Name: harvesting venom toxins from assassin bugs and other heteropteran insects
Uploaded: 2018-04-21T13:00:00
Description: Watch this Scientific Journal Video about Harvesting Venom Toxins from Assassin Bugs and Other Heteropteran Insects at JoVE.com

Vi anerkender finansiel støtte fra det australske Forskningsråd (tilskud DP130103813 og LP140100832 til G.F.K., DECRA Fellowship DE160101142 til EABU), den australske National Health & Medical Research Council (Principal Research Fellowship APP1044414 til G.F.K.), og University of Queensland (postdoc stipendium til A.A.W.).

Denne protokol i overensstemmelse med The University of Queenslands politik i ansvarlig pleje og brug af dyr i undervisning og forskning (PPL 4.20.11) samt National sundhed og Medical Research Council's australske kode for pasning og brug af dyr til videnskabelige formål (8th Edition 2013).
Forsigtig: Sørg for ikke for at være røgter ved håndtering af assassin bugs. Sørge for at beskytte øjnene ved håndtering af arter, der spytter venom defensivt. Tage sig ...

<ol>
	<li>Walker, A. A., Weirauch, C., Fry, B. G., King, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Venoms+of+heteropteran+insects:+A+treasure+trove+of+diverse+pharmacological+toolkits.">Venoms of heteropteran insects: A treasure trove of diverse pharmacological toolkits.</a> Toxins. 8 (2), 43 (2016).</li><li>Ribeiro, J. M. C., Assump&#231;&#227;o, T. C., Francischetti, I. M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+insight+into+the+sialomes+of+bloodsucking+Heteroptera.">An insight into the sialomes of bloodsucking Heteroptera.</a> Psyche (Stuttg). 2012, 1-16 (2012).</li><li>Ambrose, D. P., Maran, S. P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+protein+content+and+paralytic+potential+of+saliva+of+fed+and+prey+deprived+reduviid+Acanthaspis+pedestris+St&#229;l+(Heteroptera:+Reduviidae:+Reduviinae).">Quantification protein content and paralytic potential of saliva of fed and prey deprived reduviid Acanthaspis pedestris St&#229;l (Heteroptera: Reduviidae: Reduviinae).</a> Indian Journal of Environmental Science. 3 (1), 11-16 (1999).</li><li>Edwards, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+action+and+compostion+of+the+saliva+of+an+assassin+bug+Platymeris+rhadamanthus+Gaerst.+(Hemiptera,+Reduviidae).">The action and compostion of the saliva of an assassin bug Platymeris rhadamanthus Gaerst. (Hemiptera, Reduviidae).</a> Journal of Experimental Biology. 38, 61-77 (1961).</li><li>Zerachia, T., Bergmann, F., Shulov, A., Kaiser, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Animal and Plant Toxins. , 143-146 (1973).</li><li>Walker, A. A., Hern&#225;ndez-Vargas, M. J., Corzo, G., Fry, B. G., King, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Giant+fish-killing+water+bug+reveals+ancient+and+dynamic+venom+evolution+in+Heteroptera.">Giant fish-killing water bug reveals ancient and dynamic venom evolution in Heteroptera.</a> Cellular and Molecular Life Sciences. , (2018).</li><li>Walker, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Giant+fish-killing+water+bug+reveals+ancient+and+dynamic+venom+evolution+in+Heteroptera.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+assassin+bug+Pristhesancus+plagipennis+produces+two+distinct+venoms+in+separate+gland+lumens.">The assassin bug Pristhesancus plagipennis produces two distinct venoms in separate gland lumens.</a> Nature Communications. 9 (1), 755 (2018).</li><li>Hern&#225;ndez-Vargas, M. J., Santib&#225;&#241;ez-L&#243;pez, C. E., Corzo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+insight+into+the+triabin+protein+family+of+American+hematophagous+reduviids:+Functional,+structural+and+phylogenetic+analysis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Action+of+the+saliva+of+Triatoma+infestans+(Heteroptera:+Reduviidae)+on+sodium+channels.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+peptides+from+assassin+bugs+(Hemiptera:+Reduviidae):+isolation,+chemical+and+biological+characterization.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+milking+and+electric+shocks+for+venom+collection+from+hunter+reduviids.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paralytic+activity+of+lysophosphatidylcholine+from+saliva+of+the+waterbug+Belostoma+anurum.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Triabin,+a+highly+potent+exosite+inhibitor+of+Thrombin.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+inhibitor+of+collagen-induced+platelet+aggregation+from+the+saliva+of+Triatoma+pallidipennis.">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.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibacterial+activity+of+Rhynocoris+marginatus+(Fab.)+and+Catamirus+brevipennis+(Servile)+(Hemiptera:+Reduviidae)+venoms+against+human+pathogens.">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://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+morphology+of+the+salivary+system+in+some+reduviids+(Insecta-Heteroptera-Reduviidae).">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>Maran, S. P. M., Ambrose, D. P., Ignacimuth, A., Sen, A., Janarthanan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biotechnological Applications for Integrated Pest Management. , 125-131 (2000).</li><li>Maran, S. P. M., Selvamuthu, K., Rajan, K., Kiruba, D. A., Ambrose, D. P., Ambrose, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Insect Pest Management, A Current Scenario. , 346-361 (2011).</li><li>Pereira, M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anticoagulant+activity+of+Triatoma+infestans+and+Panstrongylus+megistus+saliva+(Hemiptera/Triatominae).">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+salivary+vasodilator+in+the+blood-sucking+bug,+Rhodnius+prolixus.">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., Guimar&#227;es, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+characterization+of+prolixin-S+(nitrophorin+2),+the+salivary+anticoagulant+of+the+blood-sucking+bug+Rhodnius+prolixus.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+salivary+gland+and+salivary+enzymes+of+the+giant+waterbugs+(Heteroptera;+Belostomatidae).">The salivary gland and salivary enzymes of the giant waterbugs (Heteroptera; Belostomatidae).</a> Comparative Biochemistry and Physiology A Molecular &amp; Integrative Physiology. 145 (1), 114-122 (2006).</li><li>Rasmussen, S., Young, B., Krimm, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+'spitting'+behaviour+in+cobras+(Serpentes:+Elapidae).">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="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Venom+spitting+by+the+green+lynx+spider,+Peucetia+viridans+(Araneae,+Oxyopidae).">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ontogenesis,+gender,+molting+influence+the+venom+yield+in+the+spider+Coremiocnemis+tropix+(Araneae,+Theraphosidae).">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+electrophoretic+analyses+of+saliva+from+the+predatory+reduviid+species+Rhynocoris+marginatus+(Fab).">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>

