicellular model organism for the study of telomere and ALT2. Visualization of repetitive sequences in the genome is critical in understanding the biology of telomeres. While telomere length can be measured by telomere restriction fragment assay or quantitative PCR, these methods only provide the averaged telomere length. On the contrary, fluorescence in situ hybridization (FISH) can provide the information of the individual telomeres in cells. Here, we provide protocols and representative results of the method to determine telomere length of C. elegans by fluorescent in situ hybridization. This method provides a simple, but powerful, in situ procedure that does not cause noticeable damage to morphology. By using fluorescently labeled peptide nucleic acid (PNA) and digoxigenin-dUTP-labeled probe, we were able to visualize two different repetitive sequences: telomere repeats and template of ALT (TALT) in C. elegans embryos and gonads.

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization FISH with PNA Probes in Caenorhabditis elegans

We report a concise procedure of fluorescence in situ hybridization (FISH) in the gonad and embryos of Caenorhabditis elegans for observing and quantifying repetitive sequences. We successfully observed and quantified two different repetitive sequences, telomere repeats and template of alternative lengthening of telomeres (TALT).

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase ...

Quantification of Proliferative and Dead Cells in Enteroids

The intestinal epithelium acts as a barrier that prevents luminal contents, such as pathogenic microbiota and toxins, from entering the rest of the body. Epithelial barrier function requires the integrity of intestinal epithelial cells. While epithelial cell proliferation maintains a continuous layer of cells that forms a barrier, epithelial damage leads to barrier dysfunction. As a result, luminal contents can across the intestinal barrier via an unrestricted pathway. Dysfunction of intestinal barrier has been associated with many intestinal diseases, such as inflammatory bowel disease. Isolated mouse intestinal crypts can be cultured and maintained as crypt-villus-like structures, which are termed intestinal organoids or &ldquo;enteroids&rdquo;. Enteroids are ideal to study the proliferation and cell death of intestinal epithelial cells in vitro. In this protocol, we describe a simple method to quantify the number of proliferative and dead cells in cultured enteroids. 5-ethynyl-2&rsquo;-deoxyuridine (EdU) and propidium iodide are used to label proliferating and dead cells in enteroids, and the proportion of proliferating and dead cells are then analyzed by flow cytometry. This is a useful tool to test the effects of drug treatment on intestinal epithelial cell proliferation and cell survival.

The presented protocol uses flow cytometry to quantify the number of proliferating and dead cells in cultured mouse enteroids. This method is helpful to evaluate the effects of drug treatment on organoid proliferation and survival.

The intestinal epithelium acts as a barrier that prevents luminal contents, such as pathogenic microbiota and toxins, from entering the rest of the body. ...

Culturing and Screening the Plant Parasitic Nematode Ditylenchus dipsaci

Plant-parasitic nematodes (PPNs) destroy over 12% of global food crops every year, which equates to roughly 157 billion dollars (USD) lost annually. With a growing global population and limited arable land, controlling PPN infestation is critical for food production. Compounding the challenge of maximizing crop yields are the mounting restrictions on effective pesticides because of a lack of nematode selectivity. Hence, developing new and safe chemical nematicides is vital to food security. In this protocol, the culture and collection of the PPN species Ditylenchus dipsaci are demonstrated. D. dipsaci is both economically damaging and relatively resistant to most modern nematicides. The current work also explains how to use these nematodes in screens for novel small molecule nematicides and reports on data collection and analysis methodologies. The demonstrated pipeline affords a throughput of thousands of compounds per week and can be easily adapted for use with other PPN species such as Pratylenchus penetrans. The techniques described herein can be used to discover new nematicides, which may, in turn, be further developed into highly selective commercial products that safely combat PPNs to help feed an increasingly hungry world.

Culturing and Screening the Plant Parasitic Nematode Ditylenchus dipsaci

The present protocol describes a reliable and straightforward method for culturing, collecting, and screening Ditylenchus dipsaci.

