Gli autori desiderano ringraziare i membri passati della Fotografico laboratorio: Ming-Min Lee, Kathryn Plichta, Victoria Miranda-Thompson e Sam-Kyu Kim per il loro contributo al miglioramento di molti di questi protocolli. Questo lavoro è stato finanziato in parte mediante la concessione National Science Foundation IOS e IOS-0840932-0724978 SP Archivio

1 In vivo Allevamento di Nematodi entomopatogeni con i loro batteri Symboitic <ol><li> Invertire una capsula di Petri in plastica 100 x 15 mm e collocare due dischi di carta da filtro (90 mm) nel coperchio del piatto. </li><li> Distribuire uniformemente 1 ml della sospensione acqua IJ (larve infettive) (ad una concentrazione di 1.000-2.000 IJ / ml) sulla carta da filtro. NOTA: IJs non devono essere sterilizzati in superficie. </li><li> Aggiungere 10 ultimo instar larve della maggior waxmoth Galleria mellonella al piatto. L&#39;obiettivo è quello di fornire circa 100-200 IJs / larva. </li><li> Coprire il coperchio con la parte inferiore della scatola di Petri (Figura 1) ed etichettare la piastra di Petri. Indicare le seguenti informazioni: nematode nome della specie (se noto); isolare il codice / designazione; trappola infezione data è stata impostata. </li><li> Porre la capsula all&#39;interno di un sacchetto di plastica sigillato liberamente (per conservare l&#39;umidità) e tenerlo al buio sia a temperatura ambiente o in un incubatore tra i 20-25 ° C. Controllare i piatti tutti i giorni NOTA: L&#39;oscurità facilita l&#39;infezione da nematodi, in quanto imita le condizioni naturali di infezione nel terreno. </li><li> Dopo 3-5 giorni, rimuovere i cadaveri con segni di infezione da nematodi ad una modifica trappola Bianco 7. NOTA: Se i cadaveri odore putrido, questo può essere un&#39;indicazione che la coltura non ha avuto successo e / o c&#39;era contaminazione. Impostare una nuova camera di infezione con un nuovo lotto di nematodi e insetti. </li></ol> 2 in vitro Allevamento di Nematodi entomopatogeni con i loro batteri simbionti <ol><li> Fegato-rene Agar Metodo di 9,19 <ol><li> Tritate il fegato e reni in piccoli pezzi (da 2 cm 3 o più piccoli) e mettere in un frullatore con NaCl, agar e 300 ml di acqua e mescolare fino a quando la carne diventa una poltiglia liquida, pasta spessa o purea. Poi, il trasferimento miscela di un pallone da 1 L Erlenmeyer. </li><li> Aggiungere gli ultimi 200 ml di acqua al vaso frullatore e decantare eventuali supporti rimasti nelBeuta. Autoclave per 15 minuti a 121 ° C. Dopo la sterilizzazione in autoclave agar lasciare raffreddare a toccare prima di versare. NOTA: dispensazione dei media non è raccomandato perché questo mezzo tende ad avere pezzi di carne. </li><li> Versare ~ 20 ml di agar su 5 (o 6) cm piastre di Petri agitando la mano o agitando la beuta tra versa per un supporto più omogeneo. Lasciare agar per solidificare e piatti conservare a 4 ° C fino al momento NOTA: Utilizzare una spatola di metallo-sterilizzati fiamma per rompere tutti i grandi pezzi di carne versato nella piastra di Petri. </li></ol></li><li> Lipid Agar Metodo 9,19 <ol><li> Preparare mezzo lipidico agar mescolando brodo nutriente, estratto di agar e il lievito e mescolare versare in un pallone da 2 L Erlenmeyer. Aggiungere acqua distillata H 2 O e MgCl 2 • 6H 2 O e autoclave per 15 minuti a 121 ° C. </li><li> Aggiungere miscela sterile di olio e di sciroppo di mais mais mix e mescolare o agitare energicamente e spesso per disperdere goccioline di olio in modo uniforme. <br/&gt; NOTA: Sterilizzare olio di mais e lo sciroppo mettendo il volume necessario in un UV cross-linker. L&#39;olio non si dissolvono nel liquido, ma assicurarsi che sia in minuscole goccioline. </li><li> Versare ~ 20 ml di agar su un 5 (o 6) cm piastre di Petri e lasciare solidificare l&#39;agar e piatti conservare a 4 ° C fino al momento dell&#39;uso. </li><li> Streak il batteri simbionti desiderato sulla piastra di agar lipidi e incubare le piastre a 28 ° C per 24-48 ore. NOTA: Stendere 100-200 ml di una notte (12-16 ore) LB sottocultura. </li><li> Il giorno dopo, aggiungere circa 0,4 ml di sospensione sterile superficie nematode (uova o larve infettive) e incubare le piastre a temperatura ambiente al buio o in un incubatore a 22 ± 3 ° C. NOTA: Non aggiungere più di 0,4 ml di sospensione nematode come si può creare un ambiente eccessivamente umido che è sfavorevole alla crescita nematode. </li><li> Monitorare culture quotidiana. Le culture dovrebbero produrre una nuova generazione di IJs in circa 12 giorni (Figura 2).