Name: observation and quantification of mating behavior in the pinewood nematode, bursaphelenchus xylophilus
Uploaded: 2016-12-25T14:00:00
Description: Watch this Scientific Journal Video about Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus at JoVE.com

Najie Zhu e Liqun Bai contribuito in maniera uguale a questo lavoro. Questo lavoro è stato sostenuto finanziariamente dal Fondo speciale per la ricerca scientifica forestale nel benessere pubblico (201.204.501) e la National Science Foundation naturale della Cina (31.170.604, 31.270.688 e 31.570.638). Ringraziamo il Dott Holighaus Gerrit e il Dr. Danielle Hickford per i loro utili suggerimenti sulla scrittura inglese.

1. Acquisizione di Virgin adulti Nematodi <ol><li> Nematode del pino isolato e condizioni di coltura NOTA: Il B. xylophilus isolare Nxy61 è stato originariamente estratto da un malato massoniana Pinus nella zona di Ningbo della provincia di Zhejiang in Cina. <ol><li> Preparare Potato Dextrose Agar (PDA, vedere la Tabella dei Materiali) piastre per la coltivazione di Botrytis cinerea. </li><li> Per l&#39;inoculazione, pugno un pezzo di tappet...

<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>

test comportamentali sono un aspetto fondamentale di ecologia chimica, la neurobiologia, la biologia molecolare e la genetica. Nematodi, e in particolare C. elegans, sono ampiamente utilizzati per gli studi in questi settori. Il comportamento di accoppiamento di C. elegans è stato studiato in precedenza 10-11. Tuttavia, B. xylophilus è gonochoristic e diverso dagli ermafroditi C. elegans, e il suo processo di accoppiamento ha diverse caratteristiche comportamentali 9...

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

Un metodo per osservare e quantificare il comportamento di accoppiamento del nematode del pino, Bursaphelenchus xylophilus, è stato istituito allo stereomicroscopio. Per migliorare l&#39;efficienza di accoppiamento di B. Xylophilus e per aumentare le possibilità di osservazione accoppiamento, adulti vergini sono stati coltivati e utilizzati per l&#39;indagine. Le uova sono stati ottenuti mantenendo i nematodi in acqua e permettendo le femmine di deporre le uova per 10 min. Le larve di secondo stadio (J2) sono stati sincronizzati incubando le uova per 24 ore a 25 ° C al buio, e il primo J4 sono stati ottenuti coltivando la J2 con muffa grigia, Botrytis cinerea, per un altro 52 h. A questo punto di tempo, la maggior parte dei nematodi J4 potrebbero essere chiaramente distinti come maschio o femmina usando la loro morfologia genitale. Il maschio e la femmina J4 sono stati raccolti e coltivate separatamente in due diverse piastre di Petri per 24 ore per ottenere nematodi adulti vergini. Un maschio e una femmina vergine vergine sono stati associati in una goccia di water nel pozzo di una diapositiva concava. Il comportamento di accoppiamento è stato girato con un videoregistratore allo stereomicroscopio. L&#39;intera durata del processo di accoppiamento era 82,8 ± 3,91 min (media ± SE) e potrebbe essere diviso in 4 diverse fasi: ricerca, contatto, che copulano, e persistente. Il verbale medi di durata erano 21,8 ± 2,0, 28,0 ± 1,9, 25,8 ± 0,7 e 7,2 ± 0,5, rispettivamente. Undici sub-comportamenti sono stati descritti: crociera, si avvicina, incontrando, toccando, hoop, localizzare, allegando, eiaculazione, la separazione, quiescenza, e il roaming. È interessante notare che, evidente la concorrenza intra-sessuale è stata osservata quando una femmina è stata raggruppata con 3 maschi o un maschio con 3 femmine. Questo protocollo è utile e prezioso, non solo in indagare il comportamento di accoppiamento di B. xylophilus, ma anche in qualità di riferimento per studi comportamentali degli altri nematodi.

Il tasso di successo di accoppiamento (R) degli adulti vergini era 86,67% in media, che era significativamente più elevata di quella degli adulti selezionati casualmente: 13,33 ± 4,65% (F = 1301,71, df = 1, p = 0,0001) (Figura 2). Questa scoperta suggerisce che è più facile osservare il comportamento di accoppiamento di B. xylophilus con gli adulti vergini che con gli adulti selezionati casualmente. <p class="jove_content" fo:keep-toget...

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.

Osservazione e quantificazione di comportamento di accoppiamento nel nematode del pino,<em&gt; Bursaphelenchus xylophilus</em

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

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