We thank Dr. Allison Richard, former director of the LSU peptide facility for the fluorescent ligand synthesis, the Interdisciplinaray Center for Biotechnology Research, UF for the ligand-lytic peptide synthesis, and the Socolovsky Microscope facility for providing access to fluorescence microscopes. Funding was provided by the SERDP Exploratory Development Program (SEED) of the Department of Defense, Department of Energy and Environmental Protection Agency, the Biotechnology AgCenter Interdisciplinary Team Program and the state of Louisiana.

Experiment 1: Extraction of termite gut protozoa under anaerobic conditions
<ol>
	<li>Use a fan box (Coy Laboratories) in a glove box to constantly circulate air through a desiccant and type D catalyst Stak-Paks to control humidity and oxygen levels and eliminate uneven temperatures. Fill the glove box with a continuous stream of nitrogen for 20 to 30 min. Monitor oxygen levels with an oxygen sensor (C-squared, Inc.) for 1 h. Use nitrogen to reach and maintain anaerobic conditions when needed.</li>
	<li>Prepare Trager U media (Trager 1934) and adjust pH to 7.0. Sparge the filter sterilized media in the glove box with a mixture of 2.5% hydrogen, 5% carbon dioxide and 92.5% nitrogen for 1 h to remove oxygen residues.</li>
	<li>Silanize all materials including microscope slides, microcentrifuge tubes, pipettes, glassware etc. used in the experiments using Sigmacote to prevent adsorption of protozoa or peptides to surfaces (Sigmacote ,Sigma, # SL-2, http://www.sigmaaldrich.com/etc/medialib/docs/ Sigma/Product_Information_Sheet/1/sl2pis.Par.0001.File.tmp/sl2pis.pdf).</li>
	<li>Since termite gut protozoa are strictly anaerobic organisms they must not be exposed to oxygen. Therefore the following steps are performed under anaerobic conditions in a glove box (see 1.1). With a forceps submerge the whole body of a termite worker in 70% ethanol and gently swirl for approximately 10 s to remove surface contaminants.</li>
	<li>Remove the worker from the ethanol and allow to dry on a clean Kimwipe for about 20 s. Use a sterile fine-tipped forceps to hold the worker abdomen and grab the tip of the abdomen with another pair of forceps to gently pull the gut upward or downward in a 45 degree angle. If the gut is pulled at a straight angle and with too much force it is likely to break apart. Place 10 guts in a drop of 100 &mu;l Trager U media on a microscope slide.</li>
	<li>Pierce the guts with a pair of sterile fine dissecting probes to release the protozoa and gently transfer the gut contents with a 200 &mu;l pipette into a 1 ml microcentrifuge tube containing 900 &mu;l Trager U media. After allowing for sedimentation of gut wall fragments (5 s), transfer 900 &mu;l of the supernatant to a fresh tube. </li>
	<li>Transfer 10 &mu;l of protozoa culture to a sigmacoted microscope slide and check the condition of protozoa under a microscope at 200 X magnification.</li>
	<li>Prepare control cultures of the aerobic protozoa Tetrahymena pyriformis, Amoeba sp., Euglena sp., and Paramecium sp. (Carolina Biological Supply Company, Burlington, NC) as well as an overnight culture of Escherichia coli in the culture media recommended by the supplier.</li>
</ol>
Experiment 2: Add ligand coupled with a fluorescent dye to the protozoa and the bacteria cultures to test for binding to the surface membranes and cell walls
We previously used phage display libraries (New England Biolabs Inc, Ipswich, MA) to identify 19 heptapeptide sequences that bind to protozoa (protocols available at http://www.neb.com/nebecomm/ManualFiles/manualE8110.pdf). A ligand with a peptide sequence (ALNLTLH) that showed similarities to putative glycoproteins known from the Trypanosoma brucei membrane was synthesized and coupled to a C-terminal fluorescent probe (EDANS, 5-((2-Aminoethyl) amino) naphthalene-1-sulfonic acid, &lambda;max = 341 nm, &lambda;em = 471 nm) via solid state peptide synthesis (SSPS) using the EDANS NovaTag resin (EMD Biosciences). Here we demonstrate that the ligand binds to the protozoa that were isolated from the termite gut and other free-living protozoa, but not to bacteria.
<ol>
	<li>Culture the protozoa as described in Exp. 1. Fix protozoa with 10 % formaldehyde at 4 &deg;C for 12 h. </li>
	<li>Centrifuge the protozoa solution (30 x g, 10 min), discard the supernatant and wash the pellet containing the fixed protozoa twice in 1ml Trager U media. Re-suspend the pellet in 1 ml Trager U media. Also, fix other protozoa, and E. coli bacteria for controls.</li>
	<li>Incubate the fixed microorganisms for 1-2 h with solution of synthesized ligand coupled to the fluorescent dye EDANS (prepared in water) at a final concentration of 50 &mu;M. The ligand dissolves better in water than Trager U media.</li>
	<li>Observe the microorganisms under a fluorescent microscope at 400 X magnification at an absorbance maximum at 341 nm and an emission in the blue region at 471 nm.</li>
</ol>
Experiment 3: Testing protozoacidal activity of the ligand coupled to a lytic peptide in vitro (protozoa culture)
