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><tbody><tr><td>&lt;em&gt;B. xylophilus&lt;/em&gt; isolate &lt;em&gt;Nxy61&lt;/em&gt;</td><td></td><td></td><td>Extracted from &lt;em&gt;Pinus massoniana&lt;/em&gt; in Ningbo area of China</td></tr><tr><td>Grey mold strain</td><td></td><td></td><td>Obtained from Forestry Academy of China</td></tr><tr><td>Baermann funnels</td><td>Sengong</td><td></td><td></td></tr><tr><td>falcon tubes</td><td>Sengong</td><td></td><td></td></tr><tr><td>centrifuge</td><td>Sengong</td><td></td><td></td></tr><tr><td>pipette</td><td>Sengong</td><td></td><td></td></tr><tr><td>sterile dd H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>Sterilized by high pressure of steam at 121 &amp;deg;C for 30 min.</td></tr><tr><td>Petri dishes</td><td>Sengong</td><td></td><td></td></tr><tr><td>PDA medium</td><td>Huankai Bio.</td><td>021050</td><td></td></tr><tr><td>incubator</td><td>Sengong</td><td></td><td></td></tr><tr><td>Laminar flow</td><td>Sengong</td><td></td><td></td></tr><tr><td>glass capillary</td><td>Sengong</td><td></td><td></td></tr><tr><td>Stereoscope</td><td>Leica</td><td>LED5000 RL</td><td></td></tr><tr><td>inverted stereo microscope</td><td>Zeiss</td><td>A1</td><td></td></tr><tr><td>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="https://cloudflare-jove-com.bdigitaluss.remotexs.co/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="https://cloudflare-jove-com.bdigitaluss.remotexs.co/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="https://cloudflare-jove-com.bdigitaluss.remotexs.co/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="https://cloudflare-jove-com.bdigitaluss.remotexs.co/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="https://cloudflare-jove-com.bdigitaluss.remotexs.co/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="https://cloudflare-jove-com.bdigitaluss.remotexs.co/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. ...

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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

A Noninvasive Method For In situ Determination of Mating Success in Female American Lobsters (Homarus americanus)

Despite being one of the most productive fisheries in the Northwest Atlantic, much remains unknown about the natural reproductive dynamics of American lobsters. Recent work in exploited crustacean populations (crabs and lobsters) suggests that there are circumstances where mature females are unable to achieve their full reproductive potential due to sperm limitation. To examine this possibility in different regions of the American lobster fishery, a reliable and noninvasive method was developed for sampling large numbers of female lobsters at sea. This method involves inserting a blunt-tipped needle into the female&#39;s seminal receptacle to determine the presence or absence of a sperm plug and to withdraw a sample that can be examined for the presence of sperm. A series of control studies were conducted at the dock and in the laboratory to test the reliability of this technique. These efforts entailed sampling 294 female lobsters to confirm that the presence of a sperm plug was a reliable indicator of sperm within the receptacle and thus, mating. This paper details the methodology and the results obtained from a subset of the total females sampled. Of the 230 female lobsters sampled from George&#39;s Bank and Cape Ann, MA (size range = 71-145 mm in carapace length), 90.3% were positive for sperm. Potential explanations for the absence of sperm in some females include: immaturity (lack of physiological maturity), breakdown of the sperm plug after being used to fertilize a clutch of eggs, and lack of mating activity. The surveys indicate that this technique for examining the mating success of female lobsters is a reliable proxy that can be used in the field to document reproductive activity in natural populations.

A Noninvasive Method For In situ Determination of Mating Success in Female American Lobsters Homarus americanus

Fishery-induced changes to exploited crustacean fisheries, such as the American lobster fishery, could potentially influence their reproductive dynamics, leading to a reduction in mating success. This study&#39;s goal was to develop a noninvasive method for ascertaining the mating success of female lobsters that may be physiologically or functionally mature.

Despite being one of the most productive fisheries in the Northwest Atlantic, much remains unknown about the natural reproductive dynamics of American ...

