The authors wish to thank past members of the Stock lab: Ming-Min Lee, Kathryn Plichta, Victoria Miranda-Thompson and Sam-Kyu Kim for their contributions to the improvement of many of these protocols. This work was funded in part by the National Science Foundation grant IOS-0840932 and IOS-0724978 to S. P. Stock

1. In vivo Rearing of Entomopathogenic Nematodes with their Symboitic Bacteria
<ol>
	<li>Invert a 100 x 15 mm plastic Petri dish and place two discs of filter paper (90 mm) in the lid of the dish.</li>
	<li>Evenly distribute 1 ml of the IJ (infective juveniles) water suspension (at a concentration of 1,000-2,000 IJ/ml) on the filter paper. 
	NOTE: IJs do not need to be surface-sterilized.</li>
	<li>Add 10 last instar larvae of the greater waxmoth Galleria mellonella to the dish. The goal is to provide approximately 100-200 IJs/larva.</li>
	<li>Cover the lid with the bottom of the Petri dish (Figure 1) and label the Petri dish. Mention the following information: nematode species name (if known); isolate code/designation; date infection trap was set.</li>
	<li>Place the dish inside a loosely sealed plastic bag (to conserve moisture) and keep it in the dark at either room temperature or in an incubator between 20-25 &#176;C. Check dishes daily 
	NOTE: Darkness facilitates nematode infection, as it mimics natural infection conditions in the soil.</li>
	<li>After 3-5 days, remove cadavers with signs of nematode infection to a modified White trap7. 
	NOTE: If the cadavers smell putrid, this may be an indication that the culturing was not successful and/or there was contamination. Set a new infection chamber with a new batch of nematodes and&#160;insects.</li>
</ol>
2. In vitro Rearing of Entomopathogenic Nematodes with their Symbiotic Bacteria
<ol>
	<li>Liver-kidney Agar Method9,19
	<ol>
		<li>Chop liver and kidney into small pieces (2 cm3 or smaller) and place into a blender with NaCl, agar and 300 ml of water and blend until the meat becomes a liquid pulp, thick paste or puree. Then, transfer mixture to a 1 L Erlenmeyer flask.</li>
		<li>Add the final 200 ml of water to the blender vessel and decant any remaining media into the Erlenmeyer flask. Autoclave for 15 min at 121 &#176;C. After autoclaving allow agar to cool to touch before pouring. 
		NOTE: Pipetting of medium is not recommended because this medium tends to have chunks of meat.</li>
		<li>Pour ~20 ml of agar onto 5 (or 6) cm Petri dishes while hand stirring or swirling the Erlenmeyer flask between pours for a more homogenous media. Allow agar to solidify and store dishes at 4 &#176;C until needed 
		NOTE: Use a flame-sterilized metal spatula to break up any large chunks of meat poured into the Petri dish.</li>
	</ol>
	</li>
	<li>Lipid Agar Method9,19
	<ol>
		<li>Prepare lipid agar medium by mixing together nutrient broth, agar and yeast extract and pour mix into a 2 L Erlenmeyer flask. Add distilled H2O and MgCl2&#8226;6H2O and autoclave for 15 min at 121 &#176;C.</li>
		<li>Add sterile mix of corn oil and corn syrup mix and stir or swirl vigorously and often to disperse oil droplets evenly. 
		NOTE: Sterilize corn oil and syrup by placing the needed volume in a UV cross-linker. The oil will not dissolve into the liquid, but make sure it is in tiny droplets.</li>
		<li>Pour ~20 ml agar onto a 5 (or 6) cm Petri dishes and allow agar to solidify and store dishes at 4 &#176;C until needed.</li>
		<li>Streak the desired symbiotic bacteria onto the lipid agar plate and incubate plates at 28 &#176;C for 24-48 hr. 
		NOTE: Spread 100-200 &#181;l of overnight (12-16 hr) LB subculture.</li>
		<li>The day after, add approximately 0.4 ml of surface sterilized nematode suspension (eggs or infective juveniles) and incubate plates at room temperature in the dark or in an incubator at 22 &#177;3 &#176;C. 
		NOTE: Do not add more than 0.4 ml of nematode suspension as it can create an excessively wet environment that is unfavorable to nematode growth.</li>
		<li>Monitor cultures daily. Cultures should produce a new generation of IJs in about 12 days (Figure 2).</li>
		<li>Transfer bottom dish with agar when nematodes are seen crawling the sides of the dish (Figure 3A) to a modified White trap (see Orozco et al.12) to harvest IJs (Figure 3B). Perform this step under a laminar flow hood to reduce contamination of the media.</li>
		<li>Collect IJs from the modified White trap upon emergence and rinse IJ suspension to remove any medium debris. Store IJ in sterilized distilled water at 10 &#176;C (in a cold-temperature incubator or cold room) or use immediately if needed. 
