Name: Efficient Venom Extraction Method from Trichogramma Parasitoid Wasps (Video) | JoVE
Uploaded: 2023-10-06T13:50:00
Description: Watch this Scientific Journal Video about Efficient Venom Extraction Method from Trichogramma Parasitoid Wasps at JoVE.com

We acknowledge financial support from the Natural Science Foundation of Hainan Province (Grant no. 323QN262), the National Natural Science Foundation of China (Grant no. 31701843 and 32172483), the Jiangsu Agriculture Science and Technology Innovation Fund (Grant No. CX(22)3012 and CX(21)3008), the &#34;Shuangchuang Doctor&#34; Foundation of Jiangsu Province (Grant No. 202030472), and the Nanjing Agricultural University startup fund (Grant No. 804018).

1. Insect rearing
<ol>
	<li>Feed Corcyra cephalonica on corn flour at a temperature of 26 &#177; 1&#176;C and a relative humidity of 40% &#177; 10%.</li>
	<li>Breed T. dendrolimi strain from the Jilin insectary indoors using the eggs of Corcyra cephalonica as hosts. Feed wasp adults 10% sucrose water in Drosophila tubes at a temperature of 26 &#177; 1 &#176;C, relative humidity of 70% &#177; 10%, light (L): dark (D) period of 14 h: 10 h.</li>
</ol>
<p class=...

Artificial Host Parasitization by Trichogramma dendrolimi and Venom Extraction

<ol>
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Here, we present a method for extracting venom from T. dendrolimi using artificial hosts. The key points in the venom collection experiment are as follows. (1) During preparation, T. dendrolimi must be anesthetized rapidly with an appropriate concentration of CO2. If the CO2 concentration is too low, it will be insufficient to anesthetize the Trichogramma quickly. Conversely, if the concentration is too high, Trichogramma may die, reducing their ability to parasit...

