We acknowledge financial support from the Australian Research Council (Grants DP130103813 and LP140100832 to G.F.K., DECRA Fellowship DE160101142 to EABU), the Australian National Health &amp; Medical Research Council (Principal Research Fellowship APP1044414 to G.F.K.), and The University of Queensland (Postdoctoral Fellowship to A.A.W.).

This protocol complies with The University of Queensland&#39;s policy set out in Responsible Care and Use of Animals in Teaching and Research (PPL 4.20.11) as well as the National Health and Medical Research Council&#39;s Australian code for the care and use of animals for scientific purposes (8th Edition 2013).
Caution: Take care not to be envenomated when handling assassin bugs. Take care to protect the eyes when handling species that spit venom defensively. Take care throughout not to injure the experimental animals. This includes monitoring of pressure on restraints such as rubber bands and ensuring that the proboscis is not broken.
NOTE: Optionally, anaesthetize animals by exposure to CO2 for 0.5-2 min or cooling to 4-10 &#176;C prior to venom harvesting in Aims 1-3 to facilitate safe transfer and restraint. Anaesthetization is not strictly required but may facilitate safe restraint of agile or strong specimens. However, animals must be awake to allow venom harvesting. Keep downstream applications in mind when deciding whether or not to add protease inhibitors.
1. Harvesting Venom Toxins by Electrostimulation
<ol>
	<li>Obtain live specimens from which to harvest toxins.</li>
	<li>Use pre-prepared plastic tweezers with positive and negative electrodes mounted on either tip. Connect electrified tweezers to an electrostimulator or a source of constant voltage that allows adjustment of the voltage.
	<ol>
		<li>For small (~10 mm) and large (~25 mm) assassin bugs, use peak voltages of 15 and 25 V respectively.</li>
		<li>For larger heteropterans such as giant water bugs, use up to 40 V.</li>
	</ol>
	</li>
	<li>Restrain live bugs by strapping them to a platform, using a rubber band over the thorax.</li>
	<li>Place the tip of the proboscis in a suitable collecting tip. For assassin bugs, use a P200 pipette tip. For giant water bugs, cut the extremity off a P200 tip to increase the size of the aperture.
	<ol>
		<li>Gently lift the proboscis with a closed pair of clean tweezers and push the open aperture of the collection tip over the end of the proboscis.</li>
		<li>If desired, uptake ~5 &#181;L ultrapure water prior to placing the proboscis into the collecting tip. This reduces losses of venom remaining inside of the tip, though the harvested venom will be diluted.</li>
	</ol>
	</li>
	<li>Apply electrostimulation. Dip the stimulating electrodes in conductive gel, such as 2.5 M NaCl/50% glycerol. Apply the electrodes to the thorax. For belostomatids, apply the two electrodes to the dorsal posterior surface of the head.</li>
	<li>Store venom to prevent autodegradation. After venom is extruded, quickly transfer it to a tube at -20 &#176;C or -60 &#176;C, or a tube containing protease inhibitor cocktail.</li>
	<li>Repeat steps 1.5 and 1.6 until sufficient venom is acquired or no further venom is forthcoming.</li>
</ol>
2. Harvesting of Venom Toxins by Harassment
<ol>
	<li>Prepare animals for venom harvesting and place the tip of the proboscis in a collection vestibule, as described in subsections 1.1 and 1.3-1.4.</li>
	<li>If venom is extruded spontaneously, go to step 2.3. If not, harass the animals by gently touching it on the legs, abdomen and antennae with tweezers until venom is produced.</li>
	<li>Quickly transfer venom to a tube at -20 &#176;C or -60 &#176;C, or a tube containing protease inhibitor cocktail, if desired.</li>
</ol>
3. Harvesting of Venom Toxins by Harassment from Venom &#34;Spitting&#34; Species
<ol>
	<li>Anaesthetize, or partly anaesthetize, the insect before removing it from its enclosure to prevent any premature defensive spitting.</li>
	<li>Provoke venom spitting behavior. Contain and reposition the insect using the deep lid of a standard 90 x 16 mm Petri dish. Hold the lid slightly posterior and 1-4 cm above the insect to prevent flight. Most insects will spit several times, often in quick succession. Ensure all venom is collected on the underside of the dish.</li>
	<li>Collect the venom on the underside of the Petri dish by rinsing with 10 &#181;L of ultrapure water. Quickly transfer it to a tube at -20 &#176;C or -60 &#176;C, or a tube containing protease inhibitor cocktail.</li>
</ol>
4. Harvesting Venom Toxins by Gland Dissection
<ol>
	<li>Sacrifice animals. Heavily anaesthetize or kill animals using &#62;5 min exposure to CO2. Pipe pure CO2 directly into the air-holes of the animal&#39;s housing enclosure.</li>
	<li>Pin insect to dissection tray. For assassin bugs, dissect through the ventral surface (4.3). For giant water bugs, dissect through the dorsal surface (4.4).</li>
	<li>Ventral dissection
	<ol>
		<li>Insert three pins into the posterior abdomen to hold the insect down without puncturing the venom glands.</li>
		<li>Cut a short midline incision in the ventral surface of the abdomen using a miniature scalpel. Use miniature scissors to extend the midline incision anteriorly to the head, taking care to cut the exoskeleton only and not damage internal structures.</li>
		<li>To expose the internal structures, make multiple lateral cuts extending from the midline incision to the side of the insect. Then, pin back each flap of ventral exoskeleton to reveal internal structures.</li>
		<li>For large assassin bugs, make four lateral incisions, in the mid-abdomen, anterior abdomen, between first and second legs, and in front of the first leg.</li>
	</ol>
	</li>
	<li>Dorsal dissection
	<ol>
		<li>Remove the wings near the base. Insert three pins into the posterior abdomen to hold the insect down without puncturing the venom glands.</li>
		<li>Cut a midline incision from the head to the abdomen using miniature scissors and scalpel, taking care to cut the exoskeleton only and not damage internal structures.</li>
		<li>Force apart the two halves of the insect. Place several pins laterally along the length of the insect to leave the internal cavity exposed.</li>
		<li>Remove the flight muscles using tweezers.</li>
	</ol>
	</li>
	<li>Flood the dissection tray. Add PBS until the bug is submerged to allow internal structures float up and be more easily visualized.</li>
	<li>Using tweezers and micro-scissors, carefully remove connective and nervous tissue and trachea. The venom glands appear as elongated, translucent structures extending along each side of the alimentary canal.
	<ol>
		<li>Identify the main gland by its characteristic appearance, with anterior and posterior lobes and two ducts meeting at the hilus.</li>
		<li>If desired, identify the accessory gland by tracing the duct from the hilus. Free the main gland by cutting the two ducts emanating from the hilus.</li>
	</ol>
	</li>
	<li>Harvest the desired gland lumens. Transfer the gland to a microcentrifuge on ice containing 30 &#181;L of PBS or PBS plus protease inhibitor cocktail. Lance the glands with a clean sharp pin.
	<ol>
		<li>Vortex for 10 s and centrifuge (1 min, 5,000 &#215; g, 4 &#176;C) to empty the gland lumens. Remove the glandular tissue using tweezers.</li>
	</ol>
	</li>
	<li>Clarify the toxin extract. Centrifuge (5 min, 17,000 &#215; g, 4&#176;C) to remove any solid particles, retaining the supernatant and discarding the pellet. Store at -20&#176;C or -60 &#176;C to prevent autoproteolytic degradation.</li>
</ol>