A conjugate of the ligand and the lytic peptide Hecate (Mutwiri et al. 2000) was previously synthesized at the LSU Protein Facility.
<ol>
	<li>Silanize materials and prepare termite gut protozoa culture in the anaerobic glove box as described in Exp. 1.</li>
	<li>Prepare cultures of aerobe control microorganisms (e.g., E. coli and the free-living protozoa species T. pyriformis).</li>
	<li>In the anaerobic environment of the glove box, pipette 6 aliquots of 198 &mu;l of protozoa culture into 0.5 ml Eppendorf tubes. Add 2 &mu;l of a 100 &mu;M solution of ligand-lytic peptide to half of the aliquots (end concentration 1 &mu;M) of the termite gut protozoa culture. Add 2 &mu;l of water to the other half of the aliquots (controls).</li>
	<li>On the benchtop, prepare similar aliquots of E. coli and T. pyriformis with 1 &mu;M lytic peptide, ligand-lytic peptide or water.</li>
	<li>After 1 h, transfer 10 &mu;l of each protozoa culture to the slides. Compare the survival of treated protozoa to that of controls under a microscope at 200 X magnification.</li>
	<li>After 1 h, plate 100 &mu;l of approx. 10-4 dilution of the E. coli cultures on BHI and incubate at 37&deg;C overnight. Compare the numbers of colony forming units on the plates.</li>
</ol>
Experiment 4: Injection of the ligand coupled to a fluorescent dye in into the termite hindgut
<ol>
	<li>Pull needles (Model GD-1, 1 X 900 mm) using a Narishige PC-10 glass micropipette puller with a dual stage heat level (65 and 48) to obtain a tip size of 20-30 &mu;m. Confirm the tip size by measuring it under a microscope using a micrometer.</li>
	<li>Fill one needle with ca. 30 &mu;l of 50 &mu;M of fluorescently marked ligand suspended in water using an attached syringe. Fill another needle with water for the control. Attach a micropipette (0.5 &mu;l capacity and 32 mm length, Drummond Scientific Company) to a holder in a micromanipulator. Attach a needle to the injection system holder in a second micromanipulator. Set initial injection parameters to approximately 1 s pulse length and 10-12 psi injection. Advance the needle slowly into the micropipette and inject the solution using a pedal-driven high-speed electronic injection system with a pulse length control attached to a controlled nitrogen gas flow, which ensures that a constant volume is reproducibly injected. After injection, remove the micropipette and record the length of the injected solution using a Vernier caliper. Calculate the injected volume from the known parameters of the micropipette. Adjust the pressure of nitrogen gas and the pulse length to expel 0.3 &mu;l solution in a single injection. </li>
	<li>Squeeze the end of a termite worker abdomen with soft forceps to remove any excreta present in the rectum. Immobilize termite worker by chilling them on ice for 5 min.</li>
 <li>Make receivers for holding termite workers by cutting off 100 &mu;l pipette tips using a scalpel blade. Cut the tip to a length of 10 - 12 mm and use according to the size of termites.</li>
	<li>Attach the receiver to the micromanipulator. Place a worker on a Petri dish on its dorsal side and aspirate the worker head first into the receiver using a nitrogen suction pump so that the terminus of the worker protrudes from the receiver. </li>
	<li>Holding the termite in the receiver, carefully advance the filled needle using the micromanipulator to insert it into worker anus. Inject 0.3 &mu;l of the solution (fluorescently marked ligand or water for controls).</li>
	<li>Place the workers injected with ligand or water into separate petri dishes with damp filter paper and keep them at 26&plusmn;2 &deg;C with 78% R.H.</li>
	<li>Extirpate guts from the injected termites and collect the protozoa after 24 h as shown above in Exp. 1. Fix and observe the protozoa as shown in Exp. 2.</li>
</ol>
Experiment 5: Testing protozoacidal activity of the ligand coupled to a lytic peptide in vivo (injection into termite hindgut)
<ol>
	<li>Silanize materials as described in Exp. 1.</li>
	<li>Prepare a 500 &mu;M solution of the ligand-lytic peptide in water.</li>
	<li>Follow steps 3.1) through 3.4) to prepare glass needles, receivers and termite workers.</li>
	<li>Following the methods described in 3.5) inject 0.3 &mu;l of ligand-lytic peptide solution (treatment) or water (control) into the hindgut of 20 termite workers.</li>
	<li>Hold termites for 24 h, extirpate guts from several workers and observe gut contents under the microscope as described in 3.6). and 3.7).</li>
	<li>As soon a death of protozoa in the termite gut is confirmed, keep remaining treated termites and controls in Petri dishes with moist filter paper and observe mortality daily.</li>
</ol>
Representative Results:
Experiment 1: Usually, foregut, midgut and hindgut are obtained in one piece when the procedure is followed correctly (Figure 1a). The protozoa reside in high density in the anaerobic portions of the hindgut and can be released by piercing the hindgut with forceps (Figure 1b 1&amp;2). The largest protozoa species in the gut of the Formosan subterranean termite is the spindle-shaped P. grassii, which is 200-300 &mu;m long and 150 &mu;m wide and can be seen with the naked eye. The second largest species is the pear-shaped H. hartmanni (50-140 &mu;m long and 30-80 &mu;m wide). The smallest species is the cone-shaped S. leidyi (15-50 &mu;m long and 8-30 &mu;m wide; Lai et al. 1983). The protozoa species are shown in Figure 2.