Den mest afgørende skridt i høst assassin bug venom er at vælge den passende metode afhængig af i forbindelse med undersøgelsen. Hver af de tre metoder præsenteres for høst heteropteran venoms har fordele og ulemper afhængigt af downstream applikationer.
Inducerende bugs at udvise venom fra Snabel (protokoller 1-3) undgår forurening af venom ved glandulær væv. Derudover disse metoder er ikke-dødelige og kan gentages mange gange i løbet af en bug's liv. Elektrostimulation normalt g...

Heteropteran venoms er potent bioaktive stoffer1. For eksempel, letter venom/spyt sekreter af blod-fodring tæger som kysse bugs (Triatominae) og væggelus (Cimicidae), fodring af forstyrre hæmostase2. Toksiner i disse venoms målrette flere veje herunder koagulation, trombocytaggregation og vasokonstriktion, samt smerter og kløe veje. Venoms fra de fleste andre heteropteran arter er tilpasset til at lette prædation snarere end blod-fodring. Deres venoms forårsage lammelse, død og væv flydendegørelse når injiceres hvirvelløse dyr3,4. Når injiceres i hvirveldyr, kan deres gift også har drastiske virkninger. For eksempel, forårsager injektion af venom fra assassin bug Holotrichius indre i hvirveldyr smerte, muskellammelse og blødning; mus røgter ved denne bug dør hurtigt på grund af respiratorisk lammelse5.
Transkriptom og proteom undersøgelser har afsløret nogle heteropteran venoms protein sammensætning. Venoms predaceous arter er rig på proteaser, andre enzymer, og peptider og proteiner af ukendt struktur og funktion6,7,8. Tægen venom er rig på triabin protein familie, hvis medlemmer dybt påvirker koagulation, trombocytaggregation og vasokonstriktion2,9. Det vides dog ikke, hvilke toksiner ligge til grund for de fleste bioactivities af venom. For eksempel, venom af tægen Triatoma infestans er blevet rapporteret at være smertestillende og hæmmer natrium kanaler10, men de ansvarlige komponenter mangler at blive belyst. Ligeledes, det er ikke kendt hvad komponent(er) af assassin bug venom forårsage lammelse eller smerte. En forudsætning for at identificere de ansvarlige for særlige venom bioactivities samt kendetegner struktur og funktion af roman venom toksiner, giftstoffer er at få gift.
Venom er opnået fra heteropterans af elektrostimulation5,6,7,8,11,12,13, provokation af defensiv svar4,8, mekanisk klemme i thorax12,14,15,16, dissekere ud gift kirtler8,17 ,18,19,20,21,22, og anvendelsen af agonister muskarine acetylcholin receptor23. At dømme de potentielle fordele og ulemper ved enhver metode, der er kompliceret af morfologi af heteropteran gift kirtler, som består af en vigtigste kirtel med to separate lumen, forreste vigtigste kirtel (AMG) og posterior vigtigste kirtel (PMG), samt en tilhørende tilbehør kirtel (AG). Disse forskellige kirtel rum producere forskellige protein sekreter, som kan være specialiserede for forskellige biologiske funktioner herunder bytte capture, forsvar og ekstra mundtlig fordøjelsen8,17. I peiratine og ectrichodiine assassin bugs, har AMG været forbundet med bytte opsamling og PMG med ekstra mundtlig fordøjelsen17. Men i harpactorine bug Pristhesancus plagipennis PMG er specialiseret til bytte opsamling og fordøjelsen mens AMG er hypotese for at udskille defensive venom8. AG er blevet beskrevet som havende lille sekretoriske funktion i assassin bugs8 eller som en større site af protease opbevaring i gigantiske vand bugs23. Klart, yderligere arbejde er nødvendigt at klarlægge funktionen af hver kirtel rum blandt forskellige heteropteran undergrupper og bestemme funktionen af de fleste venom toksiner. I denne rapport beskriver vi protokoller for høst venom toksiner fra heteropterans mod dette mål.

