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>
	<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. 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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, ...

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.

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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

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

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

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

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

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) ...