<ol><li>Walker, A. A., Weirauch, C., Fry, B. G., King, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Venoms+of+heteropteran+insects%3A+A+treasure+trove+of+diverse+pharmacological+toolkits%5BTitle%5D)+AND+%22Toxins%22%5BJournal%5D)">Venoms of heteropteran insects: A treasure trove of diverse pharmacological toolkits.</a> Toxins. 8 (2), 43(2016).</li>
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<li>Ambrose, D. P., Maran, S. P. M. <a target="_blank" href="https://www.researchgate.net/publication/320234828_Quantification_protein_content_and_paralytic_potential_of_saliva_of_fed_and_prey_deprived_reduviid_Acanthaspis_pedestris_Stal_Heteroptera_Reduviidae_Reduviinae">Quantification protein content and paralytic potential of saliva of fed and prey deprived reduviid Acanthaspis pedestris Stål (Heteroptera: Reduviidae: Reduviinae).</a> Indian Journal of Environmental Science. 3 (1), 11-16 (1999).</li>
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<li>Walker, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Giant+fish-killing+water+bug+reveals+ancient+and+dynamic+venom+evolution+in+Heteroptera%5BTitle%5D)+AND+%22Cell.+Mol.+Life+Sci%22%5BJournal%5D)">Giant fish-killing water bug reveals ancient and dynamic venom evolution in Heteroptera.</a> Cell. Mol. Life Sci. , (2018).</li>
<li>Walker, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+assassin+bug+Pristhesancus+plagipennis+produces+two+distinct+venoms+in+separate+gland+lumens%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">The assassin bug Pristhesancus plagipennis produces two distinct venoms in separate gland lumens.</a> Nature Communications. 9 (1), 755(2018).</li>
<li>Hernández-Vargas, M. J., Santibáñez-López, C. E., Corzo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+insight+into+the+triabin+protein+family+of+American+hematophagous+reduviids%3A+Functional%2C+structural+and+phylogenetic+analysis%5BTitle%5D)+AND+%22Toxins%22%5BJournal%5D)">An insight into the triabin protein family of American hematophagous reduviids: Functional, structural and phylogenetic analysis.</a> Toxins. 8 (2), 44(2016).</li>
<li>Dan, A., Pereira, M. H., Pesquero, J. L., Diotaiuti, L., Beirao, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Action+of+the+saliva+of+Triatoma+infestans+%28Heteroptera%3A+Reduviidae%29+on+sodium+channels%5BTitle%5D)+AND+%22Journal+of+Medical+Entomology%22%5BJournal%5D)">Action of the saliva of Triatoma infestans (Heteroptera: Reduviidae) on sodium channels.</a> Journal of Medical Entomology. 36 (6), 875-879 (1999).</li>
<li>Corzo, G., Adachi-Akahane, S., Nagao, T., Kusui, Y., Nakajima, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Novel+peptides+from+assassin+bugs+%28Hemiptera%3A+Reduviidae%29%3A+isolation%2C+chemical+and+biological+characterization%5BTitle%5D)+AND+%22FEBS+Letters%22%5BJournal%5D)">Novel peptides from assassin bugs (Hemiptera: Reduviidae): isolation, chemical and biological characterization.</a> FEBS Letters. 499 (3), 256-261 (2001).</li>
<li>Sahayaraj, K., Kumar, S. M., Anandh, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+milking+and+electric+shocks+for+venom+collection+from+hunter+reduviids%5BTitle%5D)+AND+%22Entomon%22%5BJournal%5D)">Evaluation of milking and electric shocks for venom collection from hunter reduviids.</a> Entomon. 31 (1), 65-68 (2006).</li>
<li>Silva-Cardoso, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Paralytic+activity+of+lysophosphatidylcholine+from+saliva+of+the+waterbug+Belostoma+anurum%5BTitle%5D)+AND+%22Journal+of+Experimental+Biology%22%5BJournal%5D)">Paralytic activity of lysophosphatidylcholine from saliva of the waterbug Belostoma anurum.</a> Journal of Experimental Biology. 213 (19), 3305-3310 (2010).</li>
<li>Noeske-Jungblut, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Triabin%2C+a+highly+potent+exosite+inhibitor+of+Thrombin%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">Triabin, a highly potent exosite inhibitor of Thrombin.</a> Journal of Biological Chemistry. 270 (48), 28629-28634 (1995).</li>
<li>Noeske-Jungblut, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+inhibitor+of+collagen-induced+platelet+aggregation+from+the+saliva+of+Triatoma+pallidipennis%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">An inhibitor of collagen-induced platelet aggregation from the saliva of Triatoma pallidipennis.</a> Journal of Biological Chemistry. 269 (7), 5050-5053 (1994).</li>