<table border="0" cellpadding="0" cellspacing="0" width="912">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Electostimulator</td>
			<td style="width:203px;">Grass Technologies</td>
			<td style="width:187px;">S48 Square Pulse Stimulator</td>
			<td style="width:307px;">Electrostimulator allowing pulsed electrostimulation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Featherlight tweezers</td>
			<td style="width:203px;">Australian Entomological Supplies</td>
			<td style="width:187px;">E122B</td>
			<td>For handling live venomous insects</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Protease inhibitor cocktail</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:187px;">4693124001</td>
			<td>For preventing autoproteolytic digestion of venom</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dissection equipment</td>
			<td style="width:203px;">Australian Entomological Supplies</td>
			<td style="width:187px;">E152Micro</td>
			<td>For fine dissections</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Insect pins</td>
			<td style="width:203px;">Australian Entomological Supplies</td>
			<td style="width:187px;">E162</td>
			<td>For fine dissections</td>
		</tr>
	</tbody>
</table>

harvesting venom toxins from assassin bugs and other heteropteran insects

Heteropteran insekter som assassin bugs (Reduviidae) og gigantiske vand bugs (Belostomatidae) nedstammer fra en fælles predaceous og giftige forfader, og størstedelen af bevarede heteropterans bevarer denne trofiske strategi. Nogle heteropterans har skiftet til fodring på hvirveldyr blod (såsom kysse bugs, Triatominae; og væggelus, Cimicidae) mens andre have vendt tilbage til fodring på planter (de fleste Pentatomomorpha). Dog er med undtagelse af spyt bruges af kysse bugs til at lette blod-fodring, lidt kendt om heteropteran venoms sammenlignet med venoms edderkopper, skorpioner og slanger.
En hindring for karakterisering af heteropteran venom toksiner er struktur og funktion af venom/labial kirtler, som er både morfologisk komplekse og udføre flere biologiske roller (forsvar, bytte opsamling og ekstra mundtlig fordøjelsen). I denne artikel vil beskrive vi tre metoder vi har med succes brugt til at indsamle heteropteran venoms. Først, vi præsenterer elektrostimulation som en praktisk måde at indsamle venom, der er ofte dødelig, når injiceres bytte dyr, og som overflødiggør kontaminering af kirtelvæv. For det andet viser vi, at blide chikane af dyr er tilstrækkeligt til at producere venom ekstrudering fra Snabel og/eller venom spytte i nogle grupper af heteropterans. For det tredje, vi beskriver metoder til at høste venom toksiner ved dissektion af bedøvet dyr at få gift kirtler. Denne metode er et supplement til andre metoder, som det kan tillade høstning af toksiner fra taxa hvor elektrostimulation og chikane er ineffektive. Disse protokoller vil sætte forskerne til at høste toksiner fra heteropteran insekter til struktur-funktion karakterisering og mulige anvendelser inden for medicin og landbrug.