</li><li> Trasferire piatto fondo con agar quando nematodi sono visti strisciare i lati del piatto (Figura 3A) ad una trappola Bianco modificata (vedi Orozco et al. 12) per raccogliere IJs (Figura 3B). Eseguire questo passaggio sotto una cappa a flusso laminare per ridurre la contaminazione dei media. </li><li> Raccogliere IJs dalla trappola Bianco modificato sulla nascita e risciacquare sospensione IJ per rimuovere eventuali detriti mezzo. Conservare IJ in acqua distillata sterilizzata a 10 ° C (in un incubatore a freddo-temperatura o cella frigorifera) o utilizzare immediatamente, se necessario. NOTA: A seconda l&#39;obiettivo dello studio, il seme piatti sia con nematodi simbionti-colonizzati o aposymbiotic. </li></ol></li></ol> 3. in vitro allevamento di Aposymbiotic (Symbiont-gratuito) Nematodi entomopatogeni <ol><li> Infettare 10-20 G. mellonella larve con IJs delle specie di nematodi desiderati (vedi capitolo 1). Dopo 3 o 4 giorni (in questo momento IJs avrebbe maturatoagli adulti di prima generazione) raccogliere cadaveri. NOTA: Il tempo a scadenza e numero di femmine necessari per ottenere un adeguato numero di uova può variare da specie. Infettare più G. mellonella larve se necessario e iniziare dissezioni già alla 48 ore dopo l&#39;infezione per valutare se sono in fase di sviluppo destra. </li><li> Mettete ogni cadavere singolarmente in una capsula di Petri con una soluzione salina (tampone M9 18). Sezionare un cadavere tirando la sua testa con una pinza sottile punta. </li><li> Raccogliere almeno 20 gravido (con le uova all&#39;interno) femmine con un ago sottile (L-forma preferibilmente) e metterli in un vetro da orologio contenente 10 ml di tampone M9 (Figura 4A). </li><li> Preparare una soluzione axenizing considerando i seguenti ingredienti: 6,75 ml di acqua distillata, 1,25 ml 5 N NaOH, e 2,25 ml di soluzione di candeggina disponibile in commercio diluito al 3% di ipoclorito. NOTA: NaOH è una soluzione di base-guanti, occhiali e altro adeguato di protezioneabbigliamento deve essere indossato. NOTA: Preparare soluzione axenizing fresco ogni volta, come candeggina degrada alla luce. Preparare diversi lotti di soluzione axenizing ed eseguire la sterilizzazione di superficie e la raccolta delle uova in più vetri da orologio. </li><li> Risciacquare femmine almeno tre volte con riempimento del buffer M9 vetro d&#39;orologio con soluzione tampone e succhiare buffer con una pipetta. Assicurarsi che le femmine rimangono nel vetro d&#39;orologio. Ripetere questa operazione altre due volte. </li><li> Aggiungere immediatamente la soluzione axenizing e permettono le femmine di rimanere in questa soluzione per non più di 10-15 minuti. Osservare i tessuti nematode cominciano a disintegrarsi, mentre le uova (che sono resistenti alla soluzione axenizing) rimangono intatti. </li><li> Interrompere manualmente corpi femminili per accelerare il processo, tagliandoli con un ago o un bisturi (Figura 4B, C). Rimuovere le uova da loro pipettando in provette da 1,5 ml microcentrifuga, evitando l&#39;aggiunta di qualsiasi tessuto non disciolto. NOTA: Utilizzare come meventuali microprovette se necessario per raccogliere le uova e lavorare rapidamente, come la vitalità delle uova diminuisce per un periodo prolungato di esposizione alla soluzione di axenizing. </li><li> Tubi vortex per 15 secondi utilizzando l&#39;impostazione &quot;alta velocità&quot; per rimuovere ulteriormente i tessuti nematodi attaccati alle uova. In alternativa, se un vortice non è disponibile, pipettare su e giù per le soluzioni più volte. </li><li> Uova pellet di centrifugazione a 18.000 g per circa 2 min. Aumentare la velocità se le uova non fanno sufficientemente pellet. Rimuovere la soluzione axenizing e risciacquare due volte riempiendo la provetta con acqua distillata sterile e centrifugare ancora una volta a 900 xg per 1 min. </li><li> Dopo l&#39;ultimo lavaggio, risospendere le uova in 300-500 ml di acqua distillata sterile e metterli in una sterile 3 centimetri scatola di Petri o vetro da orologio sterilizzati. Ricordate che le uova sono ormai sterilizzato superficie, in modo da utilizzare pipette sterili e / o suggerimenti pippetter e acqua per mantenere la pulizia delle uova. </li><li>Esaminare le uova sotto un microscopio da dissezione (30-50X ingrandimento) per confermare che sono intatti (Figura 4D) e trasferirli in una piastra 5 cm e agar fegato-rene (vedere paragrafo 2.1) NOTA: Non aggiungere più di 400 microlitri della sospensione uovo sulle piastre come farà l&#39;agar eccessivamente bagnato. </li><li> Le targhette di identificazione con le informazioni appropriate e memorizzarli in posizione verticale al buio a 28 ° C. Schiusa delle uova dovrebbe essere evidente durante la notte. NOTA: L&#39;acqua può condensare sul coperchio del piatto ma evapora dopo 1 o 2 giorni. A questo punto, porre il recipiente in sacchetto di plastica per trattenere l&#39;umidità del agar ed evitare la sua essiccazione. </li><li> Osservare piatti periodicamente e scartare tutti i piatti che mostrano segni di contaminazione batterica o fungina. Una volta IJs sono visti migrare il muro della capsula di Petri, togliere il coperchio e trasferire il piatto fondo con agar ad una modifica trappola Bianco 12. Considerare condizioni sterili quando si trasferisce il piatto nel Modified trappola bianco per evitare la contaminazione dei mezzi esposti. </li><li> Continua il monitoraggio delle trappole bianche e raccolto IJ, se necessario. NOTA: nematodi Aposymbiotic continueranno a migrare in acqua della trappola Bianca per molti giorni e addirittura settimane. </li></ol>