Under optimal culture conditions the three species of protozoa isolated from the gut of Formosan subterranean termites will stay alive and healthy for at least 72 h in anaerobic Trager U media (Figure 3a). However, if culture conditions are not optimal protozoa will die fast. If there are oxygen residues in the media, the movement of protozoa will cease immediately. If osmotic pressure is too high or membrane integrity is compromised the surface membrane of the protozoa will bulge out and the cells rupture (Figure 3b). If osmotic pressure is too low or membranes are compromised, protozoa will shrivel and shrink (Figure 3c).
Experiment 2: We confirmed that the ligand coupled to a fluorescent probe bound to all three species of protozoa from the hindgut of Formosan subterranean termites in detectable densities. Ligand binding occurs on the whole cell surface (Figure 4). Binding sites are concentrated in the anterior region of the protozoa on the axostyle (a sheet of microtubules) and nucleus in P. grassii.
We observed some patchy autofluorescence of wood particles ingested by the protozoa. However, autofluorescence is usually easy to discern from specific binding of the ligand, since there is no autofluorescence of the surface, the axostyle and the nucleus (Figure 4).
We also detected fluorescence in all tested free-living aerobic protozoa species (Figure 5), which suggests that the ligand binds to structures generic to protozoa. However, no ligand binding was observed for E. coli.
Experiment 3: One &mu;M of ligand-lytic peptide killed all three species of protozoa from the gut of Formosan subterranean termite workers and the free-living T. pyriformis in vitro in less than 10 min, while controls stayed alive. Figure 6 shows the progressive loss of membrane integrity of the termite gut protozoa treated with ligand-lytic peptide. Membranes bulge and rupture, protozoa shrivel and die. No difference was observed in the number of E. coli colonies between treatments of ligand-lytic peptide and water. Lytic peptide with no ligand, however, reduced the number of E. coli colonies considerably (Figure 7). This suggests that attachment of the ligand to some degree protects non target microorganisms from lysis.
Experiment 4: When 0.3 &mu;l 50 &mu;M of the fluorescently marked ligand was injected into the hindgut of termite workers, binding to P. grassii, S. leidyi and H. hartmanni was confirmed via fluorescence microscopy similar to Exp. 2 (Figure 4). Termite gut tissue did not show fluorescence.
Experiment 5: Injection of 0.3 &mu;l 500 &mu;M ligand-lytic peptide killed all three species of protozoa in the gut of Formosan subterranean termites within 24h. Termites died within 10 days after loss of their symbiotic protozoa. Previously, Husseneder and Collier (2009) injected the same concentration of lytic peptide into termite guts. Without the attached ligand, it took more time until the protozoa in the gut (72 h) and the termites were dead (six weeks). This suggests that the ligand increases protozoacidal efficiency of lytic peptides, most likely by binding the lytic peptides to the protozoa. 
<img src="/files/ftp_upload/2190/2190fig1.jpg" alt="Figure 1" /> Figure 1. a: Formosan subterranean termite gut on a slide showing the main sections of the gut (fore-, mid-, hindgut); 
b 1&amp; 2: Hindgut is pierced with forceps to release the gut content containing the protozoa.
<img src="/files/ftp_upload/2190/2190fig2.jpg" alt="Figure 2" /> Figure 2. The three species of flagellate protozoa found in the hindgut of the Formosan subterranean termite: a) Pseudotrichonympha grassii, b) Holomastigotoides hartmanni, and c) Spirotrichonympha leidyi.
<img src="/files/ftp_upload/2190/2190fig3.jpg" alt="Figure 3" /> Figure 3. Protozoa in culture, a) Healthy protozoa, b) Protozoa with bulging membranes, c) Shriveled protozoa.
<img src="/files/ftp_upload/2190/2190fig4.jpg" alt="Figure 4" /> Figure 4. Confirmation of the binding of the ligand coupled to a fluorescent probe to termite gut protozoa (from top to bottom: P. grassii, H. hartmanni, S. leydi), treated with fluorescently marked ligand and untreated controls (showing autofluorescence).
<img src="/files/ftp_upload/2190/2190fig5.jpg" alt="Figure 5" /> Figure 5. Ligand binding to free-living aerobic protozoa, a) Tetrahymena, b) Amoeba, c) Euglena, and d) Paramecium.
<img src="/files/ftp_upload/2190/2190fig6.jpg" alt="Figure 6" /> Figure 6. Treatment of protozoa with a) water (control) and b) 1 &mu;M ligand-lytic peptide.
<img src="/files/ftp_upload/2190/2190fig7.jpg" alt="Figure 7" /> Figure 7. E. coli colonies on plates (10-4 dilution): a)Treated with water (control), b)Treated with 1 &mu;M ligand-lytic peptide, c)Treated with 1 &mu;M lytic peptide.