<li>Sahayaraj, K., Borgio, J. F., Muthukumar, S., Anandh, G. P. <a target="_blank" href="http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1678-91992006000300011">Antibacterial activity of Rhynocoris marginatus (Fab.) and Catamirus brevipennis (Servile) (Hemiptera: Reduviidae) venoms against human pathogens.</a> Journal of Venomous Animals and Toxins Including Tropical Diseases. 12 (3), 487-496 (2006).</li>
<li>Haridass, E. T., Ananthakrishnan, T. N. <a target="_blank" href="http://agris.fao.org/agris-search/search.do?recordID=US201301326115">Functional morphology of the salivary system in some reduviids (Insecta-Heteroptera-Reduviidae).</a> Proceedings of the Indian Academy of Sciences. Animal Sciences. 90 (2), 145-160 (1981).</li>
<li>Ignacimuth, A., Sen, A., Janarthanan, S. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aMaran%2C+SPM+%22Biotechnological+Applications+for+Integrated+Pest+Management%22">Biotechnological Applications for Integrated Pest Management</a>. , Oxford Publishing. 125-131 (2000).</li>
<li>Maran, S. P. M., Selvamuthu, K., Rajan, K., Kiruba, D. A., Ambrose, D. P. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aMaran%2C+SPM+%22Insect+Pest+Management%2C+A+Current+Scenario%22">Insect Pest Management, A Current Scenario</a>. Ambrose, D. P. , Entomology Research Unit. 346-361 (2011).</li>
<li>Pereira, M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anticoagulant+activity+of+Triatoma+infestans+and+Panstrongylus+megistus+saliva+%28Hemiptera%2FTriatominae%29%5BTitle%5D)+AND+%22Acta+Tropica%22%5BJournal%5D)">Anticoagulant activity of Triatoma infestans and Panstrongylus megistus saliva (Hemiptera/Triatominae).</a> Acta Tropica. 61, 255-261 (1996).</li>
<li>Ribeiro, J. M., Marinotti, O., Gonzales, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+salivary+vasodilator+in+the+blood-sucking+bug%2C+Rhodnius+prolixus%5BTitle%5D)+AND+%22British+Journal+of+Pharmacology%22%5BJournal%5D)">A salivary vasodilator in the blood-sucking bug, Rhodnius prolixus.</a> British Journal of Pharmacology. 101 (4), 932-936 (1990).</li>
<li>Ribeiro, J. M., Schneider, M., Guimarães, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Purification+and+characterization+of+prolixin-S+%28nitrophorin+2%29%2C+the+salivary+anticoagulant+of+the+blood-sucking+bug+Rhodnius+prolixus%5BTitle%5D)+AND+%22Biochem+Journal%22%5BJournal%5D)">Purification and characterization of prolixin-S (nitrophorin 2), the salivary anticoagulant of the blood-sucking bug Rhodnius prolixus.</a> Biochem Journal. 308 (1), 243-249 (1995).</li>
<li>Swart, C. C., Deaton, L. E., Felgenhauer, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+salivary+gland+and+salivary+enzymes+of+the+giant+waterbugs+%28Heteroptera%3B+Belostomatidae%29%5BTitle%5D)+AND+%22Comparative+Biochemistry+and+Physiology+A+Molecular+%26+Integrative+Physiology%22%5BJournal%5D)">The salivary gland and salivary enzymes of the giant waterbugs (Heteroptera; Belostomatidae).</a> Comparative Biochemistry and Physiology A Molecular & Integrative Physiology. 145 (1), 114-122 (2006).</li>
<li>Rasmussen, S., Young, B., Krimm, H. <a target="_blank" href="http://onlinelibrary.wiley.com/doi/10.1111/j.1469-7998.1995.tb02743.x/full">On the 'spitting' behaviour in cobras (Serpentes: Elapidae).</a> Journal of Zoology. 237 (1), 27-35 (1995).</li>
<li>Fink, L. S. <a target="_blank" href="https://bugguide.net/node/view/1371711">Venom spitting by the green lynx spider, Peucetia viridans (Araneae, Oxyopidae).</a> Journal of Arachnology. 12, 372-373 (1984).</li>
<li>Herzig, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ontogenesis%2C+gender%2C+molting+influence+the+venom+yield+in+the+spider+Coremiocnemis+tropix+%28Araneae%2C+Theraphosidae%29%5BTitle%5D)+AND+%22Journal+of+Venomous+Research%22%5BJournal%5D)">Ontogenesis, gender, molting influence the venom yield in the spider Coremiocnemis tropix (Araneae, Theraphosidae).</a> Journal of Venomous Research. 1, 76-83 (2010).</li>
<li>Sahayaraj, K., Subramanium, M., Rivers, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biochemical+and+electrophoretic+analyses+of+saliva+from+the+predatory+reduviid+species+Rhynocoris+marginatus+%28Fab%29%5BTitle%5D)+AND+%22Acta+Biochimica+Polonica%22%5BJournal%5D)">Biochemical and electrophoretic analyses of saliva from the predatory reduviid species Rhynocoris marginatus (Fab).</a> Acta Biochimica Polonica. 60 (1), 91-97 (2013).</li>
</ol>