<ol>
	<li>Akhurst, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+and+functional+dimorphism+in+Xenorhabdus+spp.,+bacteria+symbiotically+associated+with+the+insect+pathogenic+nematodes,+Neoaplectana+and+Heterorhabditis.">Morphological and functional dimorphism in Xenorhabdus spp., bacteria symbiotically associated with the insect pathogenic nematodes, Neoaplectana and Heterorhabditis.</a> J. Gen. Microbiol. 121, 303-309 (1980).</li><li>Dunphy, G. B., Webster, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+monoxenic+culture+of+Neoaplectana+carpocapsae+DD+136+and+Heterorhabditis+heliothidis.">The monoxenic culture of Neoaplectana carpocapsae DD 136 and Heterorhabditis heliothidis.</a> Revue Nematol. 12, 113-123 (1989).</li><li>Boemare, N., Gaugler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology,+taxonomy+and+systematics+of+Photorhabdus+and+Xenorhabdus.">Biology, taxonomy and systematics of Photorhabdus and Xenorhabdus.</a> Entomopathogenic Nematology. , (2002).</li><li>Boemare, N. E., Akhurst, R. A., Dworkin, M., Falkow, S., Rosenberg, E., Schleifer, K. -. H., Stackebrandt, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Genera+Photorhabdus+and+Xenorhabdus.">The Genera Photorhabdus and Xenorhabdus.</a> The Prokaryotes. , 473-488 (2002).</li><li>Ehlers, R. U., Wulff, A., Peters, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenicity+of+axenic+Steinernema+feltiae.+Xenorhabdus+bovienii.+and+the+bacto-helminthic+complex+to+larvae+of+Tipula+oleracea+(Diptera)+and+Galleria+mellonella+(Lepidoptera).">Pathogenicity of axenic Steinernema feltiae. Xenorhabdus bovienii. and the bacto-helminthic complex to larvae of Tipula oleracea (Diptera) and Galleria mellonella (Lepidoptera).</a> Journal Invertebrate Pathology. 69, 212-217 (1997).</li><li>Gaugler, R., Kaya, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Entomopathogenic Nematodes in Biological Control. Boca. , (1990).</li><li>Heungens, K., Cowles, C. E., Goodrich-Blair, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Xenorhabdus+nematophila+genes+required+for+mutualistic+colonization+of+Steinernema+carpocapsae+nematodes.">Identification of Xenorhabdus nematophila genes required for mutualistic colonization of Steinernema carpocapsae nematodes.</a> Molecular Microbiology. 45, 1337-1353 (2002).</li><li>Kaya, H. K., Gaugler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entomopathogenic+nematodes.">Entomopathogenic nematodes.</a> Annual Review of Entomology. 38, 181-206 (1993).</li><li>Kaya, H. K., Stock, S. P., Lackey, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+in+insect+nematology.">Techniques in insect nematology.</a> Manual of techniques in insect pathology. , 281-324 (1997).</li><li>Koppenh&#246;fer, H., Nguyen, K. B., Hunt, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+Symbionts+of+Steinernema+and+Heterorhabditis.">Bacterial Symbionts of Steinernema and Heterorhabditis.</a> Entomopathogenic Nematodes: Systematics, Phylogeny and Bacterial Symbionts. , 735-759 (2007).</li><li>Lunau, S., Stoessel, S., Schmidt Peisker, A. J., Ehlers, R. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+monoxenic+inocula+for+scaling+up+in+vitro+cultures+of+the+entomopathogenic+nematodes+Steinernema+spp+and+Heterorhabditis+spp.">Establishment of monoxenic inocula for scaling up in vitro cultures of the entomopathogenic nematodes Steinernema spp and Heterorhabditis spp.</a> Nematologica. 39, 385-399 (1993).</li><li>Orozco, R. A., Lee, M., Stock, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soil+sampling+and+isolation+of+entomopathogenic+nematodes+(Steinernematidae,+Heterorhabditidae).">Soil sampling and isolation of entomopathogenic nematodes (Steinernematidae, Heterorhabditidae).</a> J. Vis. Exp. (89), (2014).</li><li>Poinar, G. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+presence+of+Achromobacter+nematophilus+in+the+infective+stage+of+a+Neoaplectana+sp+(Steinernematidae:+Nematoda).">The presence of Achromobacter nematophilus in the infective stage of a Neoaplectana sp (Steinernematidae: Nematoda).</a> Nematologica. 12, 105-108 (1966).</li><li>Poinar, G. O., Thomas, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Significance+of+Achromobacter+nematophilus+sp.+nov.+(Achromobacteriaceae:+Eubacteriales)+associated+with+a+nematode.">Significance of Achromobacter nematophilus sp. nov. (Achromobacteriaceae: Eubacteriales) associated with a nematode.</a> Int. Bull. Bacteriol. Nomencl. Taxon. 15, 249-252 (1966).</li><li>Sicard, M., Brugirard-Ricaud, K., Pag&#232;s, S., Lanois, A., Boemare, N. E., Breh&#233;li, M., Givaudan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+infection+during+the+tripartite+interaction+between+Xenorhabdus+nematophila,+its+nematode+vector,+and+insect+hosts.">Stages of infection during the tripartite interaction between Xenorhabdus nematophila, its nematode vector, and insect hosts.</a> Appl. Environ. Microbiol. 70, 6473-6480 (2004).</li><li>Sicard, M., Hinsinger, J., LeBrun, N., Pag&#232;s, S., Boemare, N., Moulia, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interspecific+competition+between+entomopathogenic+nematodes+(Steinernema)+is+modified+by+their+bacterial+symbionts+(Xenorhabdus).">Interspecific competition between entomopathogenic nematodes (Steinernema) is modified by their bacterial symbionts (Xenorhabdus).</a> BMC Evolutionary Biology. 6, 68-78 (2006).</li><li>Snyder, H., Stock, S. P., Kim, S. K., Flores-Lara, Y., Forst, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+the+colonization+and+release+processes+of+Xenorhabdus+nematophila+and+the+morphology+and+ultrastructure+of+the+bacterial+receptacle+of+its+nematode+host,+Steinernema+carpocapsae.">New insights into the colonization and release processes of Xenorhabdus nematophila and the morphology and ultrastructure of the bacterial receptacle of its nematode host, Steinernema carpocapsae.</a> Applied and environmental microbiology. 73, 5338-5346 (2007).</li><li>Stiernagle, T., ed, C. .. e. l. e. g. a. n. s. .. W. o. r. m. B. o. o. k. .. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+C.+elegans.">Maintenance of C. elegans.</a> WormBook. , (2006).</li><li>Stock, S. P., Goodrich-Blair, H., Lacey, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nematode+parasites,+pathogens+and+associated+of+insects+and+invertebrates+of+economic+importance.">Nematode parasites, pathogens and associated of insects and invertebrates of economic importance.</a> Manual of Techniques in Invertebrate Pathology. , 373-426 (2012).</li></ol>