<ol>
	<li>Hansel, W., Leuschner, C., Enright, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conjugates+of+lytic+peptides+and+LHRH+or+&#946;CG+target+and+cause+necrosis+of+prostate+cancers+and+metastases.">Conjugates of lytic peptides and LHRH or &#946;CG target and cause necrosis of prostate cancers and metastases.</a> Mol. Cell. Endocrinol. 269, 26-33 (2007).</li><li>Husseneder, C., Collier, R. E., Bourtzis, K., Miller, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paratransgenesis+for+termite+control.">Paratransgenesis for termite control.</a> Insect Symbiosis. 3, 361-376 (2009).</li><li>Husseneder, C., Grace, J. K., Oishi, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+genetically+engineered+bacteria+(Escherichia+coli)+to+monitor+ingestion,+loss+and+transfer+of+bacteria+in+termites.">Use of genetically engineered bacteria (Escherichia coli) to monitor ingestion, loss and transfer of bacteria in termites.</a> Curr. Microbiol. 50, 119-123 (2005).</li><li>Husseneder, C., Grace, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+engineered+termite+gut+bacteria+deliver+and+spread+foreign+genes+in+termite+colonies.">Genetically engineered termite gut bacteria deliver and spread foreign genes in termite colonies.</a> Appl. Microbiol. Biotechnol. 68, 360-367 (2005).</li><li>Javadpour, M. M., Juban, M. M., Lo, W. C., Bishop, S. M., Alberty, J. B., Cowell, S. M., Becker, C. L., Mc Laughlin, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+novo+antimicrobial+peptides+with+low+mammalian+cell+toxicity.">De novo antimicrobial peptides with low mammalian cell toxicity.</a> J. Med. Chem. 39, 3107-3113 (1996).</li><li>Lai, P. Y., Tamashiro, M., Fuji, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abundance+and+distribution+of+the+three+species+of+symbiotic+protozoa+in+the+hindgut+of+Coptotermes+formosanus+(Isoptera).">Abundance and distribution of the three species of symbiotic protozoa in the hindgut of Coptotermes formosanus (Isoptera).</a> Proc. Haw. Entomol. Soc. 24, 271-276 (1983).</li><li>Leuschner, C., Hansel, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+disrupting+lytic+peptides+for+cancer+treatments.">Membrane disrupting lytic peptides for cancer treatments.</a> Curr. Pharm. Des. 10, 2299-2310 (2004).</li><li>Mutwiri, G. K., Henk, W. G., Enright, F. M., Corbeil, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+the+Antimicrobial+Peptide,+d-Hecate,+on+Trichomonads.">Effect of the Antimicrobial Peptide, d-Hecate, on Trichomonads.</a> J. Parasitol. 86, 1355-1359 (2000).</li><li>Trager, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cultivation+of+a+cellulose-digesting+flagellate,+Trichomonas+termopsidis,+and+of+certain+other+termite+protozoa.">The cultivation of a cellulose-digesting flagellate, Trichomonas termopsidis, and of certain other termite protozoa.</a> Biol. Bull. 66, 182-190 (1934).</li></ol>