		NOTE: Depending on the goal of the study, seed the plates with either symbiont-colonized or aposymbiotic nematodes.</li>
	</ol>
	</li>
</ol>
3. In vitro Rearing of Aposymbiotic (Symbiont-free) Entomopathogenic Nematodes
<ol>
	<li>Infect 10-20 G. mellonella larvae with IJs of the desired nematode species (see section 1). After 3 or 4 days (at this time IJs should have matured to first generation adults) collect cadavers. 
	NOTE: Time to maturity and number of females needed to obtain an adequate number of eggs will vary by species. Infect more G. mellonella larvae if necessary and start dissections as early as the 48 hr post-infection to assess whether they are in the right developmental stage.</li>
	<li>Place each cadaver individually in a Petri dish with saline solution (M9 buffer18). Dissect a cadaver by pulling its head with a fine tip forceps.</li>
	<li>Collect at least 20 gravid (with eggs inside) females with a fine needle (L-shape preferably) and place them in a watch glass containing 10 ml of M9 buffer (Figure 4A).</li>
	<li>Prepare an axenizing solution by considering the following ingredients: 6.75 ml distilled water, 1.25 ml 5 N NaOH, and 2.25 ml commercially available bleach solution diluted to 3% hypochlorite. 
	NOTE: NaOH is a basic solution&#8212;gloves, goggles and other appropriate protective clothing should be worn. 
	NOTE: Prepare fresh axenizing solution each time, as bleach degrades in light. Prepare several batches of axenizing solution and perform the surface sterilization and harvesting of eggs in multiple watch glasses.</li>
	<li>Rinse females at least three times with M9 buffer filling watch glass with buffer solution and sucking up buffer with a pipette. Make sure the females remain in the watch glass. Repeat this step two more times.</li>
	<li>Immediately add the axenizing solution and allow the females to remain in this solution for no more than 10&#8211;15 min. Observe the nematode tissues beginning to disintegrate while the eggs (which are resistant to the axenizing solution) remain intact.</li>
	<li>Manually disrupt female bodies to speed up the process, by cutting them with a needle or a scalpel (Figure 4B, C). Remove eggs by pipetting them into 1.5 ml microcentrifuge tubes, avoiding adding any undissolved tissue. 
	NOTE: Use as many microcentrifuge tubes as necessary to gather eggs and work quickly, as the viability of the eggs will decrease over a prolonged period of exposure to axenizing solution.</li>
	<li>Vortex tubes for 15 sec using the &#8220;high speed setting&#8221; to further remove nematode tissues attached to the eggs. Alternatively, if a vortex is unavailable, pipet up and down the solution several times.</li>
	<li>Pellet eggs by centrifugation at approximately 18,000 g for 2 min. Increase speed if eggs do not sufficiently pellet. Remove axenizing solution and rinse twice by filling the microcentrifuge tube with sterile distilled water and centrifuge one more time at 900 x g for 1 min.</li>
	<li>After the last wash, resuspend eggs in 300&#8211;500 &#181;l of sterile distilled water and place them in a sterile 3 cm Petri dish or sterilized watch glass. Remember that eggs are now surface-sterilized, so use sterile pipettes and/or pippetter tips and water to maintain cleanness of the eggs.</li>
	<li>Examine eggs under a dissecting microscope (30-50X magnification) to confirm they are intact (Figure 4D) and transfer them to a 5 cm plate with liver-kidney agar (see section 2.1) 
	NOTE: Do not add more than 400 &#181;l of the egg suspension on the plates as it will make the agar excessively wet.</li>
	<li>Label plates with appropriate information and store them upright in the dark at 28 &#176;C. Hatching of eggs should be evident overnight. 
	NOTE: Water may condense on the lid of the dish but it will evaporate after 1 or 2 days. At this time, place the dish in plastic bag to retain moisture of the agar and avoid its drying.</li>
	<li>Observe dishes periodically and discard any dishes that show signs of fungal or bacterial contamination. Once IJs are seen migrating up the wall of the Petri dish, remove the lid and transfer the bottom dish with agar to a modified White trap12. Consider sterile conditions when transferring the dish into the modified White trap to avoid contamination of the exposed media.</li>
	<li>Continue monitoring the White Traps and harvest IJ as needed. 
	NOTE: Aposymbiotic nematodes will continue to migrate into the water of the White trap for many days and even weeks.</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>