Nessun conflitto di interessi dichiarati.

Utilizzando un ospite adatto è un fattore chiave per il successo in allevamento vivo di EPN. Di solito, sia steinernematids e heterorhabditids possono riprodursi e completare il loro ciclo di vita in larve della maggiore tarma della cera, Galleria mellonella (Lepidoptera: Pyralidae). Tuttavia, altre specie di insetti provenienti da diverse famiglie e / o gli ordini possono essere considerati. Alcune delle specie di nematodi attualmente descritte sono noti per avere specificità per un partico...

Nematodi entomopatogeni (EPN) Steinernema e Heterorhabditis spp. (Steinernematidae, Heterorhabditidae) e dei loro simbionti batterici, Xenorhabdus e Photorhabdus spp (Enterobacteriaceae) sono considerati un modello emergente di animali terrestri-microbici relazioni simbiotiche 2-4,6,10,19. Xenorhabdus e Photorhabdus spp. sono nutrito come simbionti nell&#39;intestino della sola fase di vita libera dei nematodi, noto anche come il giovanile infettiva (IJ) o stadio 3 ° infettivo giovanile 8,10,13. La coppia batterio-nematode è patogeno per una vasta gamma di insetti ed è stato implementato con successo in programmi di lotta biologica e lotta integrata in tutto il mondo 6,8. Qui mostriamo una selezione di in vivo e in vitro tecniche che sono spesso esercitati per l&#39;allevamento di EPN in condizioni di laboratorio. In metodi in vivo contemplano un host insetto per l&#39;allevamento dei nematodi. Di solito, stadi immaturi di vari ordini di insetti (ad esempio Lepidotteri, Coleotteri, Ditteri, ecc.) Sono considerati host adatti. In vivo i metodi sono di solito presi in considerazione per la manutenzione delle culture nematodi in laboratorio. Questo metodo potrebbe non essere adatto se si considera la produzione di massa dei nematodi. Grandi quantità di insetti ospiti possono essere richiesti a tale scopo chiedendo più tempo e costi supplementari connesse con l&#39;allevamento degli insetti. Nematodi entomopatogeni possono anche essere coltivate in vitro su diversi supporti. A seconda l&#39;obiettivo dello studio, metodi in vitro può o prendere in considerazione l&#39;incorporazione dei batteri simbiotici nei media. In questa presentazione, descriviamo due metodi comunemente utilizzati per la propagazione della EPN. Gli ingredienti dei media forniscono una fonte di nutrienti per il batterio simbiotico unMetodi in vitro trovare una fonte di steroli per i nematodi. offrono il vantaggio l&#39;allevamento di EPN senza un host insetto. In origine, molti dei media in vitro sviluppati sono stati utilizzati per la moltiplicazione di EPN quando adatti insetti ospiti non sono disponibili. Tuttavia, negli ultimi anni, in vitro metodi di allevamento sono diventati largamente impiegato nel campo della ricerca al fine di comprendere il rapporto mutualistico tra EPN ed i loro batteri simbionti 17,19. Le tecniche descritti in questa presentazione corrispondono a quelli descritti da vari autori e raffinato dal laboratorio Stock, University of Arizona (Tucson, AZ, USA). Queste tecniche sono distinti dal corpo di tecniche che vengono utilizzati nella produzione di massa di tali organismi a fini di gestione nocivo.