No conflicts of interest declared.

Ligand-lytic peptides have been successfully used to effectively target and destroy cancer cells (Hansel and Leuschner 2004, Hansel et al. 2007). Based on this concept, we developed a heptapeptide ligand that binds to the surface of protozoa in the gut of Formosan subterranean termites and coupled it to a lytic peptide with the goal to destroy these obligate cellulose-digesting symbionts in the gut of termites to achieve termite control (Husseneder and Collier 2009).
We successfully ...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Sigmacote</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>SL-2</td>
<td>&nbsp;</td>
<tr>
<td>EDANS</td>
<td>&nbsp;</td>
<td>Novabiochem</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Anaerobic glove box</td>
<td>&nbsp;</td>
<td>Coy Laboratories, Inc.</td>
<td>Custom made</td>
<td>&nbsp;</td>
<tr>
<td>Intellus environmental controller</td>
<td>&nbsp;</td>
<td>Percival</td>
<td>I36NL</td>
<td>&nbsp;</td>
<tr>
<td>PC-10 Glass micropipette puller</td>
<td>&nbsp;</td>
<td>Narishige Scientific Instrument Lab</td>
<td>PC-10</td>
<td>&nbsp;</td>
<tr>
<td>Glass needles (Model GD-1, 1 X 900 mm)</td>
<td>&nbsp;</td>
<td>Narishige Scientific Instrument Lab</td>
<td>GD-1</td>
<td>&nbsp;</td>
<tr>
<td>Leitz micromanipulators</td>
<td>&nbsp;</td>
<td>Vermont Optechs, Inc.</td>
<td>ACS01</td>
<td>&nbsp;</td>
<tr>
<td>Microinjector</td>
<td>&nbsp;</td>
<td>Tritech Research, Inc.</td>
<td>MINJ-1</td>
<td>&nbsp;</td>
<tr>
<td>Microcaps</td>
<td>&nbsp;</td>
<td>Drummond Scientific Company</td>
<td>1-000-0005</td>
<td>&nbsp;</td>
<tr>
<td>LEICA fluorescence imaging system</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>DMRxA2</td>
<td>&nbsp;</td>
<tr>
<td>LEICA dissecting scope</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>MZ16</td>
<td>&nbsp;</td>
<tr>
<td>LEICA microscope</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>DMLB</td>
<td>&nbsp;</td>
<tr>
<td>Olympus dissecting scope</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>SZ61</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

testing protozoacidal activity of ligand-lytic peptides against termite gut protozoa in vitro (protozoa culture) and in vivo (microinjection into termite hindgut)

We are developing a novel approach to subterranean termite control that would lead to reduced reliance on the use of chemical pesticides. Subterranean termites are dependent on protozoa in the hindguts of workers to efficiently digest wood. Lytic peptides have been shown to kill a variety of protozoan parasites (Mutwiri et al. 2000) and also protozoa in the gut of the Formosan subterranean termite, Coptotermes formosanus (Husseneder and Collier 2009). Lytic peptides are part of the nonspecific immune system of eukaryotes, and destroy the membranes of microorganisms (Leuschner and Hansel 2004). Most lytic peptides are not likely to harm higher eukaryotes, because they do not affect the electrically neutral cholesterol-containing cell membranes of higher eukaryotes (Javadpour et al. 1996). Lytic peptide action can be targeted to specific cell types by the addition of a ligand. For example, Hansel et al. (2007) reported that lytic peptides conjugated with cancer cell membrane receptor ligands could be used to destroy breast cancer cells, while lytic peptides alone or conjugated with non-specific peptides were not effective. Lytic peptides also have been conjugated to human hormones that bind to receptors on tumor cells for targeted destruction of prostate and testicular cancer cells (Leuschner and Hansel 2004).
In this article we present techniques used to demonstrate the protozoacidal activity of a lytic peptide (Hecate) coupled to a heptapeptide ligand that binds to the surface membrane of protozoa from the gut of the Formosan subterranean termite. These techniques include extirpation of the gut from termite workers, anaerobic culture of gut protozoa (Pseudotrichonympha grassii, Holomastigotoides hartmanni, Spirotrichonympha leidyi), microscopic confirmation that the ligand marked with a fluorescent dye binds to the termite gut protozoa and other free-living protozoa but not to bacteria or gut tissue. We also demonstrate that the same ligand coupled to a lytic peptide efficiently kills termite gut protozoa in vitro (protozoa culture) and in vivo (microinjection into hindgut of workers), but is less bacteriacidal than the lytic peptide alone. The loss of protozoa leads to the death of the termites in less than two weeks.
In the future, we will genetically engineer microorganisms that can survive in the termite hindgut and spread through a termite colony as "Trojan Horses" to express ligand-lytic peptides that would kill the protozoa in the termite gut and subsequently kill the termites in the colony. Ligand-lytic peptides also could be useful for drug development against protozoan parasites.

