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/pubmed?term=((Bursaphelenchus+sp.+in+the+wood+of+dead+pine+trees%5BTitle%5D)+AND+%22J.+Jap.+For.+Soc%22%5BJournal%5D)">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/pubmed?term=((Bursaphelenchus+xylophilus+was+discovered+in+Sun+Yet-sen%27s+mausoleum+in+Nanjing%5BTitle%5D)+AND+%22J.+Jiangsu+Fore.+Sci.+Tech.+%28in+Chinese%29%22%5BJournal%5D)">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/pubmed?term=((First+report+of+Bursaphelenchus+xylophilus+in+Portugal+and+in+Europe%5BTitle%5D)+AND+%22Nematology%22%5BJournal%5D)">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/pubmed?term=((Incidence+of+the+pine+wood+nematode+Bursaphelenchus+xylophlius+Steiner+%26+Buhrer%2C+1934+%28Nickle%2C+1970%29+in+Spain%5BTitle%5D)+AND+%22Nematology%22%5BJournal%5D)">Incidence of the pine wood nematode Bursaphelenchus xylophlius Steiner & 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/pubmed?term=((Pine+wilt+disease%3A+a+threat+to+European+forestry%5BTitle%5D)+AND+%22Euro.+J.+Plant+Path%22%5BJournal%5D)">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/pubmed?term=((Bursaphelenchus+xylophilus%3A+opportunities+in+comparative+genomics+and+molecular+host-parasite+interactions%5BTitle%5D)+AND+%22Mol.+Plant.+Path%22%5BJournal%5D)">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/pubmed?term=((Fecundity+and+reproduction+of+Bursaphelenchus+lignicolus%5BTitle%5D)+AND+%22Jap.+J.+Nemat%22%5BJournal%5D)">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/pubmed?term=((Description+of+Bursaphelenchus+lignicolus.+n.+sp.+%28Nematoda%3A+Aphelenchoididae%29+from+pine+wood+and+histophathology+of+nematode-infested+trees%5BTitle%5D)+AND+%22Nematologica%22%5BJournal%5D)">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/pubmed?term=((Behavioural+features+of+Bursaphelenchus+xylophilus+in+the+mating+process%5BTitle%5D)+AND+%22Nematology%22%5BJournal%5D)">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/pubmed?term=((Evidence+of+a+mate+finding+cue+in+the+hermaphrodite+nematode+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+USA%22%5BJournal%5D)">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/pubmed?term=((Male+mating+behaviour%5BTitle%5D)+AND+%22The+C.+elegans+Research+Community+Wormbook%22%5BJournal%5D)">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/pubmed?term=((Sex+attraction+and+mating+in+Bursaphelenchus+okinawaensis+and+B.+xylophilus%5BTitle%5D)+AND+%22J.+Nematol%22%5BJournal%5D)">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 Bursaphele...

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 wit...

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

Research

JoVE Journal

Biology

9.4K 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

Technique for Studying Arthropod and Microbial Communities within Tree Tissues

Phloem tissues of pine are habitats for many thousands of organisms. Arthropods and microbes use phloem and cambium tissues to seek mates, lay eggs, rear young, feed, or hide from natural enemies or harsh environmental conditions outside of the tree. Organisms that persist within the phloem habitat are difficult to observe given their location under bark. We provide a technique to preserve intact phloem and prepare it for experimentation with invertebrates and microorganisms. The apparatus is called a &#8216;phloem sandwich&#8217; and allows for the introduction and observation of arthropods, microbes, and other organisms. This technique has resulted in a better understanding of the feeding behaviors, life-history traits, reproduction, development, and interactions of organisms within tree phloem. The strengths of this technique include the use of inexpensive materials, variability in sandwich size, flexibility to re-open the sandwich or introduce multiple organisms through drilled holes, and the preservation and maintenance of phloem integrity. The phloem sandwich is an excellent educational tool for scientific discovery in both K-12 science courses and university research laboratories.

We provide a technique to preserve intact tree phloem and prepare it for observation. We create an apparatus called a phloem sandwich that allows for the introduction and observation of arthropods, microbes, and other organisms that inhabit phloem tissues.

Phloem tissues of pine are habitats for many thousands of organisms.  Arthropods and microbes use phloem and cambium tissues to seek mates, lay eggs, rear ...

Environment

Assaying Predatory Feeding Behaviors in Pristionchus and Other Nematodes

This protocol provides multiple methods for the analysis and quantification of predatory feeding behaviors in nematodes. Many nematode species including Pristionchus pacificus display complex behaviors, the most striking of which is the predation of other nematode larvae. However, as these behaviors are absent in the model organism Caenorhabditis elegans, they have thus far only recently been described in detail along with the development of reliable behavioral assays 1. These predatory behaviors are dependent upon phenotypically plastic but fixed mouth morphs making the correct identification and categorization of these animals essential. In P. pacificus there are two mouth types, the stenostomatous and eurystomatous morphs 2, with only the wide mouthed eurystomatous containing an extra tooth and being capable of killing other nematode larvae. Through the isolation of an abundance of size matched prey larvae and subsequent exposure to predatory nematodes, assays including both "corpse assays" and "bite assays" on correctly identified mouth morph nematodes are possible. These assays provide a means to rapidly quantify predation success rates and provide a detailed behavioral analysis of individual nematodes engaged in predatory feeding activities. In addition, with the use of a high-speed camera, visualization of changes in pharyngeal activity including tooth and pumping dynamics are also possible.

