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

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...

<ol>
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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 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 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...

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

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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