Watch this Scientific Journal Video about Testing Protozoacidal Activity of Ligand-lytic Peptides Against Termite Gut Protozoa in vitro Protozoa Culture and in vivo Microinjection into Termite Hindgut

Testing Protozoacidal Activity of Ligand-lytic Peptides Against Termite Gut Protozoa in vitro Protozoa Culture and in vivo Microinjection into Termite Hindgut

We present procedures for demonstrating that ligands bind to the surface membrane of the cellulose-digesting protozoa in the gut of Formosan subterranean termites using fluorescent microscopy and that ligands coupled with lytic peptides kill these protozoa in vitro (anaerobic protozoa culture) and in vivo (injection into the termite hindgut).

Testing Protozoacidal Activity of Ligand-lytic Peptides Against Termite Gut Protozoa in vitro (Protozoa Culture) and in vivo (Microinjection into Termite Hindgut)

We are developing a novel approach to subterranean termite control that would lead to reduced reliance on the use of chemical pesticides. Subterranean ...

testing-protozoacidal-activity-ligand-lytic-peptides-against-termite

Research

JoVE Journal

Biology

14.8K vistas.  Louisiana State University Agricultural Center. Se presentan los procedimientos para demostrar que los ligandos se unen a la membrana de la superficie de la celulosa para digerir estos protozoos en el intestino de las termitas subterráneas de Formosa utilizando microscopía de fluorescencia y los ligandos que, junto con los péptidos líticos matar protozoarios In vitro (Cultivo de anaerobios protozoos) y In vivo (Inyección en el intestino grueso de termitas).

Video: Testing Protozoacidal Activity of Ligand-lytic Peptides Against Termite Gut Protozoa in vitro Protozoa Culture and in vivo Microinjection into Termite Hindgut

Extracting DNA from the Gut Microbes of the Termite (Zootermopsis Angusticollis) and Visualizing Gut Microbes

Termites are among the few animals known to have the capacity to subsist solely by consuming wood.  The termite gut tract contains a dense and species-rich microbial population that assists in the degradation of lignocellulose predominantly into acetate, the key nutrient fueling termite metabolism (Odelson &amp; Breznak, 1983).  Within these microbial populations are bacteria, methanogenic archaea and, in some ("lower") termites, eukaryotic protozoa. Thus, termites are excellent research subjects for studying the interactions among microbial species and the numerous biochemical functions they perform to the benefit of their host.  The species composition of microbial populations in termite guts as well as key genes involved in various biochemical processes has been explored using molecular techniques (Kudo et al., 1998; Schmit-Wagner et al., 2003; Salmassi &amp; Leadbetter, 2003). These techniques depend on the extraction and purification of high-quality nucleic acids from the termite gut environment.  The extraction technique described in this video is a modified compilation of protocols developed for extraction and purification of nucleic acids from environmental samples (Mor  et al., 1994; Berthelet et al., 1996; Purdy et al., 1996; Salmassi &amp; Leadbetter, 2003; Ottesen et al. 2006) and it produces DNA from termite hindgut material suitable for use as template for polymerase chain reaction (PCR).

Extracting DNA from the Gut Microbes of the Termite Zootermopsis Angusticollis and Visualizing Gut Microbes

This video illustrates the technique for extracting DNA from the species of microbes resident in the termite hindgut. The preparation of a wet mount slide, which is useful for visualizing the gut microbial community is also illustrated, and a tour through the species-rich gut environment is given.

Termites are among the few animals known to have the capacity to subsist solely by consuming wood.  The termite gut tract contains a dense and ...

Investigating the Microbial Community in the Termite Hindgut - Interview

Jared Leadbetter explains why the termite-gut microbial community is an excellent system for studying the complex interactions between microbes.     The symbiotic relationship existing between the host insect and lignocellulose-degrading gut microbes is explained, as well as the industrial uses of these microbes for degrading plant biomass and generating biofuels.

Jared Leadbetter explains why the termite-gut microbial community is an excellent system for studying the complex interactions between microbes. The symbiotic relationship existing between the host insect and lignocellulose-degrading gut microbes is explained, as well as the industrial uses of these microbes for degrading plant biomass and generating biofuels.

Jared Leadbetter explains why the termite-gut microbial community is an excellent system for studying the complex interactions between microbes.     The ...

Layers of Symbiosis - Visualizing the Termite Hindgut Microbial Community

Jared Leadbetter takes us for a nature walk through the diversity of life resident in the termite hindgut - a microenvironment containing 250 different species found nowhere else on Earth.  Jared reveals that the symbiosis exhibited by this system is multi-layered and involves not only a relationship between the termite and its gut inhabitants, but also involves a complex web of symbiosis among the gut microbes themselves.