Plant-parasitic nematodes (PPNs) destroy over 12% of global food crops every year, which equates to roughly 157 billion dollars (USD) lost annually. With ...

Application of RNA Interference in the Pinewood Nematode, Bursaphelenchus xylophilus

The pinewood nematode, Bursaphelenchus xylophilus, is one of the most destructive invasive species worldwide, causing the wilting and eventual death of pine trees. Despite the recognition of their economic and environmental significance, it has thus far been impossible to study the detailed gene functions of plant-parasitic nematodes (PPNs) using conventional forward genetics and transgenic methods. However, as a reverse genetics technology, RNA interference (RNAi) facilitates the study of the functional genes of nematodes, including B. xylophilus. 
This paper outlines a new protocol for RNAi of the ppm-1 gene in B. xylophilus, which has been reported to play crucial roles in the development and reproduction of other pathogenic nematodes. For RNAi, the T7 promoter was linked to the 5&#8242;-terminal of the target fragment by polymerase chain reaction (PCR), and double-stranded RNA (dsRNA) was synthesized by in vitro transcription. Subsequently, dsRNA delivery was accomplished by soaking the nematodes in a dsRNA solution mixed with synthetic neurostimulants. Synchronized juveniles of B. xylophilus (approximately 20,000 individuals) were washed and soaked in dsRNA (0.8 &#181;g/mL) in the soaking buffer for 24 h in the dark at 25 &#176;C.
The same quantity of nematodes was placed in a soaking buffer without dsRNA as a control. Meanwhile, another identical quantity of nematodes was placed in a soaking buffer with green fluorescent protein (gfp) gene dsRNA as a control. After soaking, the expression level of the target transcripts was determined using real-time quantitative PCR. The effects of RNAi were then confirmed using microscopic observation of the phenotypes and a comparison of the body size of the adults among the groups. The current protocol can help advance research to better understand the functions of the genes of B. xylophilus and other parasitic nematodes toward developing control strategies through genetic engineering.

Application of RNA Interference in the Pinewood Nematode, Bursaphelenchus xylophilus

Here, we introduce a detailed soaking method of RNA interference in Bursaphelenchus xylophilus to facilitate the study of gene functions.

The pinewood nematode, Bursaphelenchus xylophilus, is one of the most destructive invasive species worldwide, causing the wilting and eventual death of ...

Observation of Photobehavior in Chlamydomonas reinhardtii

For the survival of the motile phototrophic microorganisms, being under proper light conditions is crucial. Consequently, they show photo-induced behaviors (or photobehavior) and alter their direction of movement in response to light. Typical photobehaviors include photoshock (or photophobic) response and phototaxis. Photoshock is a response to a sudden change in light intensity (e.g., flash illumination), wherein organisms transiently stop moving or move backward. During phototaxis, organisms move toward the light source or in the opposite direction (called positive or negative phototaxis, respectively). The unicellular green alga Chlamydomonas reinhardtii is an excellent organism to study photobehavior because it rapidly changes its swimming pattern by modulating the beating of cilia (a.k.a., flagella) after photoreception. Here, various simple methods are shown to observe photobehaviors in C. reinhardtii. Research on C. reinhardtii&#39;s photobehaviors has led to the discovery of common regulatory mechanisms between eukaryotic cilia and channelrhodopsins, which may contribute to a better understanding of ciliopathies and the development of new optogenetics methods.

Observation of Photobehavior in Chlamydomonas reinhardtii

Most swimming photoautotrophic organisms show photo-induced behavioral changes (photobehavior). The present protocol observes the said photobehavior in the model organism Chlamydomonas reinhardtii.

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

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Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis

Quantification of γH2AX Foci in Response to Ionising Radiation

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization (FISH) with PNA Probes in Caenorhabditis elegans

Quantification of Proliferative and Dead Cells in Enteroids

Culturing and Screening the Plant Parasitic Nematode Ditylenchus dipsaci

Application of RNA Interference in the Pinewood Nematode, Bursaphelenchus xylophilus

Observation of Photobehavior in Chlamydomonas reinhardtii