The authors have nothing to disclose.

The most critical step in harvesting assassin bug venom is selecting the appropriate method depending on the purposes of the study. Each of the three methods presented for harvesting heteropteran venoms has advantages and disadvantages depending on downstream applications.
Inducing bugs to expel venom from the proboscis (Protocols 1-3) avoids contamination of venom by glandular tissues. In addition, these methods are non-lethal and can be repeated many times over the course of a bug's life. El...

Heteropteran venoms are potently bioactive substances1. For example, the venom/saliva secretions of blood-feeding Heteroptera such as kissing bugs (Triatominae) and bed bugs (Cimicidae) facilitates feeding by disrupting hemostasis2. Toxins in these venoms target multiple pathways including coagulation, platelet aggregation and vasoconstriction, as well as the pain and itch pathways. Venoms from most other heteropteran species are adapted to facilitate predation rather than blood-feeding. Their venoms cause paralysis, death and tissue liquefaction when injected into invertebrates3,4. When injected into vertebrates, their venom may also have drastic effects. For example, injection of venom from the assassin bug Holotrichius innesi into vertebrates causes pain, muscle paralysis and hemorrhage; mice envenomated by this bug die quickly due to respiratory paralysis5.
Transcriptomic and proteomic studies have revealed the protein composition of some heteropteran venoms. Venoms of predaceous species are rich in proteases, other enzymes, and peptides and proteins of unknown structure and function6,7,8. Kissing bug venom is rich in the triabin protein family, whose members profoundly affect coagulation, platelet aggregation, and vasoconstriction2,9. However, it is not known which toxins underlie most bioactivities of venom. For example, venom of the kissing bug Triatoma infestans has been reported to be analgesic and inhibit sodium channels10, but the components responsible remain to be elucidated. Likewise, it is not known what component(s) of assassin bug venom cause paralysis or pain. A prerequisite for identifying the toxins responsible for particular venom bioactivities, and for characterizing the structure and function of novel venom toxins, is obtaining venom.
Venom has been obtained from heteropterans by electrostimulation5,6,7,8,11,12,13, provocation of defensive responses4,8, mechanically squeezing the thorax12,14,15,16, dissecting out venom glands8,17,18,19,20,21,22, and application of agonists of the muscarinic acetylcholine receptor23. Judging the potential advantages and disadvantages of any method is complicated by the morphology of heteropteran venom glands, which consist of a main gland with two separate lumens, the anterior main gland (AMG) and posterior main gland (PMG), as well as an associated accessory gland (AG). These different gland compartments produce different protein secretions, which may be specialized for different biological functions including prey capture, defense and extra-oral digestion8,17. In peiratine and ectrichodiine assassin bugs, the AMG has been associated with prey capture and the PMG with extra-oral digestion17. However, in the harpactorine bug Pristhesancus plagipennis the PMG is specialized for prey capture and digestion whereas the AMG is hypothesized to secrete defensive venom8. The AG has been described as having little secretory function in assassin bugs8 or as a major site of protease storage in giant water bugs23. Clearly, further work is required to clarify the function of each gland compartment among various heteropteran subgroups, and to determine the function of most venom toxins. In this report we describe protocols for harvesting venom toxins from heteropterans toward this goal.