Jared Leadbetter takes us for a nature walk through the diversity of life resident in the termite hindgut - a microenvironment containing 250 different species found nowhere else on Earth. Jared reveals that the symbiosis exhibited by this system is multi-layered and involves not only a relationship between the termite and its gut inhabitants, but also involves a complex web of symbiosis among the gut microbes themselves.

Jared Leadbetter takes us for a nature walk through the diversity of life resident in the termite hindgut - a microenvironment containing 250 different ...

Testing the Physiological Barriers to Viral Transmission in Aphids Using Microinjection

Potato loafroll virus (PLRV), from the family Luteoviridae infects solanaceous plants. It is transmitted by aphids, primarily, the green peach aphid. When an uninfected aphid feeds on an infected plant it contracts the virus through the plant phloem. Once ingested, the virus must pass from the insect gut to the hemolymph (the insect  blood ) and then must pass through the salivary gland, in order to be transmitted back to a new plant. An aphid may take up different viruses when munching on a plant, however only a small fraction will pass through the gut and salivary gland, the two main barriers for transmission to infect more plants. In the lab, we use physalis plants to study PLRV transmission. In this host, symptoms are characterized by stunting and interveinal chlorosis (yellowing of the leaves between the veins with the veins remaining green). The video that we present demonstrates a method for performing aphid microinjection on insects that do not vector PLVR viruses and tests whether the gut is preventing viral transmission.
The video that we present demonstrates a method for performing Aphid microinjection on insects that do not vector PLVR viruses and tests whether the gut or salivary gland is preventing viral transmission.

Aphids are effective transmitters of plant viruses. Aphid microinjection of virus, the procedure we will show you today, is a technique allowing researchers to inject virus directly into the hemocoel of the aphid, bypassing the gut, one of the 2 major barriers for virus transmission in a circulative manner. The same technique is also used to inject dsRNA for RNAi.

Potato loafroll virus (PLRV), from the family Luteoviridae infects solanaceous plants. It is transmitted by aphids, primarily, the green peach aphid. When ...

In vivo and in vitro Studies of Adaptor-clathrin Interaction

A major endocytic pathway initiates with the formation of clathrin-coated vesicles (CCVs) that transport cargo from the cell surface to endosomes1-6. CCVs are distinguished by a polyhedral lattice of clathrin that coats the vesicle membrane and serves as a mechanical scaffold. Clathrin coats are assembled during vesicle formation from individual clathrin triskelia , the soluble form of clathrin composed of three heavy and three light chain subunits7,8. Because the triskelion does not have the ability to bind to the membrane directly, clathrin-binding adaptors are critical to link the forming clathrin lattice to the membrane through association with lipids and/or membrane proteins9. Adaptors also package transmembrane protein cargo, such as receptors, and can interact with each other and with other components of the CCV formation machinery9.
Over twenty clathrin adaptors have been described, several are involved in clathrin mediated endocytosis and others localize to the trans Golgi network or endosomes9. With the exception of HIP1R (yeast Sla2p), all known clathrin adaptors bind to the N-terminal -propeller domain of the clathrin heavy chain9. Clathrin adaptors are modular proteins consisting of folded domains connected by unstructured flexible linkers. Within these linker regions, short binding motifs mediate interactions with the clathrin N-terminal domain or other components of the vesicle formation machinery9. Two distinct clathrin-binding motifs have been defined: the clathrin-box and the W-box9. The consensus clathrin-box sequence was originally defined as L[L/I][D/E/N][L/F][D/E]10 but variants have been subsequently discovered11. The W-box conforms to the sequence PWxxW (where x is any residue).
Sla1p (Synthetic Lethal with Actin binding protein-1) was originally identified as an actin associated protein and is necessary for normal actin cytoskeleton structure and dynamics at endocytic sites in yeast cells12. Sla1p also binds the NPFxD endocytic sorting signal and is critical for endocytosis of cargo bearing the NPFxD signal13,14. More recently, Sla1p was demonstrated to bind clathrin through a motif similar to the clathrin box, LLDLQ, termed a variant clathrin-box (vCB), and to function as an endocytic clathrin adaptor15. In addition, Sla1p has become a widely used marker for the endocytic coat in live cell fluorescence microscopy studies16. Here we use Sla1p as a model to describe approaches for adaptor-clathrin interaction studies. We focus on live cell fluorescence microscopy, GST-pull down, and co-immunoprecipitation methods.