<table><tbody><tr><td colspan="4">For &lt;em&gt;In vivo&lt;/em&gt; Infections</td></tr><tr><td>100 x 15 Petri dishes</td><td>VWR</td><td>25384-088</td><td></td></tr><tr><td>Filter paper, 9 cm grade 1 cellulose</td><td>Whatman/VWR</td><td>28450-081</td><td>Grade 1 filter paper recommended</td></tr><tr><td>Insect hosts</td><td>Timberline</td><td>http://www.timberlinefisheries.com/ProductDetails.asp?ProductCode=WAXLG</td><td>&lt;em&gt;Galleria mellonella&lt;/em&gt; are recommended</td></tr><tr><td colspan="4">For Liver-kidney Agar (for 500 ml)</td></tr><tr><td>60 x 15 mm Petri dishes</td><td>VWR</td><td>25384-092</td><td></td></tr><tr><td>Beef liver, 50 g</td><td>Locally sourced; butcher or supermarket</td><td></td><td>Remaining may be stored frozen and thawed for future use</td></tr><tr><td>Beef kidney, 50 g</td><td>Locally sourced; butcher or supermarket</td><td></td><td>Remaining may be stored frozen and thawed for future use</td></tr><tr><td>Sodium chloride 2.5 g (0.5% final concentration)</td><td>Acros/VWR</td><td>200002-434</td><td>any research grade NaCl can be used</td></tr><tr><td>Agar, 7.5 g (1.5% agar, final concentration)</td><td>HiMedia/VWR</td><td>95026-642</td><td>any media grade agar can be used</td></tr><tr><td>500 ml distilled H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td colspan="4">For Lipid Agar (for 1 L)</td></tr><tr><td>100 x 15 Petri dishes</td><td>VWR</td><td>25384-088</td><td></td></tr><tr><td>Nutrient broth, 8 g</td><td>BD/VWR</td><td>90002-660</td><td></td></tr><tr><td>Yeast extract, 5 g</td><td>EMD/VWR</td><td>EM1.03753.0500</td><td></td></tr><tr><td>Magnesium chloride hexahydrate 10 ml (0.2 g/ml)</td><td>EMD/VWR</td><td>EM-MX0045-1</td><td></td></tr><tr><td>Corn oil, 4 ml</td><td>Any brand</td><td>Locally source; supermarket</td><td>Any brand of corn oil can be used</td></tr><tr><td>Corn syrup, 96 ml combine 7 ml corn syrup in 89 ml heated H&lt;sub&gt;2&lt;/sub&gt;O and swirl for homogeneity</td><td>Karo</td><td>Locally sourced; supermarket</td><td></td></tr><tr><td>Agar, 15 g</td><td>HiMedia/VWR</td><td>95026-642</td><td>any media grade agar can be used</td></tr><tr><td>Distilled H&lt;sub&gt;2&lt;/sub&gt;O, 890 ml</td><td></td><td></td><td></td></tr></tbody></table>

in vivo and in vitro rearing of entomopathogenic nematodes (steinernematidae and heterorhabditidae)

Nematodi entomopatogeni (EPN) (Steinernematidae e Heterorhabditidae) hanno una partnership mutualistico con Gram-negativi Gamma-Proteobacteria della famiglia Enterobacteriaceae batteri. Xenorhabdus sono associati con steinernematids nematodi, mentre Photorhabdus sono simbionti di heterorhabditids. Insieme nematodi e batteri formano un complesso potente insetticida che uccide una vasta gamma di specie di insetti in una intima e specifico. Qui, dimostriamo in vivo e in vitro tecniche comunemente utilizzate nell&#39;allevamento di questi nematodi in condizioni di laboratorio. Inoltre, queste tecniche rappresentano passaggi fondamentali per la creazione di successo di culture EPN e costituiscono anche la base per altri test biologici che utilizzano questi organismi per la ricerca. La produzione di aposymbiotic nematodi (senza simbionti) è spesso fondamentale per un approfondito e approccio multiforme allo studio della simbiosi. Questoprotocollo non richiede l&#39;aggiunta di antibiotici e può essere realizzato in un breve lasso di tempo con attrezzature di laboratorio standard. Nematodi prodotti in questo modo sono relativamente robusti, anche se la loro sopravvivenza in deposito possono variare a seconda delle specie utilizzate. Le tecniche descritti in questa presentazione corrispondono a quelli descritti da vari autori e raffinato dal Laboratorio di P. Archivio, University of Arizona (Tucson, AZ, USA). Queste tecniche sono distinti dal corpo di tecniche che vengono utilizzati nella produzione di massa di tali organismi a fini di gestione nocivo.

Il metodo di allevamento in vivo utilizza insetti vivi come host per la crescita e la riproduzione dei nematodi. Camere di infezione sono un metodo efficiente per esporre insetti IJs. Questa è l&#39;unica fase del ciclo di vita dei nematodi &#39;che i vettori simbionti batterici da un insetto host all&#39;altro. Figura 1 mostra il set up per una camera un&#39;infezione così come i materiali necessari per costruire questa camera. In vitro metodi di allevamento consentono EPN a crescer...

Watch this Scientific Journal Video about In vivo and In vitro Rearing of Entomopathogenic Nematodes Steinernematidae and Heterorhabditidae at JoVE.com

In vivo and In vitro Rearing of Entomopathogenic Nematodes Steinernematidae and Heterorhabditidae

L&#39;obiettivo di questa presentazione è quello di dimostrare in vivo e in vitro tecniche per l&#39;allevamento di nematodi entomopatogeni. Metodi in vivo considerano l&#39;allevamento di questi nematodi con un host insetto, mentre i metodi in vitro utilizzano rich media agar.