<table><tbody><tr><td>Electostimulator</td><td>Grass Technologies</td><td>S48 Square Pulse Stimulator</td><td>Electrostimulator allowing pulsed electrostimulation</td></tr><tr><td>Featherlight tweezers</td><td>Australian Entomological Supplies</td><td>E122B</td><td>For handling live venomous insects</td></tr><tr><td>Protease inhibitor cocktail</td><td>Sigma</td><td>4693124001</td><td>For preventing autoproteolytic digestion of venom</td></tr><tr><td>Dissection equipment</td><td>Australian Entomological Supplies</td><td>E152Micro</td><td>For fine dissections</td></tr><tr><td>Insect pins</td><td>Australian Entomological Supplies</td><td>E162</td><td>For fine dissections</td></tr></tbody></table>

harvesting venom toxins from assassin bugs and other heteropteran insects

Heteropteran insects such as assassin bugs (Reduviidae) and giant water bugs (Belostomatidae) descended from a common predaceous and venomous ancestor, and the majority of extant heteropterans retain this trophic strategy. Some heteropterans have transitioned to feeding on vertebrate blood (such as the kissing bugs, Triatominae; and bed bugs, Cimicidae) while others have reverted to feeding on plants (most Pentatomomorpha). However, with the exception of saliva used by kissing bugs to facilitate blood-feeding, little is known about heteropteran venoms compared to the venoms of spiders, scorpions and snakes.
One obstacle to the characterization of heteropteran venom toxins is the structure and function of the venom/labial glands, which are both morphologically complex and perform multiple biological roles (defense, prey capture, and extra-oral digestion). In this article, we describe three methods we have successfully used to collect heteropteran venoms. First, we present electrostimulation as a convenient way to collect venom that is often lethal when injected into prey animals, and which obviates contamination by glandular tissue. Second, we show that gentle harassment of animals is sufficient to produce venom extrusion from the proboscis and/or venom spitting in some groups of heteropterans. Third, we describe methods to harvest venom toxins by dissection of anaesthetized animals to obtain the venom glands. This method is complementary to other methods, as it may allow harvesting of toxins from taxa in which electrostimulation and harassment are ineffective. These protocols will enable researchers to harvest toxins from heteropteran insects for structure-function characterization and possible applications in medicine and agriculture.

Some heteropteran species, such as the harpactorine P. plagipennis and the reduviine Platymeris rhadamanthus, reliably yield large quantities (5-20 &#181;L) of venom in response to electrostimulation (Table 1). In general, most peiratine, reduviine, and harpactorine bugs yield venom in response to this method. Among stenopodaine bugs, electrostimulation elicited venom from Oncocephalus sp. but not Thodelmus sp. The holoptiline and emesi...

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Harvesting Venom Toxins from Assassin Bugs and Other Heteropteran Insects

Although many insects in the suborder Heteroptera (Insecta: Hemiptera) are venomous, their venom composition and the functions of their venom toxins are mostly unknown. This protocol describes methods to harvest heteropteran venoms for further characterization, using electrostimulation, harassment, and gland dissection.

Heteropteran insects such as assassin bugs (Reduviidae) and giant water bugs (Belostomatidae) descended from a common predaceous and venomous ancestor, ...

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13.1K Views.  The University of Queensland. Although many insects in the suborder Heteroptera (Insecta: Hemiptera) are venomous, their venom composition and the functions of their venom toxins are mostly unknown. This protocol describes methods to harvest heteropteran venoms for further characterization, using electrostimulation, harassment, and gland dissection.

Video: Harvesting Venom Toxins from Assassin Bugs and Other Heteropteran Insects

Helminth Collection and Identification from Wildlife

Wild animals are commonly parasitized by a wide range of helminths. The four major types of helminths are &#34;roundworms&#34; (nematodes), &#34;thorny-headed worms&#34; (acanthocephalans), &#34;flukes&#34; (trematodes), and &#34;tapeworms&#34; (cestodes). The optimum method for collecting helminths is to examine a host that has been dead less than 4-6 hr since most helminths will still be alive. A thorough necropsy should be conducted and all major organs examined. Organs are washed over a 106 &#956;m sieve under running water and contents examined under a stereo microscope. All helminths are counted and a representative number are fixed (either in 70% ethanol, 10% buffered formalin, or alcohol-formalin-acetic acid). For species identification, helminths are either cleared in lactophenol (nematodes and small acanthocephalans) or stained (trematodes, cestodes, and large acanthocephalans) using Harris&#39; hematoxylin or Semichon&#39;s carmine. Helminths are keyed to species by examining different structures (e.g. male spicules in nematodes or the rostellum in cestodes). The protocols outlined here can be applied to any vertebrate animal. They require some expertise on recognizing the different organs and being able to differentiate helminths from other tissue debris or gut contents. Collection, preservation, and staining are straightforward techniques that require minimal equipment and reagents. Taxonomic identification, especially to species, can be very time consuming and might require the submission of specimens to an expert or DNA analysis.

Wild animals are commonly parasitized by a wide range of helminths.&#160; The four major types of helminths are &#8220;roundworms&#8221; (nematodes), &#8220;thorny-headed worms&#8221; (acanthocephalans), &#8220;flukes&#8221; (trematodes), and &#8220;tapeworms&#8221; (cestodes).&#160; Here we describe how helminths are collected from a vertebrate animal and how they are preserved and taxonomically identified.&#160;

Wild animals are commonly parasitized by a wide range of helminths. The four major types of helminths are &#34;roundworms&#34; (nematodes), ...