In vivo and in vitro Studies of Adaptor-clathrin Interaction

Clathrin-mediated endocytosis depends on adaptor proteins that coordinate cargo selection and clathrin coat assembly. Here we describe procedures to study adaptor-clathrin physical interaction and live cell imaging approaches using as a model the yeast endocytic adaptor protein Sla1p.

A major endocytic pathway initiates with the formation of clathrin-coated vesicles (CCVs) that transport cargo from the cell surface to endosomes1-6. CCVs ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of neurulation, migrates extensively towards various organs, and differentiates into many types of derivatives (neurons, glia, cartilage and bone, pigmented and endocrine cells). In this protocol, we describe how to dissect the premigratory cranial NC from Xenopus laevis embryos, in order to study NC development in vivo and in vitro. The frog model offers many advantages to study early development; abundant batches are available, embryos develop rapidly, in vivo gain and loss of function strategies allow manipulation of gene expression prior to NC dissection in donor and/or host embryos. The NC explants can be plated on fibronectin and used for in vitro studies. They can be cultured for several days in a serum-free defined medium. We also describe how to graft NC explants back into host embryos for studying NC migration and differentiation in vivo.

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

This protocol describes how to dissect premigratory cranial neural crest (NC) from Xenopus laevis neurulas. These explants can be plated on fibronectin and cultured in vitro, or grafted back into host embryos. This technique allows studying the mechanisms of NC epithelium-to-mesenchyme transition, migration, and differentiation.

The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of ...

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

In order to efficiently stimulate an innate immune response, DNA must be of sufficient length and purity. We present a method where double stranded DNA (dsDNA) which has the requisite characteristics to stimulate the cytoplasmic DNA sensing pathways can be generated cheaply and with ease. By the concatemerization of short, synthetic oligonucleotides (which lack CpG motifs), dsDNA can be generated to be of sufficient length to activate the cytosolic DNA sensing pathway. This protocol involves blunt end ligation of the oligonucleotides in the presence of polyethylene glycol (PEG), which provides an environment for efficient ligation to occur. The dsDNA concatemers can be used, following purification by phenol/chloroform extraction, to simulate the innate immune response in vitro by standard transfection protocols. This DNA can also be used to stimulate innate immunity in vivo by intradermal injection into the ear pinna of a mouse, for example. By standardizing the concatemerization process and the subsequent stimulation protocols, a reliable and reproducible activation of the innate immune system can be produced.

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

The aim of the protocol is to use optimal methods for stimulating the cytoplasmic DNA sensing pathways in cells and in vivo. This is achieved by improving the generation of long, double-stranded DNA during blunt-end ligation. Cells or mice are then transfected using a lipid transfection reagent.

In order to efficiently stimulate an innate immune response, DNA must be of sufficient length and purity. We present a method where double stranded DNA ...

In Vivo Microinjection and Electroporation of Mouse Testis

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular transfection technique for testicular mouse cells allows the study of unique processes in spermatogenesis.
The following protocol focuses on transfection of testicular mouse cells with plasmid constructs. Specifically, we used the reporter vector pEGFP-C1, which expresses enhanced green fluorescent protein (eGFP) and also the pDsRed2-N1 vector expressing red fluorescent protein (DsRed2). Both encoded reporter genes were under the control of the human cytomegalovirus immediate-early promoter (CMV).
For performing gene transfer into mouse testes, the reporter plasmid constructs are injected into testes of living mice. To that end, the testis of an anaesthetized animal is exposed and the site of microinjection is prepared. Our preferred place of injection is the efferent duct, with the ultimately connected rete testis as the anatomical transport route of the spermatozoa between the testis and the epididymis. In this way, the filling of the seminiferous tubules after microinjection is excellently managed and controlled due to the use of stained DNA solutions. After observing a sufficient filling of the testis by its colored tubule structure, the organ is electroporated. This enables the transfer of the DNA solution into the testicular&#160;cells. Following 3 days of incubation, the testis is removed and investigated under the microscope for green or red&#160;fluorescence, illustrating transfection success.
Generally, this protocol can be employed for delivering DNA- or RNA- constructs into living mouse testis in order to (over)express or knock down genes, facilitating in vivo gene function analysis. Furthermore, it is suitable for studying reporter constructs or putative gene regulatory elements. Thus, the main advantages of the electroporation technique are fast performance in combination with low effort as well as the moderate technical equipment and skills required compared to alternative techniques.

In Vivo Microinjection and Electroporation of Mouse Testis

This article describes microinjection and electroporation of mouse testis in&#160;vivo as a&#160;transfection technique for testicular mouse cells to study unique processes of spermatogenesis. The presented protocol involves steps of glass capillary preparation, microinjection via the efferent duct, and transfection by electroporation.

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular ...