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

Summary

Here, we present a protocol for extracting venom from Trichogramma dendrolimi using an artificial host created with polyethylene film and amino acid solution.

Abstract

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' immunity, modulate hosts' 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.

Introduction

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'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.

Protocol

1. Insect rearing

1. Feed Corcyra cephalonica on corn flour at a temperature of 26 ± 1°C and a relative humidity of 40% ± 10%.
2. 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 ± 1 °C, relative humidity of 70% ± 10%, light (L): dark (D) period of 14 h: 10 h.

2. Preparation of polyethylene plastic film egg cards

1. Take a polyethylene plastic film with a length of 16 cm, a width of 12 cm, and a thickness of 20 µm. Press out 30 semicircular protrusions with a diameter of 2-3 mm and a height of approximately 3 mm using a glass grinding rod according to the standard PCR plate layout of 96 holes.
   NOTE: The process of pressing out 30 semicircular protrusions using a glass grinding rod needs to be done paying attention to the pressure because too hard a press will puncture the plastic and contaminate the extracted venomless grinding rod.
2. Disinfect the pressed polyethylene plastic film by exposing both sides to ultraviolet (UV) light for 1 h.
3. Add a small amount of 10% polyvinyl alcohol to the semicircular surface.

3. Trichogramma dendrolimi parasitism

1. After CO₂ anesthesia, place T. dendrolimi female wasps into a collection box, and the number of wasps was ~3000.
2. Place the convex side of the film egg card toward the collection box and secure the edges with a rubber band.
3. Add 4 µL of amino acid solution (6 g/L leucine, 4 g/L phenylalanine, 4.25 g/L histidine) to each semicircular protrusion. Cover it with a flat polyethylene plastic film 16 cm long and 12 cm wide. Use a rubber band to tightly cover the collection box with two sheets of plastic.
4. Let T. dendrolimi wasps parasitize freely for 4-8 h and provide 10% sucrose water through wetted cotton.

4. Collecting T. dendrolimi venom

1. Obtain the parasitized amino acid solution from the inner protrusion of the artificial egg card and transfer it to the cap of 1.5 mL tubes.
2. Cover the tube cap with a 10 µm nylon net with a 25 mm diameter, fasten the nylon net and centrifuge tube tightly. Place the centrifuge tube upright for short centrifugation using a mini-centrifuge (1360 x g) for 10 s and collect the filtered solution (~100 µL of T. dendrolimi venom).
3. Measure the concentration of collected T. dendrolimi venom using a Bicinchoninic acid (BCA) assay kit (Table of Materials).
4. Store the venom at -80 °C for further analyses.

5. SDS-PAGE analyses

1. Add 30 µL of T. dendrolimi venom to 10 µL of 4x sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer (Table of Materials) and heat at 95 °C for 10 min.
2. Perform SDS-PAGE gel run at 130 V for 120 min.
3. Stain and decolorize the SDS-PAGE gel using the protein staining apparatus (Table of Materials).

Results

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 µg/µL to 0.46 µg/µL, while the negative control of amino acid solution only had a protein concentration of 0.03 µg/µL to 0.05 µg/µL. The concentration of venom protein collected by this method is much higher than that of negative...

Discussion

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...

Materials

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