Quantification of Heavy Metals and Other Inorganic Contaminants on the Productivity of Microalgae

Increasing demand for renewable fuels has researchers investigating the feasibility of alternative feedstocks, such as microalgae. Inherent advantages include high potential yield, use of non-arable land and integration with waste streams. The nutrient requirements of a large-scale microalgae production system will require the coupling of cultivation systems with industrial waste resources, such as carbon dioxide from flue gas and nutrients from wastewater. Inorganic contaminants present in these wastes can potentially lead to bioaccumulation in microalgal biomass negatively impact productivity and limiting end use. This study focuses on the experimental evaluation of the impact and the fate of 14 inorganic contaminants (As, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb, Sb, Se, Sn, V and Zn) on Nannochloropsis salina growth. Microalgae were cultivated in photobioreactors illuminated at 984 &#181;mol m-2 sec-1 and maintained at pH 7 in a growth media polluted with inorganic contaminants at levels expected based on the composition found in commercial coal flue gas systems. Contaminants present in the biomass and the medium at the end of a 7 day growth period were analytically quantified through cold vapor atomic absorption spectrometry for Hg and through inductively coupled plasma mass spectrometry for As, Cd, Co, Cr, Cu, Mn, Ni, Pb, Sb, Se, Sn, V and Zn. Results show N. salina is a sensitive strain to the multi-metal environment with a statistical decrease in biomass yieldwith the introduction of these contaminants. The techniques presented here are adequate for quantifying algal growth and determining the fate of inorganic contaminants.

Integration of microalgal cultivation with industrial flue gas will ultimately introduce heavy metals and other inorganic compounds into the growth media. This study presents a procedure used to determine the end fate and impact of heavy metals and inorganic contaminants on the growth of Nannochloropsis salina grown in photobioreactors.

Increasing demand for renewable fuels has researchers investigating the feasibility of alternative feedstocks, such as microalgae. Inherent advantages ...

Assaying Predatory Feeding Behaviors in Pristionchus and Other Nematodes

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

Assaying Predatory Feeding Behaviors in Pristionchus and Other Nematodes

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

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

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment during manufacturing and transport. Because of their environmental persistence, they are widely distributed in the oceans and on beaches all over the world. They can act as a vector of potentially toxic organic compounds (e.g., polychlorinated biphenyls) and might consequently negatively affect marine organisms. Their possible impacts along the food chain are not yet well understood. In order to assess the hazards associated with the occurrence of plastic pellets in the marine environment, it is necessary to develop methodologies that allow for rapid determination of associated organic contaminant levels. The present protocol describes the different steps required for sampling resin pellets, analyzing adsorbed organochlorine pesticides (OCPs) and identifying the plastic type. The focus is on the extraction of OCPs from plastic pellets by means of a pressurized fluid extractor (PFE) and on the polymer chemical analysis applying Fourier Transform-InfraRed (FT-IR) spectroscopy. The developed methodology focuses on 11 OCPs and related compounds, including dichlorodiphenyltrichloroethane (DDT) and its two main metabolites, lindane and two production isomers, as well as the two biologically active isomers of technical endosulfan. This protocol constitutes a simple and rapid alternative to existing methodology for evaluating the concentration of organic contaminants adsorbed on plastic pieces.

Microplastics act as vector of potentially toxic organic contaminants with unpredictable effects. This protocol describes an alternative methodology for assessing the levels of organochlorine pesticides adsorbed on plastic pellets and identifying the polymer chemical structure. The focus is on pressurized fluid extraction and attenuated total reflectance Fourier transform infrared spectroscopy.

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment ...

Extraction and Characterization of Surfactants from Atmospheric Aerosols

Surface-active compounds, or surfactants, present in atmospheric aerosols are expected to play important roles in the formation of liquid water clouds in the Earth&#39;s atmosphere, a central process in meteorology, hydrology, and for the climate system. But because specific extraction and characterization of these compounds have been lacking for decades, very little is known on their identity, properties, mode of action and origins, thus preventing the full understanding of cloud formation and its potential links with the Earth&#39;s ecosystems.
In this paper we present recently developed methods for 1) the targeted extraction of all the surfactants from atmospheric aerosol samples and for the determination of 2) their absolute concentrations in the aerosol phase and 3) their static surface tension curves in water, including their Critical Micelle Concentration (CMC). These methods have been validated with 9 references surfactants, including anionic, cationic and non-ionic ones. Examples of results are presented for surfactants found in fine aerosol particles (diameter &#60;1 &#956;m) collected at a coastal site in Croatia and suggestions for future improvements and other characterizations than those presented are discussed.

Methods are presented for the targeted extraction of surfactants present in atmospheric aerosols and the determination of their absolute concentrations and surface tension curves in water, including their Critical Micelle Concentration (CMC).

Surface-active compounds, or surfactants, present in atmospheric aerosols are expected to play important roles in the formation of liquid water clouds in ...