In vivo e in vitro Allevamento di Nematodi entomopatogeni (Steinernematidae e Heterorhabditidae)

Entomopathogenic nematodes (EPN) (Steinernematidae and Heterorhabditidae) have a mutualistic partnership with Gram-negative Gamma-Proteobacteria in the ...

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Research

JoVE Journal

Bioengineering

In vivo and In vitro Rearing of Entomopathogenic Nematodes (Steinernematidae and Heterorhabditidae)

20.5K Views.  University of Arizona.  L'obiettivo di questa presentazione è quello di dimostrare in vivo e in vitro tecniche per l'allevamento di nematodi entomopatogeni. Metodi in vivo considerano l'allevamento di questi nematodi con un host insetto, mentre i metodi in vitro utilizzano rich media agar. 

Video: In vivo and In vitro Rearing of Entomopathogenic Nematodes Steinernematidae and Heterorhabditidae

Soil Sampling and Isolation of Entomopathogenic Nematodes (Steinernematidae, Heterorhabditidae)

Entomopathogenic nematodes (a.k.a. EPN) represent a group of soil-inhabiting nematodes that parasitize a wide range of insects. These nematodes belong to two families: Steinernematidae and Heterorhabditidae. Until now, more than 70 species have been described in the Steinernematidae and there are about 20 species in the Heterorhabditidae. The nematodes have a mutualistic partnership with Enterobacteriaceae bacteria and together they act as a potent insecticidal complex that kills a wide range of insect species.
Herein, we focus on the most common techniques considered for collecting EPN from soil. The second part of this presentation focuses on the insect-baiting technique, a widely used approach for the isolation of EPN from soil samples, and the modified White trap technique which is used for the recovery of these nematodes from infected insects. These methods and techniques are key steps for the successful establishment of EPN cultures in the laboratory and also form the basis for other bioassays that consider these nematodes as model organisms for research in other biological disciplines. The techniques shown in this presentation correspond to those performed and/or designed by members of S. P. Stock laboratory as well as those described by various authors.

Soil Sampling and Isolation of Entomopathogenic Nematodes Steinernematidae, Heterorhabditidae

Entomopathogenic nematodes are soil-inhabiting roundworms that parasitize a wide range of insects. We demonstrate sampling methods used for the isolation of these nematodes from the soil using two techniques: the insect baiting and the modified White trap, for the recovery of nematodes from soil samples and infected insect cadavers, respectively.

Campionamento e Isolamento di Nematodi entomopatogeni Suolo (Steinernematidae, Heterorhabditidae)

Entomopathogenic nematodes (a.k.a. EPN) represent a group of soil-inhabiting nematodes that parasitize a wide range of insects. These nematodes belong to ...

Ricerca

Ambiente

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the intestine the primary location for disease. The nematode intestine structurally and functionally mimics mammalian intestines and is transparent making it amenable to microscopic study of colonization. Here we show that pathogens can cause disease and death. We are able to identify microbial mutants that show altered virulence. Its conserved innate response to biotic stresses makes C. elegans an excellent system to probe facets of host innate immune interactions. We show that hosts with mutations in the dual oxidase gene cannot produce reactive oxygen species and are unable to resist microbial insult. We further demonstrate the versatility of the presented survival assay by showing that it can be used to study the effects of inhibitors of microbial growth. This assay may also be used to discover fungal virulence factors as targets for the development of novel antifungal agents, as well as provide an opportunity to further uncover host-microbe interactions. The design of this assay lends itself well to high throughput whole-genome screens, while the ability to cryo-preserve worms for future use makes it a cost-effective and attractive whole animal model to study.

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

Here, we present the nematode Caenorhabditis elegans as a versatile host model to study microbial interaction.

Il Nematode Caenorhabditis Elegans - un modello Versatile In Vivo per studiare le interazioni ospite-microbo

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the ...

Genetica

High and Low Throughput Screens with Root-knot Nematodes Meloidogyne spp.

Root-knot nematodes (genus Meloidogyne) are obligate plant parasites. They are extremely polyphagous and considered one of the most economically important plant parasitic nematodes. The microscopic second-stage juvenile (J2), molted once in the egg, is the infective stage. The J2s hatch from the eggs, move freely in the soil within a film of water, and locate root tips of suitable plant species. After penetrating the plant root, they migrate towards the vascular cylinder where they establish a feeding site and initiate feeding using their stylets. The multicellular feeding site is comprised of several enlarged multinuclear cells called 'giant cells' which are formed from cells that underwent karyokinesis (repeated mitosis) without cytokinesis. Neighboring pericycle cells divide and enlarge in size giving rise to a typical gall or root knot, the characteristic symptom of root-knot nematode infection. Once feeding is initiated, J2s become sedentary and undergo three additional molts to become adults. The adult female lays 150-250 eggs in a gelatinous matrix on or below the surface of the root. From the eggs new infective J2s hatch and start a new cycle. The root-knot nematode life cycle is completed in 4-6 weeks at 26-28&deg;C.
Here we present the traditional protocol to infect plants, grown in pots, with root-knot nematodes and two methods for high-throughput assays. The first high-throughput method is used for plants with small seeds such as tomato while the second is for plants with large seeds such as cowpea and common bean. Large seeds support extended seedling growth with minimal nutrient supplement. The first high throughput assay utilizes seedlings grown in sand in trays while in the second assay plants are grown in pouches in the absence of soil. The seedling growth pouch is made of a 15.5 x 12.5cm paper wick, folded at the top to form a 2-cm-deep trough in which the seed or seedling is placed. The paper wick is contained inside a transparent plastic pouch. These growth pouches allow direct observation of nematode infection symptoms, galling of roots and egg mass production, under the surface of a transparent pouch. Both methods allow the use of the screened plants, after phenotyping, for crossing or seed production. An additional advantage of the use of growth pouches is the small space requirement because pouches are stored in plastic hanging folders arranged in racks.