Environment

Field Experiments of Pollination Ecology: The Case of Lycoris sanguinea var. sanguinea

Plant-pollinator interactions have been studied for approximately one hundred years. During that time, many field methods have been developed to clarify the pollination effectiveness of each pollinator for visited flowers. Pollinator observations have been one of the most common methods to identify pollinators, and bagging and cage experiments have been conducted to show the effectiveness of specific pollinators. In a previous study of Lycoris sanguinea var. sanguinea, its effective pollinators, the visitation frequencies of each floral visitor, and its reproductive strategies were not identified. This study reports the observation that small bees visited flowers that were partially opened (breaking buds). To the best of our knowledge, this phenomenon has not been reported previously. Further, this study investigates the hypothesis that small bees can pollinate at that flowering stage. This study demonstrates the basic methods of field experiments in pollination ecology using L. sanguinea var. sanguinea. Pollinator observations and digital video showed the visitation frequencies of each floral visitor. Bagging and cage experiments revealed that these flowers could be pollinated fully and that breaking-bud pollination could be important for the pollination of this plant species. The advantages and disadvantages of each method are discussed, and recent developments, including laboratory experiments, are described.

Field Experiments of Pollination Ecology: The Case of Lycoris sanguinea var. sanguinea

To reveal the pollinator effectiveness of a given plant species, multiple methods of field experiments have been developed. This study demonstrates the basic methods of field experiments for pollination ecology using the case study of Lycoris sanguinea var. sanguinea and the novel pollination mechanism, breaking-bud pollination.

Plant-pollinator interactions have been studied for approximately one hundred years. During that time, many field methods have been developed to clarify ...

Induction and Evaluation of Inbreeding Crosses Using the Ant, Vollenhovia Emeryi

The genetic and molecular components of the sex-determination cascade have been extensively studied in the honeybee, Apis mellifera, a hymenopteran model organism. However, little is known about the sex-determination mechanisms found in other non-model hymenopteran taxa, such as ants. Because of the complex nature of the life cycles that have evolved in hymenopteran species, it is difficult to maintain and conduct experimental crosses between these organisms in the laboratory. Here, we describe the methods for conducting inbreeding crosses and for evaluating the success of those crosses in ant Vollenhovia emeryi. Inducing inbreeding in the laboratory using V. emeryi, is relatively simple because of the unique biology of the species. Specifically, this species produces androgenetic males, and female reproductives exhibit wing polymorphism, which simplifies identification of the phenotypes in genetic crosses. In addition, evaluating the success of inbreeding is straightforward as males can be produced continuously by inbreeding crosses, while normal males only appear during a well-defined reproductive season in the field. Our protocol allow for using V. emeryi as a model to investigate the genetic and molecular basis of the sex determination system in ant species.

Induction and Evaluation of Inbreeding Crosses Using the Ant, Vollenhovia Emeryi

In this protocol, methods for conducting inbreeding crosses, and for evaluating the success of those crosses, are described for the ant Vollenhovia emeryi. These protocols are important for experiments aimed at understanding the genetic basis of sex determination systems in Hymenoptera.

The genetic and molecular components of the sex-determination cascade have been extensively studied in the honeybee, Apis mellifera, a hymenopteran model ...

Genetics

A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster

An individual's sexual drive is influenced by genotype, experience and environmental conditions. How these factors interact to modulate sexual behaviors remains poorly understood. In Drosophila melanogaster, environmental cues, such as food availability, affect mating activity offering a tractable system to investigate the mechanisms modulating sexual behavior. In D. melanogaster, environmental cues are often sensed via the chemosensory gustatory and olfactory systems. Here, we present a method to test the effect of environmental chemical cues on mating behavior. The assay consists of a small mating arena containing food medium and a mating couple. The mating frequency for each couple is continuously monitored for 24 h. Here we present the applicability of this assay to test environmental compounds from an external source through a pressurized air system as well as manipulation of the environmental components directly in the mating arena. The use of a pressurized air system is especially useful to test the effect of very volatile compounds, while manipulating components directly in the mating arena can be of value to ascertain a compound's presence. This assay can be adapted to answer questions about the influence of genetic and environmental cues on mating behavior and fecundity as well as other male and female reproductive behaviors.

A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster

We demonstrate an assay to analyze the environmental and genetic cues that influence mating behavior in the fruit fly Drosophila melanogaster.

An individual's sexual drive is influenced by genotype, experience and environmental conditions. How these factors interact to modulate sexual behaviors ...

Behavior

Individual Culturing of Tigriopus Copepods and Quantitative Analysis of Their Mate-guarding Behavior

Copepods of the genus Tigriopus, which are common zooplankton in rocky tide pools, show precopulatory mate-guarding behavior where a male clasps a potential mate to form a pair. While this phenomenon has attracted interest of researchers, methods for its analysis have not been well described. Here we describe procedures for: 1) individual culturing and staging of Tigriopus juveniles and adults, and 2) video-based analysis of their mate-guarding behavior. The culturing method enables experimental control of paring experience of animals as well as the ability to track their development before behavioral tests. The analysis method allows quantitative evaluation of several aspects of the mate-guarding behavior, including capturing attempts by males and swimming trajectory of mate-guarding pairs. Although these methods were originally established for ethological studies on Tigriopus, with proper modifications they can also be applied to studies of other zooplankton in different research fields, such as physiology, toxicology, and ecological genetics.

Individual Culturing of Tigriopus Copepods and Quantitative Analysis of Their Mate-guarding Behavior

Mate-guarding behavior plays an important role in reproduction of intertidal copepods of the genus Tigriopus. However, methods for studying this behavior have not been well described. Here we describe methods for: 1) individual culture of virgin Tigriopus animals, and 2) quantitative analysis of their mate-guarding behavior.

Copepods of the genus Tigriopus, which are common zooplankton in rocky tide pools, show precopulatory mate-guarding behavior where a male clasps a ...

Methods to Assay Drosophila Behavior

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases1. We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials2-4. The rapid iterative negative geotaxis (RING) assay5 has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously using large number of animals, with the high-throughput approach making it more amenable for screening experiments.

Methods to Assay Drosophila Behavior

Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular ...

Neuroscience

Dissection for Identifying Migratory Insects via Ovarian Reproductive Analysis

Dissection and Grading of Ovarian Development in Wild-Type Female Insects

Migratory insect pests pose serious challenges to food production and security all over the world. The migratory pests can be monitored and captured using searchlight traps. One of the most important techniques for migratory pest forecasting is to identify the migratory species. However, in most cases, it is difficult to get the information just by appearance. Therefore, using knowledge acquired by systematic analysis of the female reproductive system can help to understand the combined anatomic morphology of the ovarian mating sac and ovary developmental grading of wild-type migratory insects captured with searchlight traps. To demonstrate the applicability of this method, ovarian development status and egg grain development stages were directly assessed in Helicoverpa armigera, Mythimna separata, Spodoptera litura, and Spodoptera exigua for the ovarian anatomy, and the ovarian mating sacs were studied in Agrotis ipsilon, Spaelotis valida, Helicoverpa armigera, Athetis lepigone, Mythimna separata, Spodoptera litura, Mamestra brassicae, and Spodoptera exigua, to explore their relationships. This work shows the specific dissection method to predict wild-type migratory insects, comparing the unique reproductive system of different migratory insects. Then, both tissues, namely, the ovary and mating sacs, were further investigated. This method helps to predict the dynamics and the structural development of reproductive systems in wild-type female migratory insects.

Author Spotlight: Analysis of Ovarian Anatomy in Migratory Insects to Overcome Experimental Challenges 

The protocol demonstrates a simple and easy dissection method, suitable for wild-type migratory female insects captured with searchlight traps. This technique can significantly clarify the same species by comparing both reproductive tissues, namely the mating sac and ovarian development of wild-type female insects.

Migratory insect pests pose serious challenges to food production and security all over the world. The migratory pests can be monitored and captured using ...

Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized Drosophila

Mating experiments using Drosophila have contributed greatly to the understanding of sexual selection and behavior. Experiments often require simple, easy and cheap methods to distinguish between individuals in a trial. A standard technique for this is CO2 anaesthesia and then labelling or wing clipping each fly. However, this is invasive and has been shown to affect behavior. Other techniques have used coloration to identify flies. This article presents a simple and non-invasive method for labelling Drosophila that allows them to be individually identified within experiments, using food coloring. This method is used in trials where two males compete to mate with a female. Dyeing allowed quick and easy identification. There was, however, some difference in the strength of the coloration across the three species tested. Data is presented showing the dye has a lower impact on mating behavior than CO2 in Drosophila melanogaster. The impact of CO2 anaesthesia is shown to depend on the species of Drosophila, with D. pseudoobscura and D. subobscura showing no impact, whereas D. melanogaster males had reduced mating success. The dye method presented is applicable to a wide range of experimental designs.

Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized Drosophila

This protocol describes a simple, cost effective way to individually identify Drosophila or other insects. Demonstration data investigating mating success across three species of Drosophila show that this method is comparable or better than the use of CO2 anaesthesia.

Mating experiments using Drosophila have contributed greatly to the understanding of sexual selection and behavior. Experiments often require simple, easy ...

Measuring and Altering Mating Drive in Male Drosophila melanogaster

Despite decades of investigation, the neuronal and molecular bases of motivational states remain mysterious. We have recently developed a novel, reductionist, and scalable system for in-depth investigation of motivation using the mating drive of male Drosophila&#160;melanogaster (Drosophila), the methods for which we detail here. The behavioral paradigm centers on the finding that male mating drive decreases alongside fertility over the course of repeated copulations and recovers over ~3 d. In this system, the powerful neurogenetic tools available in the fly converge with the genetic accessibility and putative wiring diagram available for sexual behavior. This convergence allows rapid isolation and interrogation of small neuronal populations with specific motivational functions. Here we detail the design and execution of the satiety assay that is used to measure and alter courtship motivation in the male fly. Using this assay, we also demonstrate that low male mating drive can be overcome by stimulating dopaminergic neurons. The satiety assay is simple, affordable, and robust to influences of genetic background. We expect the satiety assay to generate many new insights into the neurobiology of motivational states.

Measuring and Altering Mating Drive in Male Drosophila melanogaster

This article describes a behavioral assay that uses male mating drive in Drosophila melanogaster to study motivation. Using this method, researchers can utilize advanced fly neurogenetic techniques to uncover the genetic, molecular, and cellular mechanisms that underlie this motivation.

Despite decades of investigation, the neuronal and molecular bases of motivational states remain mysterious. We have recently developed a novel, ...

Using the Fluorescent Dye, Rhodamine B, to Study Mating Competitiveness in Male Aedes aegypti Mosquitoes

The success of sterile or incompatible insect technique-based population suppression programs depends on the ability of released males to compete for wild-type females and induce sterility in the target population. Hence, laboratory assessment of male mating competitiveness is essential for evaluating the release strain&#8217;s fitness before field release. Conventionally, such an assay is performed by determining the proportion of viable eggs produced by the females after being simultaneously exposed to two sets of males (wild-type and release strains) for copulation. However, this process is time-consuming and laborious due to the need to first blood-feed the females for egg production and then hatch and enumerate the hatched eggs to determine egg viability.&#160;
Moreover, this method cannot discern the degree of competitiveness between two sterile or Wolbachia-infected mosquito lines as wild-type female mosquitoes will only produce non-viable eggs upon mating with both. To circumvent these limitations, this paper describes a more direct method of measuring male mosquito mating competitiveness in laboratory settings using the fluorescent dye, rhodamine B (RhB), which can be used to mark males by feeding them in sucrose solution containing RhB. After the mating assay, the presence of fluorescing sperms in the spermathecae of a female can be used to determine her mating partner. This method is cost-effective, reduces the experimental time by 90% and allows comparison of mating fitness between two sterile or Wolbachia-infected lines.&#160;

Using the Fluorescent Dye, Rhodamine B, to Study Mating Competitiveness in Male Aedes aegypti Mosquitoes

Here, we present a protocol for studying mating competitiveness of male Aedes aegypti using fluorescent dye as a marker. Female mosquitoes are exposed to both marked and unmarked males for copulation. Post mating, their spermathecae are examined under a fluorescence microscope to determine their mating partner.

The success of sterile or incompatible insect technique-based population suppression programs depends on the ability of released males to compete for ...

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

Summary

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Chapters in this video

A Noninvasive Method For In situ Determination of Mating Success in Female American Lobsters (Homarus americanus)

Field Experiments of Pollination Ecology: The Case of Lycoris sanguinea var. sanguinea

Induction and Evaluation of Inbreeding Crosses Using the Ant, Vollenhovia Emeryi

A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster

Individual Culturing of Tigriopus Copepods and Quantitative Analysis of Their Mate-guarding Behavior

Methods to Assay Drosophila Behavior

Dissection and Grading of Ovarian Development in Wild-Type Female Insects

Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized Drosophila

Measuring and Altering Mating Drive in Male Drosophila melanogaster

Using the Fluorescent Dye, Rhodamine B, to Study Mating Competitiveness in Male Aedes aegypti Mosquitoes