Methodology for Developing Life Tables for Sessile Insects in the Field Using the Whitefly, Bemisia tabaci, in Cotton As a Model System

Life tables provide a means of measuring the schedules of birth and death from populations over time. They also can be used to quantify the sources and rates of mortality in populations, which has a variety of applications in ecology, including agricultural ecosystems. Horizontal, or cohort-based, life tables provide for the most direct and accurate method of quantifying vital population rates because they follow a group of individuals in a population from birth to death. Here, protocols are presented for conducting and analyzing cohort-based life tables in the field that takes advantage of the sessile nature of the immature life stages of a global insect pest, Bemisia tabaci. Individual insects are located on the underside of cotton leaves and are marked by drawing a small circle around the insect with a non-toxic pen. This insect can then be observed repeatedly over time with the aid of hand lenses to measure development from one stage to the next and to identify stage-specific causes of death associated with natural and introduced mortality forces. Analyses explain how to correctly measure multiple mortality forces that act contemporaneously within each stage and how to use such data to provide meaningful population dynamic metrics. The method does not directly account for adult survival and reproduction, which limits inference to dynamics of immature stages. An example is presented that focused on measuring the impact of bottom-up (plant quality) and top-down (natural enemies) effects on the mortality dynamics of B. tabaci in the cotton system.

Methodology for Developing Life Tables for Sessile Insects in the Field Using the Whitefly, Bemisia tabaci, in Cotton As a Model System

Life tables allow quantification of the sources and rates of mortality in insect populations and contribute to understanding, predicting and manipulating population dynamics in agroecosystems. Methods for conducting and analyzing cohort-based life tables in the field for an insect with sessile immature life stages are presented.

Life tables provide a means of measuring the schedules of birth and death from populations over time. They also can be used to quantify the sources and ...

Artificial Host Parasitization by Trichogramma dendrolimi and Venom Extraction

Extracting Venom from the Parasitoid Wasp Trichogramma dendrolimi Using an Artificial Host

Parasitoid wasps are a diverse group of hymenopteran insects that serve as invaluable resources for pest biocontrol. To ensure successful parasitism, parasitoid wasps inject venom into their hosts to suppress their hosts&#39; immunity, modulate hosts&#39; development, metabolism, and even behavior. With over 600,000 estimated species, the diversity of parasitoid wasps surpasses that of other venomous animals, such as snakes, cone snails, and spiders. Parasitoid wasp venom is an underexplored source of bioactive molecules with potential applications in pest control and medicine. However, collecting parasitoid venom is challenging due to the inability to use direct or electrical stimulation and the difficulty in dissection because of their small size. Trichogramma is a genus of tiny (~0.5 mm) egg parasitoid wasps that are widely used for the biological control of lepidopteran pests in both agriculture and forests. Here, we report a method for extracting venom from T. dendrolimi using artificial hosts. These artificial hosts are created with polyethylene film and amino acid solutions and then inoculated with Trichogramma wasps for parasitism. The venom was subsequently collected and concentrated. This method enables the extraction of large amounts of Trichogramma venom while avoiding contamination from other tissues caused by dissection, a common issue in venom reservoir dissection protocols. This innovative approach facilitates the study of Trichogramma venom, paving the way for new research and potential applications.

Author Spotlight: Efficient Venom Extraction Method from Trichogramma Parasitoid Wasps 

Here, we present a protocol for extracting venom from Trichogramma dendrolimi using an artificial host created with polyethylene film and amino acid solution.

Parasitoid wasps are a diverse group of hymenopteran insects that serve as invaluable resources for pest biocontrol. To ensure successful parasitism, ...

Biochemistry

Microdissection of Black Widow Spider Silk-producing Glands

Modern spiders spin high-performance silk fibers with a broad range of biological functions, including locomotion, prey capture and protection of developing offspring 1,2. Spiders accomplish these tasks by spinning several distinct fiber types that have diverse mechanical properties. Such specialization of fiber types has occurred through the evolution of different silk-producing glands, which function as small biofactories. These biofactories manufacture and store large quantities of silk proteins for fiber production. Through a complex series of biochemical events, these silk proteins are converted from a liquid into a solid material upon extrusion.
Mechanical studies have demonstrated that spider silks are stronger than high-tensile steel 3. Analyses to understand the relationship between the structure and function of spider silk threads have revealed that spider silk consists largely of proteins, or fibroins, that have block repeats within their protein sequences 4. Common molecular signatures that contribute to the incredible tensile strength and extensibility of spider silks are being unraveled through the analyses of translated silk cDNAs. Given the extraordinary material properties of spider silks, research labs across the globe are racing to understand and mimic the spinning process to produce synthetic silk fibers for commercial, military and industrial applications. One of the main challenges to spinning artificial spider silk in the research lab involves a complete understanding of the biochemical processes that occur during extrusion of the fibers from the silk-producing glands.
Here we present a method for the isolation of the seven different silk-producing glands from the cobweaving black widow spider, which includes the major and minor ampullate glands [manufactures dragline and scaffolding silk] 5,6, tubuliform [synthesizes egg case silk] 7,8, flagelliform [unknown function in cob-weavers], aggregate [makes glue silk], aciniform [synthesizes prey wrapping and egg case threads] 9 and pyriform [produces attachment disc silk] 10. This approach is based upon anesthetizing the spider with carbon dioxide gas, subsequent separation of the cephalothorax from the abdomen, and microdissection of the abdomen to obtain the silk-producing glands. Following the separation of the different silk-producing glands, these tissues can be used to retrieve different macromolecules for distinct biochemical analyses, including quantitative real-time PCR, northern- and western blotting, mass spectrometry (MS or MS/MS) analyses to identify new silk protein sequences, search for proteins that participate in the silk assembly pathway, or use the intact tissue for cell culture or histological experiments.