High and Low Throughput Screens with Root-knot Nematodes Meloidogyne spp.

Two distinct methods to screen plants with root-knot nematodes are described. The described approaches include high-throughput screens with nematodes in a nondestructive manner facilitating the use of these plants in breeding programs.

Schermi throughput elevato e basso con Nematodi galligeni Meloidogyne spp.</em

Root-knot nematodes (genus Meloidogyne) are obligate plant parasites. They are extremely polyphagous and considered one of the most economically important ...

Immunologia e infezioni

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.

Rapido isolamento dei nematodi selvatici di Baermann Funnel

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

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on earth, nematodes and bacteria form a wide array of symbiotic associations that range from beneficial to pathogenic 1-3. One such association is the mutually beneficial relationship between Xenorhabdus bacteria and Steinernema nematodes, which has emerged as a model system of symbiosis 4. Steinernema nematodes are entomopathogenic, using their bacterial symbiont to kill insects 5. For transmission between insect hosts, the bacteria colonize the intestine of the nematode's infective juvenile stage 6-8. Recently, several other nematode species have been shown to utilize bacteria to kill insects 9-13, and investigations have begun examining the interactions between the nematodes and bacteria in these systems 9.
We describe a method for visualization of a bacterial symbiont within or on a nematode host, taking advantage of the optical transparency of nematodes when viewed by microscopy. The bacteria are engineered to express a fluorescent protein, allowing their visualization by fluorescence microscopy. Many plasmids are available that carry genes encoding proteins that fluoresce at different wavelengths (i.e. green or red), and conjugation of plasmids from a donor Escherichia coli strain into a recipient bacterial symbiont is successful for a broad range of bacteria. The methods described were developed to investigate the association between Steinernema carpocapsae and Xenorhabdus nematophila 14. Similar methods have been used to investigate other nematode-bacterium associations 9,15-18and the approach therefore is generally applicable.
The method allows characterization of bacterial presence and localization within nematodes at different stages of development, providing insights into the nature of the association and the process of colonization 14,16,19. Microscopic analysis reveals both colonization frequency within a population and localization of bacteria to host tissues 14,16,19-21. This is an advantage over other methods of monitoring bacteria within nematode populations, such as sonication 22or grinding 23, which can provide average levels of colonization, but may not, for example, discriminate populations with a high frequency of low symbiont loads from populations with a low frequency of high symbiont loads. Discriminating the frequency and load of colonizing bacteria can be especially important when screening or characterizing bacterial mutants for colonization phenotypes 21,24. Indeed, fluorescence microscopy has been used in high throughput screening of bacterial mutants for defects in colonization 17,18, and is less laborious than other methods, including sonication 22,25-27and individual nematode dissection 28,29.

To study the mutualism between Xenorhabdus bacteria and Steinernema nematodes, methods were developed to monitor bacterial presence and location within nematodes. The experimental approach, which can be applied to other systems, entails engineering bacteria to express the green fluorescent protein and visualizing, using fluorescence microscopy bacteria within the transparent nematode.

Visualizzare batteri nei nematodi usando la microscopia a fluorescenza

 
Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on ...

Biologia

Selective Cleaning of Wild Caenorhabditis Nematodes to Enrich for Intestinal Microbiome Bacteria

Caenorhabditis elegans (C. elegans)&#160;has proven to be an excellent model for studying host-microbe interactions and the microbiome, especially in the context of the intestines. Recently, ecological sampling of wild Caenorhabditis nematodes has discovered a diverse array of associated microbes, including bacteria, viruses, fungi, and microsporidia. Many of these microbes have interesting colonization or infection phenotypes that warrant further study, but they are often unculturable. This protocol presents a method to enrich the desired intestinal microbes in C. elegans and related nematodes and reduce the presence of the many contaminating microbes adhering to the cuticle. This protocol involves forcing animals into the dauer stage of development and using a series of antibiotic and detergent washes to remove external contamination. As dauer animals have physiological changes that protect nematodes from harsh environmental conditions, any intestinal microbes will be protected from these conditions. But, for enrichment to work, the microbe of interest must be maintained when animals develop into dauers. When the animals leave the dauer stage, they are singly propagated into individual lines. F1 populations are then selected for desired microbes or infection phenotypes and against visible contamination. These methods will allow researchers to enrich unculturable microbes in the intestinal lumen, which make up part of the natural microbiome of C. elegans and intracellular intestinal pathogens. These microbes can then be studied for colonization or infection phenotypes and their effects on the host fitness.

Selective Cleaning of Wild Caenorhabditis Nematodes to Enrich for Intestinal Microbiome Bacteria

Wild Caenorhabditis nematodes are associated with many microbes, often in the gut lumen or infecting the intestine. This protocol details a method to enrich unculturable microbes colonizing the intestine, taking advantage of the resistance of the dauer cuticle.

