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 mo....

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....

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 control, which shows that this method c.......

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

List of materials used in this article

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