Here we describe an efficient strategy to remove the silk-producing glands from the abdomen of female black widow spiders. This procedure allows the rapid isolation of the seven distinct silk-producing glands in a highly purified fashion, an important process for investigators studying spider silk production and fiber assembly.

Modern spiders spin high-performance silk fibers with a broad range of biological functions, including locomotion, prey capture and protection of ...

Biology

Extraction of Venom and Venom Gland Microdissections from Spiders for Proteomic and Transcriptomic Analyses

Venoms are chemically complex secretions typically comprising numerous proteins and peptides with varied physiological activities. Functional characterization of venom proteins has important biomedical applications, including the identification of drug leads or probes for cellular receptors. Spiders are the most species rich clade of venomous organisms, but the venoms of only a few species are well-understood, in part due to the difficulty associated with collecting minute quantities of venom from small animals. This paper presents a protocol for the collection of venom from spiders using electrical stimulation, demonstrating the procedure on the Western black widow (Latrodectus hesperus). The collected venom is useful for varied downstream analyses including direct protein identification via mass spectrometry, functional assays, and stimulation of venom gene expression for transcriptomic studies. This technique has the advantage over protocols that isolate venom from whole gland homogenates, which do not separate genuine venom components from cellular proteins that are not secreted as part of the venom. Representative results demonstrate the detection of known venom peptides from the collected sample using mass spectrometry. The venom collection procedure is followed by a protocol for dissecting spider venom glands, with results demonstrating that this leads to the characterization of venom-expressed proteins and peptides at the sequence level.

This article provides a protocol for the extraction of venom from spiders using electrical stimulation in order to 1) conduct proteomic characterization, 2) stimulate venom gland gene expression, and 3) perform functional studies of venoms. This is followed by a description of venom gland microdissections for gene expression studies.

Venoms are chemically complex secretions typically comprising numerous proteins and peptides with varied physiological activities. Functional ...

Rearing of Brazilian Yellow Scorpion for Venom Extraction

Captive Maintenance and Venom Extraction of Tityus serrulatus (Brazilian Yellow Scorpion) for Antivenom Production

Scorpion envenomation is a public health problem in several tropical and subtropical countries. Tityus serrulatus Lutz and Mello, 1922 (Brazilian yellow scorpion) are responsible for approximately 150,000 envenoming cases per year in Brazil, of which 10% require antivenom treatment to reverse life-threatening venom effects. Therefore, thousands of T. serrulatus individuals are maintained under controlled captivity conditions for venom extraction, subsequently used in the production of the national supply of scorpion antivenom. Instituto Butantan is the main antivenom-manufacturing laboratory in Brazil, providing about 70,000 vials of scorpion antivenom for the Brazilian health system. Thus, the husbandry protocols and venom extraction methodologies are key points for the success of large-scale, standardized venom production. The objective of this article is to describe the captivity protocols of T. serrulatus husbandry, encompassing the husbandry routine and the venom extraction procedures, following good manufacturing practices, and ensuring animal welfare. These practices allow for the maintenance of up to 20,000 animals in captivity, with a routine of 3,000 to 5,000 scorpions milked monthly according to antivenom manufacturing demand, achieving an average of 90% of positive extraction.

Author Spotlight: Optimizing Scorpion Venom Extraction for Antivenom Production 

Here, we present successful maintenance and venom extraction protocols for large-scale Tityus serrulatus Lutz and Mello, 1922 (Brazilian yellow scorpion) husbandry, with the aim of providing venom for subsequent scorpion antivenom production to meet the demand of the Brazilian health system.

Scorpion envenomation is a public health problem in several tropical and subtropical countries. Tityus serrulatus Lutz and Mello, 1922 (Brazilian yellow ...

Harvesting Venom Toxins from Assassin Bugs and Other Heteropteran Insects

Summary

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Helminth Collection and Identification from Wildlife

Quantification of Heavy Metals and Other Inorganic Contaminants on the Productivity of Microalgae

Assaying Predatory Feeding Behaviors in Pristionchus and Other Nematodes

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

Extraction and Characterization of Surfactants from Atmospheric Aerosols

Methodology for Developing Life Tables for Sessile Insects in the Field Using the Whitefly, Bemisia tabaci, in Cotton As a Model System

Extracting Venom from the Parasitoid Wasp Trichogramma dendrolimi Using an Artificial Host

Microdissection of Black Widow Spider Silk-producing Glands

Extraction of Venom and Venom Gland Microdissections from Spiders for Proteomic and Transcriptomic Analyses

Captive Maintenance and Venom Extraction of Tityus serrulatus (Brazilian Yellow Scorpion) for Antivenom Production