No conflicts of interest declared.

Using a suitable host is a key factor for the successful in vivo rearing of EPN. Usually, both steinernematids and heterorhabditids can reproduce and successfully complete their life cycle in larvae of the greater wax moth, Galleria mellonella (Lepidoptera: Pyralidae). However, other insect species from different families and/or orders can be considered. A few of the currently described nematode species are known to have specificity for a particular insect host. For example, S. kushidai and...

Entomopathogenic nematodes (EPN) Steinernema and Heterorhabditis spp. (Steinernematidae, Heterorhabditidae) and their bacterial symbionts, Xenorhabdus and Photorhabdus spp (Enterobacteriaceae) are considered an emergent model of terrestrial animal-microbe symbiotic relationships2-4,6,10,19. Xenorhabdus and Photorhabdus spp. are harbored as symbionts in the intestine of the only free-living stage of the nematodes, also known as the infective juvenile (IJ) or 3rd stage infective juvenile8,10,13. The bacterium-nematode pair is pathogenic for a wide range of insects and has successfully been implemented in biological control and integrated pest management programs worldwide6,8.
Herein we show a selection of in vivo and in vitro techniques that are frequently exercised for the rearing of EPN under laboratory conditions. In vivo methods contemplate an insect host for the rearing of the nematodes. Usually, immature stages of various insect orders (i.e.&#160;Lepidoptera, Coleoptera, Diptera, etc.) are considered suitable hosts. In vivo methods are usually considered for maintenance of nematode cultures in the lab. This method may not be suitable when considering mass production of the nematodes. Large quantities of insect hosts may be required for this purpose demanding more time and additional costs related to the insect rearing.
Entomopathogenic nematodes can also be cultured in vitro on several media. Depending on the goal of the study; in vitro methods may or consider the incorporation of the symbiotic bacteria in the media. In this presentation, we describe two commonly used methods for the propagation of EPN. The ingredients of the media provide a source of nutrients for the symbiotic bacterium and a sterol source for the nematodes. In vitro methods offer the advantage the rearing of EPN without an insect host.
Originally, many of the in vitro media developed were used for the multiplication of EPN when suitable insect hosts are not available. However, over the past years, in vitro rearing methods have become widely employed in research aiming to understand the mutualistic relationship between EPN and their symbiotic bacteria17,19.
The techniques detailed in this presentation correspond to those described by various authors and refined by the Stock Laboratory, University of Arizona (Tucson, AZ, USA). These techniques are distinct from the body of techniques that are used in the mass production of these organisms for pest management purposes.