Nogle heteropteran arter, som harpactorine P. plagipennis og reduviine Platymeris rhadamanthus, give pålideligt store mængder (5-20 µL) af venom svar til elektrostimulation (tabel 1). Generelt er udbytte de fleste peiratine, reduviine og harpactorine bugs venom som svar på denne metode. Blandt stenopodaine bugs fremkaldte elektrostimulation venom fra Oncocephalus sp., men ikke Thodelmus sp. Holoptiline og emesine bugs stikprøven ik...

Watch this Scientific Journal Video about Harvesting Venom Toxins from Assassin Bugs and Other Heteropteran Insects at JoVE.com

Harvesting Venom Toxins from Assassin Bugs and Other Heteropteran Insects

Selv om mange insekter i underordenen tæger (Insecta: Hemiptera) er giftige, deres venom sammensætning og funktioner af deres venom toksiner er for det meste ukendte. Denne protokol beskriver metoder til at høste heteropteran venoms for yderligere karakterisering, ved hjælp af elektrostimulation, chikane og kirtel dissektion.

Høst Venom toksiner fra Assassin Bugs og andre Heteropteran insekter

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

Assassin bugs are a group of insects that use venom to paralyze and pre-digest their prey. However, despite animal venoms being of interest as novel medicines, insecticides, and scientific tools, the toxins that they produce are almost entirely uncharacterized. One reason for this is that assassin bugs are less well-known in comparison to iconic venomous arthropods such as spiders and scorpions.Another reason is that until recently, little information has been available about how to harvest their toxins. In this video, we will present methods that will enable researchers to successfully harvest venom toxins from assassin bugs, and also related insects such as giant fish-killing water bugs. Protocol one describes how to harvest venom by electrostimulation.First, collect suitable insects. Adults and large nymphs are the easiest to work with. Using bugs two to seven days after feeding is ideal, to ensure the insects have accumulated venom stocks but do not starve.Prepare a pair of forceps with electrodes at the tips, and connect them to an electricity supply. Ideally, this supply should be an electrostimulator capable of delivering five millisecond pulses at five Hertz. Use peak voltages between 15 and 25 volts for small or large bugs, respectively.If an electrostimulator is not available, use constant voltages of five to 12 volts. Restrain the insect using rubber bands and a foam platform. Place the proboscis of the insect into a collecting tube.A P200 pipette tube is ideal for most assassin bugs. Optionally, a small amount of water can be added to the tip before harvesting, to ensure maximum recovery of venom. Apply conductive gel to the electrodes, switch on the power supply, and apply the electrified tweezers to the insect.Experiment with a range of contact points while monitoring venom expulsion from the proboscis. Store the venom at freezing temperatures to avoid autoproteolytic digestion. Repeat the electrostimulation with a new pipette tube until no more venom is forthcoming.Protocol two describes how to harvest venom by provoking a defensive reaction. Prepare and restrain a bug as described above, inserting the proboscis into a collecting tip. Gently harass the bug to provoke a defensive reaction, without injuring the animal.Sometimes, simply restraining the animal or inserting the proboscis into the collection tube can provoke a defensive reaction accompanied by venom expulsion from the proboscis. If not, gently touch the animal on the thorax, legs, abdomen, and especially the antennae, to elicit venom. In protocol three, in the written form of this article, we also describe how to harvest venom from species that spit venom defensively.Protocol four describes how to harvest venom directly from the venom glands by dissection. Anesthetize animals by exposure to carbon dioxide for 10 minutes. Insert three pins into the posterior abdomen to hold the insect down without puncturing the venom glands.Cut a short midline incision in the ventral surface of the abdomen using a miniature scalpel. Use miniature scissors to extend the midline in the anterior direction to the head, taking care to cut the exoskeleton only, and not damage internal structures. To expose the internal structures, make multiple lateral cuts extending from the midline incision to the side of the insect.The ventral exoskeleton between each two cuts can then be pinned back to reveal the internal structures. Flood the dissection tray using a saline solution until the bug is submerged, allowing the internal structures to float up and be more easily visualized. The venom glands will appear as translucent elongated structures extending on each side of the alimentary canal.Using tweezers and microscissors, carefully remove connective and nervous tissue and trachea, trying not to rupture the alimentary canal or the venom glands. The main gland is recognizable due to its characteristic structure, with anterior and posterior lobes, and two ducts meeting at the hilus. For assassin bugs, and perhaps other heteropterans, almost all toxins are stored in the lumens of the two lobes of the main gland.Therefore, after freeing the main gland from trachea and connective tissue, the two ducts leading from the hilus can be cut, and the main venom gland collected. If the accessory gland is also desired, it is usually closely opposed to the gut, and can be identified unambiguously by tracing the ducts from the hilus. In any case, take care not to break the glands prematurely and lose the venom into the surrounding saline.If the lobes of the main gland are to be separated, do this quickly after removal from the saline, and immediately proceed to harvest the contents of the gland lumens, as described in steps 4.6 and 4.7 of the written manuscript. Results will vary depending on the exact subprotocol and species of insect used. The first and second subprotocols presented in this video showed how to harvest venom toxins by electrostimulation and harassment, respectively.Both of these methods typically yield a concentrated liquid containing approximately 50 to 250 milligrams a mil of protein, and over 100 protein-based components, including enzymes, pore-forming toxins, and peptides. The principal advantage of both of these protocols is that they are quick, non-lethal, and venom is uncontaminated by glandular tissue. The greatest disadvantage for harvesting by electrostimulation or harassment is that the glandular source of the venom obtained is unknown, and the protein content of venom harvested may differ, depending on the protocol.Also, electrostimulation and harassment are not effective for all predatory species of heteroptera. In the last subprotocol, we showed how to dissect the engorged venom glands to harvest toxins directly. This technique also yields a cocktail of diverse proteins, although in this case the venom toxins may show some contamination with glandular tissue.However, an advantage of harvesting toxins from dissected glands is that the glandular compartment of origin is known exactly. In conclusion, we encourage researchers to select a harvesting method appropriate to their research question. For example, investigations into the biology of venom use within a species will benefit from employing multiple harvesting techniques.For bioprospecting studies, a single method for harvesting venoms from multiple species may be more economical, depending on the specific experimental design. We hope this video and the accompanying written article will facilitate researchers to harvest venom from assassin bugs and other heteropteran insects, which is a prerequisite to understanding its composition, bioactivity, and evolution.