Parasitoid wasps are parasitic hymenopteran insects that are important resources for biological control1. There is a wide variety of parasitoid wasps, with over 600,000 estimated species2. The diversity of parasitoid wasps far exceeds that of other venomous arthropods, such as snakes, cone snails, spiders, scorpions, and bees. Venom is an important parasitic factor in parasitoid wasps. For successful parasitism, venom is injected into the host, modulating the host&#39;s behavior, immunity, development, and metabolism3. Moreover, the venom of parasitoid wasps displays remarkable diversity in its molecular structures, targets, and functions, reflecting complex coevolution with their hosts. Thus, parasitoid venom is a valuable and underappreciated resource of active molecules for insecticidal or medical purposes4. Unlike the venom of snakes, cone snails, spiders, scorpions, and bees, parasitoid wasp venom cannot be collected by direct stimulation or electrical stimulation5. The current method of extraction of parasitoid wasp venom is to dissect the venom reservoir. However, parasitoid wasps are often small, and dissection of parasitoid wasps requires high technical skills. Therefore, if we can find a way to collect the venom of parasitoid wasps efficiently and conveniently, it will be of great help to research the venom of parasitoid wasps.
Trichogramma (Hymenoptera: Trichogrammatidae) is a genus of tiny (~0.5 mm long) parasitoid wasps6. These wasps are among the most widely used biocontrol agents, particularly targeting eggs of various lepidopteran pests in both agriculture and forests. For example, T. dendrolimi, one of the most widely used Trichogramma species in China, has been extensively applied to control a variety of agricultural and forestry pests, such as Dendrolimus superans, Ostrinia furnacalis, and Chilo suppressalis. Previous studies showed that Trichogramma wasps could inject their eggs into artificial hosts7. Artificial hosts can be created using materials such as wax8, agar9, Parafilm10, and plastic film11. The solution in artificial hosts that induces sufficient oviposition for Trichogramma can be simple, such as amino acids or inorganic salts12. Based on the characteristic that T. dendrolimi can parasitize artificial hosts, this study provides a new method for extracting venom from parasitoid wasps using artificial hosts. This approach aims to address the shortcomings of low yield, low purity, and susceptibility to contamination in current extraction techniques. By using this method, a large amount of high-purity venom from T. dendrolimi can be extracted, which meets the needs of scientific research and screening of bioactive molecules for insecticidal or medical purposes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 &#956;m Nylon Net</td>
			<td>Millipore</td>
			<td>NY1002500</td>
			<td>For filtering the eggs</td>
		</tr>
		<tr>
			<td>10% Polyvinyl alcohol</td>
			<td>Aladdin</td>
			<td>P139533</td>
			<td>For attractting&#160; T. dendrolimi&#160; to lay eggs</td>
		</tr>
		<tr>
			<td>10% Sucrose water</td>
			<td>Sinopharm Chemical Reagent&#160;</td>
			<td>10021463</td>
			<td>Feed Trichogramma dendrolimi</td>
		</tr>
		<tr>
			<td>4x LDS loading buffer</td>
			<td>Ace Hardware</td>
			<td>B23010301</td>
			<td>SDS-PAGE</td>
		</tr>
		<tr>
			<td>Collection box</td>
			<td>Deli</td>
			<td>8555</td>
			<td>Container for T. dendrolimi parasitism</td>
		</tr>
		<tr>
			<td>Future PAGE&#160; 4&#8211;12% (12 wells)</td>
			<td>Ace Hardware</td>
			<td>J70236502X</td>
			<td>SDS-PAGE</td>
		</tr>
		<tr>
			<td>GenScript eStain L1 protein staining apparatus</td>
			<td>GenScript</td>
			<td>L00753</td>
			<td>SDS-PAGE</td>
		</tr>
		<tr>
			<td>Glass grinding rod&#160;&#160;</td>
			<td>Applygen</td>
			<td>tb6268</td>
			<td>Semicircular protrudations&#160;</td>
		</tr>
		<tr>
			<td>L- Leucine</td>
			<td>Solarbio</td>
			<td>L0011</td>
			<td>Artificial host components</td>
		</tr>
		<tr>
			<td>L-Histidine</td>
			<td>Aladdin</td>
			<td>A2219458</td>
			<td>Artificial host components</td>
		</tr>
		<tr>
			<td>L-Phenylalanine</td>
			<td>Solarbio</td>
			<td>P0010</td>
			<td>Artificial host components</td>
		</tr>
		<tr>
			<td>Mini-Centrifuges</td>
			<td>Scilogex</td>
			<td>D1008</td>
			<td>Centrifuge</td>
		</tr>
		<tr>
			<td>MOPS-SDS running buffer</td>
			<td>Ace Hardware</td>
			<td>B23021</td>
			<td>SDS-PAGE</td>
		</tr>
		<tr>
			<td>Omni-Easy Instant BCA protein assay kit</td>
			<td>Shanghai Yamay Biomedical Technology&#160;</td>
			<td>ZJ102</td>
			<td>For esimation of venom protein concentration</td>
		</tr>
		<tr>
			<td>PCR plate layout of 96 holes</td>
			<td>Thermo Fisher</td>
			<td>AB1400L</td>
			<td>Semicircular protrudations&#160;</td>
		</tr>
		<tr>
			<td>Polyethylene plastic film</td>
			<td>Suzhou Aopang Trading &#160;</td>
			<td>001c5427</td>
			<td>Artificial egg card</td>
		</tr>
		<tr>
			<td>Prestained color protein marker(10&#8211;180 kDa)</td>
			<td>YiFeiXue Biotech</td>
			<td>YWB007</td>
			<td>SDS-PAGE</td>
		</tr>
		<tr>
			<td>Rubber band</td>
			<td>Guangzhou qianrui biology science and technology</td>
			<td>009</td>
			<td>Tighten the plastic film and the collection box</td>
		</tr>
		<tr>
			<td>Silicone rubber septa mat, 96-well, round hole</td>
			<td>Sangon Biotech</td>
			<td>F504416-0001</td>
			<td>Semicircular protrudations&#160;</td>
		</tr>
	</tbody>
</table>

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.

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

The protein concentration of representative venom samples was measured using the protein assay kit, with the results presented in Table 1. The results showed that the concentration of venom protein collected by this method ranged from 0.35 &#181;g/&#181;L to 0.46 &#181;g/&#181;L, while the negative control of amino acid solution only had a protein concentration of 0.03 &#181;g/&#181;L to 0.05 &#181;g/&#181;L. The concentration of venom protein collected by this method is much higher than that of negative...

Watch this Scientific Journal Video about Efficient Venom Extraction Method from Trichogramma Parasitoid Wasps at JoVE.com

Efficient Venom Extraction Method from Trichogramma Parasitoid Wasps (Video) | JoVE

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, ...