<table border="0" cellpadding="0" cellspacing="0" width="1827">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:485px;">Material&#160;</td>
			<td style="width:260px;">Company</td>
			<td style="width:488px;">Catalog Number</td>
			<td style="width:595px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;"></td>
			<td style="width:260px;"></td>
			<td style="width:488px;"></td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:485px;">For in vivo Infections</td>
			<td style="width:260px;"></td>
			<td style="width:488px;"></td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:485px;">100 x 15 Petri Dishes</td>
			<td style="width:260px;">VWR</td>
			<td style="width:488px;">25384-088</td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:485px;">Filter Paper, 9 cm Grade 1 Cellulose</td>
			<td style="width:260px;">Whatman/VWR</td>
			<td style="width:488px;">28450-081</td>
			<td style="width:595px;">Grade 1 filter paper recommended</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;width:485px;">Insect hosts</td>
			<td style="width:260px;">Timberline</td>
			<td style="width:488px;">http://www.timberlinefisheries.com/ProductDetails.asp?ProductCode=WAXLG</td>
			<td style="width:595px;">Galleria mellonella are recommended</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;"></td>
			<td style="width:260px;"></td>
			<td style="width:488px;"></td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:485px;">For Liver-Kidney Agar (for 500 ml)</td>
			<td style="width:260px;"></td>
			<td style="width:488px;"></td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;width:485px;">60 x 15 mm Petri Dishes</td>
			<td style="width:260px;">VWR</td>
			<td>25384-092</td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:485px;">Beef Liver, 50 g</td>
			<td style="width:260px;">Locally sourced; butcher or supermarket</td>
			<td style="width:488px;"></td>
			<td style="width:595px;">Remaining may be stored frozen and thawed for future use</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:485px;">Beef Kidney, 50 g</td>
			<td style="width:260px;">Locally sourced; butcher or supermarket</td>
			<td style="width:488px;"></td>
			<td style="width:595px;">Remaining may be stored frozen and thawed for future use</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:485px;">Sodium Chloride 2.5g ( 0.5% final concentration )</td>
			<td style="width:260px;">Acros/VWR</td>
			<td>200002-434</td>
			<td style="width:595px;">any research grade NaCl can be used</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:485px;">Agar, 7.5 g (1.5% agar, final concentration)</td>
			<td style="width:260px;">HiMedia/VWR</td>
			<td>95026-642</td>
			<td style="width:595px;">any media grade agar can be used</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">500 ml distilled H20&#160;</td>
			<td style="width:260px;"></td>
			<td style="width:488px;"></td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:260px;"></td>
			<td style="width:488px;"></td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:485px;">For Lipid Agar (for 1 L)</td>
			<td style="width:260px;"></td>
			<td style="width:488px;"></td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:485px;">100 x 15 Petri Dishes</td>
			<td style="width:260px;">VWR</td>
			<td>25384-088</td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:485px;">Nutrient Broth, 8 g</td>
			<td style="width:260px;">BD/VWR</td>
			<td>90002-660</td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:485px;">Yeast Extract, 5g</td>
			<td style="width:260px;">EMD/VWR</td>
			<td>EM1.03753.0500</td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:485px;">Magnesium Chloride Hexahydrate,&#160; 10 ml (0.2g/ml)</td>
			<td style="width:260px;">EMD/VWR</td>
			<td>EM-MX0045-1</td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:485px;">Corn Oil, 4 ml</td>
			<td style="width:260px;">Any brand</td>
			<td style="width:488px;">Locally source; supermarket</td>
			<td style="width:595px;">Any brand of corn oil can be used</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:485px;">Corn Syrup, 96 ml combine 7 ml corn syrup in 89 ml heated H20 and swirl for homogeneity</td>
			<td style="width:260px;">Karo</td>
			<td style="width:488px;">Locally sourced; supermarket</td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agar, 15 g</td>
			<td style="width:260px;">HiMedia/VWR</td>
			<td>95026-642</td>
			<td style="width:595px;">any media grade agar can be used</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Distilled H20,890 ml</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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

Entomopathogenic nematodes (EPN) (Steinernematidae and Heterorhabditidae) have a mutualistic partnership with Gram-negative Gamma-Proteobacteria in the family Enterobacteriaceae. Xenorhabdus bacteria are associated with steinernematids nematodes while Photorhabdus are symbionts of heterorhabditids. Together nematodes and bacteria form a potent insecticidal complex that kills a wide range of insect species in an intimate and specific partnership. Herein, we demonstrate in vivo and in vitro techniques commonly used in the rearing of these nematodes under laboratory conditions. Furthermore, these techniques represent key steps for the successful establishment of EPN cultures and also form the basis for other bioassays that utilize these organisms for research. The production of aposymbiotic (symbiont&#8211;free) nematodes is often critical for an in-depth and multifaceted approach to the study of symbiosis. This protocol does not require the addition of antibiotics and can be accomplished in a short amount of time with standard laboratory equipment. Nematodes produced in this manner are relatively robust, although their survivorship in storage may vary depending on the species used. The techniques detailed in this presentation correspond to those described by various authors and refined by P. Stock&#8217;s Laboratory, University of Arizona (Tucson, AZ, USA). These techniques are distinct from the body of techniques that are used in the mass production of these organisms for pest management purposes.