Pulizia selettiva dei nematodi caenorhabiditi selvatici per arricchire i batteri del microbioma intestinale

Caenorhabditis elegans (C. elegans)&#160;has proven to be an excellent model for studying host-microbe interactions and the microbiome, especially in the ...

Culturing and Genetically Manipulating Entomopathogenic Nematodes

Entomopathogenic nematodes in the genera Heterorhabditis and Steinernema are obligate parasites of insects that live in the soil. The main characteristic of their life cycle is the mutualistic association with the bacteria Photorhabdus and Xenorhabdus, respectively. The nematode parasites are able to locate and enter suitable insect hosts, subvert the insect immune response, and multiply efficiently to produce the next generation that will actively hunt new insect prey to infect. Due to the properties of their life cycle, entomopathogenic nematodes are popular biological control agents, which are used in combination with insecticides to control destructive agricultural insect pests. Simultaneously, these parasitic nematodes represent a research tool to analyze nematode pathogenicity and host anti-nematode responses. This research is aided by the recent development of genetic techniques and transcriptomic approaches for understanding the role of nematode secreted molecules during infection. Here, a detailed protocol on maintaining entomopathogenic nematodes and using a gene knockdown procedure is provided. These methodologies further promote the functional characterization of entomopathogenic nematode infection factors.

Entomopathogenic nematodes live in symbiosis with bacteria and together they successfully infect insects by undermining their innate immune system. To promote research on the genetic basis of nematode infection, methods for maintaining and genetically manipulating entomopathogenic nematodes are described.

Coltivare e manipolare geneticamente i nematodi entomopatogeni

Entomopathogenic nematodes in the genera Heterorhabditis and Steinernema are obligate parasites of insects that live in the soil. The main characteristic ...

Isolation and Selection of Entomopathogenic Fungi from Soil Samples and Evaluation of Fungal Virulence against Insect Pests

Entomopathogenic fungi (EPF) are one of the microbial control agents for integrated pest management. To control local or invasive pests, it is important to isolate and select indigenous EPF. Therefore, the soil bait method combined with the insect bait (mealworm, Tenebrio molitor) system was used in this study with some modifications. The isolated EPF were then subjected to the virulence test against the agricultural pest Spodoptera litura. Furthermore, the potential EPF strains were subjected to morphological and molecular identifications. In addition, the conidia production and thermotolerance assay were performed for the promising EPF strains and compared; these data were further substituted into the formula of effective conidia number (ECN) for laboratory ranking. The soil bait-mealworm system and the ECN formula can be improved by replacing insect species and integrating more stress factors for the evaluation of commercialization and field application. This protocol provides a quick and efficient approach for EPF selection and will improve the research on biological control agents.

Here we present a protocol based on the&#160;mealworm (Tenebrio molitor)-bait system that was used for isolating and selecting entomopathogenic fungi (EPF) from soil samples. An effective conidia number (ECN) formula is used to select high stress tolerant EPF based on physiological characteristics for pest microbial control in the field.

Isolamento e selezione di funghi entomopatogeni da campioni di suolo e valutazione della virulenza fungina contro gli insetti nocivi

Entomopathogenic fungi (EPF) are one of the microbial control agents for integrated pest management. To control local or invasive pests, it is important ...

Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans

The nematode Caenorhabditis elegans interacts with a large diversity of microorganisms in nature. In general, C. elegans is commonly found in rotten plant matter, especially rotten fruits like apples or on compost heaps. It is also associated with certain invertebrate hosts such as slugs and woodlice. These habitats are rich in microbes, which serve as food for C. elegans and which can also persistently colonize the nematode gut. To date, the exact diversity and consistency of the native C. elegans microbiota across habitats and geographic locations is not fully understood. Here, we describe a suitable approach for isolating C. elegans from nature and characterizing the microbiota of worms. Nematodes can be easily isolated from compost material, rotting apples, slugs, or attracted by placing apples on compost heaps. The prime time for finding C. elegans in the Northern Hemisphere is from September until November. Worms can be washed out of collected substrate material by immersing the substrate in buffer solution, followed by the collection of nematodes and their transfer onto nematode growth medium or PCR buffer for subsequent analysis. We further illustrate how the samples can be used to isolate and purify the worm-associated microorganisms and to process worms for 16S ribosomal RNA analysis of microbiota community composition. Overall, the described methods may stimulate new research on the characterization of the C. elegans microbiota across habitats and geographic locations, thereby helping to obtain a comprehensive understanding of the diversity and stability of the nematode's microbiota as a basis for future functional research.

Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans

Caenorhabditis elegans is one of the main model species in biology, yet almost all research is performed in the absence of its naturally associated microbes. The methods described here will help to improve our understanding of the diversity of associated microbes as a basis for future functional C. elegans research.

Isolamento e caratterizzazione del microbiota naturale del nematode modello Caenorhabditis elegans

The nematode Caenorhabditis elegans interacts with a large diversity of microorganisms in nature. In general, C. elegans is commonly found in rotten plant ...

<meta charset="utf8"></head><body>Ottimizzazione dell'infezione da nematode del pino nel 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.

<meta charset="utf8"></head><body>Autore in primo piano: Sistema di co-coltura in vitro di germogli di pino e nematode di pino per lo studio della risposta volatile dell'ospite

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).

In vivo e in vitro Allevamento di Nematodi entomopatogeni (Steinernematidae e Heterorhabditidae)

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