Assaying Predatory Feeding Behaviors in Pristionchus and Other Nematodes

Behavioral assays provide powerful tools for understanding neuronal function. Here we present several protocols for quantifying predatory feeding behavior found in the model nematode Pristionchus pacificus and its relatives. Additionally, we provide methods for analyzing predatory feeding adaptations including mouth structures and teeth.

This protocol provides multiple methods for the analysis and quantification of predatory feeding behaviors in nematodes. Many nematode species including ...

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 ...

Rapid Isolation of Wild Nematodes by Baermann Funnel

Beyond being robust experimental model organisms, Caenorhabditis elegans and its relatives are also real animals that live in nature. Studies of wild nematodes in their natural environments are valuable for understanding many aspects of biology, including the selective regimes in which distinctive genomic and phenotypic characters evolve, the genetic basis for complex trait variation, and the natural genetic diversity fundamental to all animal populations. This manuscript describes a simple and efficient method for extracting nematodes from their natural substrates, including rotting fruits, flowers, fungi, leaf litter, and soil. The Baermann funnel method, a classical nematology technique, selectively isolates active nematodes from their substrates. Because it recovers nearly all active worms from the sample, the Baermann funnel technique allows for the recovery of rare and slow-growing genotypes that co-occur with abundant and fast-growing genotypes, which might be missed in extraction methods that involve multiple generations of reproduction. The technique is also well suited to addressing metagenetic, population-genetic, and ecological questions. It captures the entire population in a sample simultaneously, allowing an unbiased view of the natural distribution of ages, sexes, and genotypes. The protocol allows for deployment at scale in the field, rapidly converting substrates into worm plates, and the authors have validated it through fieldwork on multiple continents.

This protocol outlines a method for efficiently extracting live nematodes from natural substrates in the field.

Beyond being robust experimental model organisms, Caenorhabditis elegans and its relatives are also real animals that live in nature. Studies of wild ...

Extraction and Quantification of Volatile Sex Pheromone Responses in C. elegans

Volatile Sex Pheromone Extraction and Chemoattraction Assay in Caenorhabditis elegans

Chemical communication is vital in organismal health, reproduction, and overall well-being. Understanding the molecular pathways, neural processes, and computations governing these signals remains an active area of research. The nematode Caenorhabditis elegans provides a powerful model for studying these processes as it produces a volatile sex pheromone. This pheromone is synthesized by virgin females or sperm-depleted hermaphrodites and serves as an attractant for males.
This protocol describes a detailed method for isolating the volatile sex pheromone from several C. elegans strains (WT strain N2, daf-22, and fog-2) and C. remanei. We also provide a protocol for quantifying the male chemotaxis response to the volatile sex pheromone. Our analysis utilizes measurements such as chemotaxis index (C.I.), arrival time (A.T.), and a trajectory plot to compare male responses under various conditions accurately. This method allows for robust comparisons between males of different genetic backgrounds or developmental stages. Furthermore, the experimental setup outlined here is adaptable to investigating other chemoattraction chemicals.

Author Spotlight: Examining Volatile Sex Pheromone Influence on Male C. elegans Behavior

This protocol establishes methods for extracting and quantifying responses to the volatile sex pheromone in C. elegans, providing tools to study chemical communication and navigation trajectory.

Chemical communication is vital in organismal health, reproduction, and overall well-being. Understanding the molecular pathways, neural processes, and ...

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, ...

Neuroscience

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 ...

Optimizing Pinewood Nematode Infection in Pinus pinaster

Infection of In Vivo and In Vitro Pines with the Pinewood Nematode Bursaphelenchus xylophilus and Isolation of Induced Volatiles

The pinewood nematode (PWN) is a phytoparasite that causes pine wilt disease (PWD) in conifer species. This plant parasitic nematode has heavily contributed to pine deforestation in Asian countries, e.g., Japan, China, and Korea. Over the last two decades, in Europe, Portugal and Spain have been greatly affected. Research on the mechanisms of PWN infection and/or PWD progression in susceptible host species relies on the controlled infection of pine seedlings under greenhouse conditions. This technique is laborious and mobilizes substantial economic and human resources. Additionally, it can be prone to variability that results from the genetic diversity associated with some pine species but also from the interference of external factors. As an alternative, in vitro co-cultures of pine with PWNs offer a more advantageous system for studying biochemical changes since they a) allow controlling single environmental or nutritional variables, b) occupy less space, c) require less time to obtain, and d) are&#160;free from contamination or from host genetic variation. The following protocol details the standard in vivo PWN infection of Pinus pinaster, the maritime pine, and the establishment of the novel in vitro co-cultures of pine shoots with the PWN as an improved methodology to study this phytoparasite influence on pine volatiles. PWN-induced volatiles are extracted from in vivo and in vitro infected pines by hydrodistillation and distillation-extraction, and the emitted volatiles are captured by solid phase microextraction (SPME), using fiber or packed column techniques.

Author Spotlight: In Vitro Co-Culture System of Pine Shoots and Pinewood Nematode for Studying Host Volatile Response

The protocol describes the in vivo and in vitro pinewood nematode infection of Pinus pinaster and their volatilome analysis through Gas Chromatography (GC) and GC coupled to Mass Spectrometry (GC-MS).

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

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Rapid Isolation of Wild Nematodes by Baermann Funnel

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Measuring and Altering Mating Drive in Male Drosophila melanogaster

Application of RNA Interference in the Pinewood Nematode, Bursaphelenchus xylophilus

Infection of In Vivo and In Vitro Pines with the Pinewood Nematode Bursaphelenchus xylophilus and Isolation of Induced Volatiles