The in vivo rearing method uses live insects as hosts for nematode growth and reproduction. Infection chambers are an efficient method for exposing insects IJs. This is the only stage in the nematodes&#8217; life cycle that vectors the bacterial symbionts from one insect host to another. Figure 1 shows the set up for an infection chamber as well as the materials needed to build this chamber. In vitro rearing methods allow EPN to grow without an insect host but are also implemented for t...

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

The goal of this presentation is to demonstrate in vivo and in vitro techniques for the rearing of entomopathogenic nematodes. In vivo methods consider the rearing of these nematodes with an insect host, whereas the in vitro methods utilize rich agar media.

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

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

in-vivo-vitro-rearing-entomopathogenic-nematodes-steinernematidae

Research

JoVE Journal

Bioengineering

20.4K Views.  University of Arizona. The goal of this presentation is to demonstrate in vivo and in vitro techniques for the rearing of entomopathogenic nematodes. In vivo methods consider the rearing of these nematodes with an insect host, whereas the in vitro methods utilize rich agar media.

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

Decellularization and Recellularization of Whole Livers

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to recreate or mimic the liver microstructure with grounds up approach using tissue engineering and microfabrication techniques have not been successful so far due to this design challenge. In addition, synthetic biomaterials used to create scaffolds for liver tissue engineering applications have been limited in inducing tissue regeneration and repair in large part due to the lack of specific cell binding motifs that would induce the proper cell functions2. Decellularized native tissues such blood vessels3and skin4on the other hand have found many applications in tissue engineering, and have provided a practical solution to some of the challenges. The advantage of decellularized native matrix is that it retains, to an extent, the original composition, and the microstructure, hence enhancing cell attachment and reorganization5.
In this work we describe the methods to perform perfusion-decellularization of the liver, such that an intact liver bioscaffold that retains the structure of major blood vessels is obtained. Further, we describe methods to recellularize these bioscaffolds with adult primary hepatocytes, creating a liver graft that is functional in vitro, and has the vessel access necessary for transplantation in vivo.

Perfusion decellularization is a novel technique to produce whole liver scaffolds that retains the organ's extracellular matrix composition and microarchitecture. Herein, the method of preparing whole organ scaffolds using perfusion decellularization and subsequent repopulation with hepatocytes is described. Functional and transplantable liver grafts can be generated using this technique.

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to ...

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.

This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

An In vitro System to Gauge the Thrombolytic Efficacy of Histotripsy and a Lytic Drug

Deep vein thrombosis (DVT) is a global health concern. The primary approach to achieve vessel recanalization for critical obstructions is catheter-directed thrombolytics (CDT). To mitigate caustic side effects and the long treatment time associated with CDT, adjuvant and alternative approaches are under development. One such approach is histotripsy, a focused ultrasound therapy to ablate tissue via bubble cloud nucleation. Pre-clinical studies have demonstrated strong synergy between histotripsy and thrombolytics for clot degradation. This report outlines a benchtop method to assess the efficacy of histotripsy-aided thrombolytic therapy, or lysotripsy.
Clots manufactured from fresh human venous blood were introduced into a flow channel whose dimensions and acousto-mechanical properties mimic an iliofemoral vein. The channel was perfused with plasma and the lytic&#160;recombinant tissue-type plasminogen activator. Bubble clouds were generated in the clot with a focused ultrasound source designed for the treatment of femoral venous clots. Motorized positioners were used to translate the source focus along the clot length. At each insonation location, acoustic emissions from the bubble cloud were passively recorded, and beamformed to generate passive cavitation images. Metrics to gauge treatment efficacy included clot mass loss (overall treatment efficacy), and the concentrations of D-dimer (fibrinolysis) and hemoglobin (hemolysis) in the perfusate. There are limitations to this in vitro design, including lack of means to assess in vivo side effects or dynamic changes in flow rate as the clot lyses. Overall, the setup provides an effective method to assess the efficacy of histotripsy-based strategies to treat DVT.

Histotripsy-aided lytic delivery or lysotripsy is under development for the treatment of deep vein thrombosis. An in vitro procedure is presented here to assess the efficacy of this combination therapy. Key protocols for the clot model, image guidance, and assessment of treatment efficacy are discussed.