trichogramma dendrolimi artificial host interaction and venom extraction

Parasitación artificial del huésped por Trichogramma dendrolimi y extracción de veneno

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.

Autor destacado: Método eficiente de extracción de veneno de avispas parasitoides Trichogramma 

Extracción de veneno de la avispa parasitoide Trichogramma dendrolimi utilizando un huésped artificial

The lack of efficient venom extraction methods often limits the research on the venom of parasitoid wasps, especially in tiny parasitoid wasps. This study provides an efficient method to extract the venom of Trichogramma with follow up post study of Trichogramma venom, such as the protein compensation and the venom function analysis. Previously, no methods of exchanging of parasitoid wasp venom requires the distinguishing of venom reserve.However, parasitoid wasps are tiny. Not only are the technical requirements of distinguishing venom was high, but continuation of other tissues during dissection is also common. Our normal method uses artificial host, thus avoiding such problems.In the future, we will focus on the agriculture and the medical applications of Trichogramma venom. We aim to utilize specific insecticidal venom genes for pest control. Meanwhile, we plan to investigate the untapped potential of Trichogramma venom in methodology, particularly its prospective utility in enhancing immunotherapy techniques, and in the discovery of novel antibiotic molecules.

Watch this Scientific Journal Video about Trichogramma dendrolimi  Artificial Host Interaction and Venom Extraction at JoVE.com

Trichogramma dendrolimi  Artificial Host Interaction and Venom Extraction  

Trichogramma dendrolimi Interacción artificial con el huésped y extracción de veneno

Para comenzar, tome una película de plástico de polietileno de 16 centímetros de largo, 12 centímetros de ancho y 20 micrómetros de grosor. Utilice una herramienta de molienda de vidrio para extraer 30 protuberancias semicirculares en un diseño de PCR estándar de 96 pocillos. Exponga ambos lados de la película plástica de polietileno prensada a la luz ultravioleta durante una hora para desinfectarla.Agregue una pequeña cantidad de alcohol polivinílico al 10% a la superficie semicircular. A continuación, coloque alrededor de 3.000 avispas hembras dendrolimi anestesiadas en una caja de recolección. Coloque el lado convexo de la tarjeta de huevo de película hacia la caja de recolección y asegure los bordes con una banda elástica.Ahora agregue cuatro microlitros de solución de aminoácidos a cada protuberancia. Luego cúbralo con otro trozo de película plástica de polietileno. Use una banda elástica para cubrir herméticamente la caja de recolección con dos hojas de plástico.Deje que las avispas parasiten libremente durante cuatro a ocho horas. Proporcione un 10% de agua de sacarosa a través del algodón húmedo durante la parasitación. A continuación, obtenga la solución de aminoácidos parasitados de la protuberancia interna de la tarjeta de huevo artificial y transfiérala a la tapa de tubos de 1,45 mililitros.Cubra la tapa del tubo con una red de nailon de 10 micrómetros, con un diámetro de 25 milímetros. A continuación, fije ligeramente la red de nailon en el tubo. Centrifugar el tubo superior derecho a 1,360 g durante 10 segundos.Recoja la solución filtrada que contiene el veneno. Almacene el veneno a menos 80 grados centígrados para realizar más análisis. El análisis de BCA de las muestras de veneno mostró que la concentración de proteína de veneno osciló entre 0,35 y 0,46 microgramos por microlitro.El control negativo de la solución de aminoácidos solo tuvo una concentración de proteína de 0,03 a 0,05 microgramos por microlitro. El análisis del veneno de SDS-PAGE mostró un rango de proteínas que abarca desde menos de 10 kilodaltons hasta más de 130 kilodaltons de tamaño.

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Research

JoVE Journal

Biochemistry

Trichogramma dendrolimi Artificial Host Interaction 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.

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, ...

Investigación

Bioquímica

To begin, take a polyethylene plastic film 16 centimeters long, 12 centimeters wide, and 20 micrometers thick. Use a glass grinding tool to press out 30 semi-circular protrusions in a standard 96-well PCR layout. Expose both sides of the pressed polyethylene plastic film to UV light for one hour to disinfect it.Add a small amount of 10%polyvinyl alcohol to the semi-circular surface. Next, place about 3, 000 anesthetized female trichogramma dendrolimi wasps into a collection box. Place the convex side of the film egg card towards the collection box and secure the edges with a rubber band.Now add four microliters of amino-acid solution to each protrusion. Then cover it with another piece of polyethylene plastic film. Use a rubber band to tightly cover the collection box with two sheets of plastic.Allow the wasps to parasitize freely for four to eight hours. Provide 10%sucrose water through wet cotton during parasitization. Next, obtain the parasitized amino-acid solution from the inner protrusion of the artificial egg card and transfer it to the cap of 1.45 milliliter tubes.Cover the tube cap with a 10-micrometer nylon net, having a diameter of 25 millimeters. Then fasten the nylon net in the tube lightly. Centrifuge the upper right tube at 1, 360 g for 10 seconds.Collect the filtered solution containing the venom. Store the venom at minus 80 degrees Celsius for further analyses. BCA analysis of the venom samples showed that the concentration of venom protein ranged from 0.35 to 0.46 micrograms per microliter.The negative control of amino-acid solution only had a protein concentration of 0.03 to 0.05 micrograms per microliter. SDS-PAGE analysis of the venom showed a protein range spanning from under 10 kilodaltons to over 130 kilodaltons in size.