Research

JoVE Journal

Environment

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), ...

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

Kvantificering af tungmetaller og andre uorganiske forureninger på produktivitet mikroalger

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

Assaying Predatory Feeding Behaviors in Pristionchus and Other Nematodes

Undersøgelse Aggressiv Fodring Adfærd i<em&gt; Pristionchus</em&gt; Og andre Nematoder

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

Extraction and Characterization of Surfactants from Atmospheric Aerosols

Udvinding og karakterisering af overfladeaktive fra atmosfæriske aerosoler

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

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

Metode til udvikling af liv tabeller for siddende insekter i den felt ved hjælp af Whitefly, Bemisia tabaci, i bomuld som et modelsystem

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 ...

Composition and Properties of Aquafaba: Water Recovered from Commercially Canned Chickpeas

Sammensætning og egenskaber for Aquafaba: vand inddrives fra kommercielt dåse kikærter

Chickpea and other pulses are commonly sold as canned products packed in a thick solution or a brine. This solution has recently been shown to produce ...

Double-stranded RNA Oral Delivery Methods to Induce RNA Interference in Phloem and Plant-sap-feeding Hemipteran Insects

Dobbelt-strenget RNA Oral leveringsmetoder til at fremkalde RNA-interferens i Phloem og plante-sap-fodring Hemipteran insekter

Phloem and plant sap feeding insects invade the integrity of crops and fruits to retrieve nutrients, in the process damaging food crops. Hemipteran ...

Laboratory and Field Protocol for Estimating Sheet Erosion Rates from Dendrogeomorphology

Laboratorium og felt protokol til estimering ark Erosion priser fra Dendrogeomorphology

Sheet erosion is among the crucial drivers of soil degradation. Erosion is controlled by environmental factors and human activities, which often lead to ...

Chemical Isolation, Quantification, and Separation of Skin Lipids from Reptiles

Kemiske Isolation, kvantificering og adskillelse af huden lipider fra krybdyr

Reptiles signal to conspecifics using lipids in their skin, primarily to enable mate tracking and assessment. The isolation of these lipids has utility in ...

Cortisol Extraction from Sturgeon Fin and Jawbone Matrices

Cortisol ekstraktion fra Sturgeon fin og Kæbeknogle matricer

The aims of this study were to develop a technique for the extraction of cortisol from sturgeon fins using two washing solvents (water and isopropanol) ...