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.
Subscription Required. Please recommend JoVE to your librarian.
Protocol
1. Insect rearing
- Feed Corcyra cephalonica on corn flour at a temperature of 26 ± 1°C and a relative humidity of 40% ± 10%.
- 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
- 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. - Disinfect the pressed polyethylene plastic film by exposing both sides to ultraviolet (UV) light for 1 h.
- Add a small amount of 10% polyvinyl alcohol to the semicircular surface.
3. Trichogramma dendrolimi parasitism
- After CO2 anesthesia, place T. dendrolimi female wasps into a collection box, and the number of wasps was ~3000.
- Place the convex side of the film egg card toward the collection box and secure the edges with a rubber band.
- 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.
- Let T. dendrolimi wasps parasitize freely for 4-8 h and provide 10% sucrose water through wetted cotton.
4. Collecting T. dendrolimi venom
- 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.
- 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).
- Measure the concentration of collected T. dendrolimi venom using a Bicinchoninic acid (BCA) assay kit (Table of Materials).
- Store the venom at -80 °C for further analyses.
5. SDS-PAGE analyses
- 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.
- Perform SDS-PAGE gel run at 130 V for 120 min.
- Stain and decolorize the SDS-PAGE gel using the protein staining apparatus (Table of Materials).
Subscription Required. Please recommend JoVE to your librarian.
Representative 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 can collect the venom of parasitic wasps well. In addition, there is no specific correlation between parasitism time and concentration because different batches of parasitic wasps may have different vitality.
Additionally, T. dendrolimi venom was analyzed by SDS-PAGE, revealing a venom protein range spanning from under 10 kDa to over 130 kDa in Figure 1. However, when the negative control of amino acid was analyzed by SDS-PAGE, it was found that there was no protein in it (Supplementary Figure 1), which also proved that the protein collected by this method was indeed the venom protein of parasitic wasps.
Figure 1: SDS-PAGE analysis of T. dendrolimi venom protein. Lanes 1-2: the loaded amounts of venom protein were 8 µg and 10 µg, respectively. M: Marker. Please click here to view a larger version of this figure.
| Sample | Parasitism time (h) | Concentration (μg/μL) | |
| Venom | 1 | 5 | 0.39 |
| 2 | 6 | 0.42 | |
| 3 | 5 | 0.4 | |
| 4 | 6 | 0.35 | |
| 5 | 5 | 0.46 | |
| Control | 1 | NP | 0.04 |
| 2 | NP | 0.03 | |
| 3 | NP | 0.05 | |
| 4 | NP | 0.03 | |
| 5 | NP | 0.03 | |
Table 1: The concentration information of the venom and control. The protein concentration of representative venom and control samples was measured using the BCA protein assay kit. Control: the unparasitized controls. NP: no parasitism
Supplementary Figure 1: SDS-PAGE analysis of control and venom. Control: the unparasitized control. Venom: the loaded amounts of venom protein were 10 µg. M: Marker. Please click here to download this File.
Subscription Required. Please recommend JoVE to your librarian.
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 parasitize the artificial host. (2) The sterility of the amino acid solution must be ensured, as contamination of the amino acid solution may negatively impact the parasitism efficiency. (3) The parasitization of artificial egg cards should be conducted in dark conditions to promote parasitism. (4) It is recommended to either directly carry out downstream experiments or freeze the samples to ensure the activity of venom and prevent degradation.
It is recommended to judge parasitization by visualizing deposited eggs. If the deposited eggs are not observed under the microscope, no venom may have been extracted. The limitation of the technique is that it requires a large number of parasitoid wasps. A single venom extraction requires about 3,000 parasitoid wasps, which increases the workload.
The previous method of extraction of parasitoid wasp venom was to dissect the venom reservoir. However, parasitoid wasps are tiny; for example, Trichogramma is less than 1 mm long. Not only are the technical requirements of dissecting venom reservoirs high, but contamination of other tissues during dissection is also common. The novel method using artificial hosts can improve the efficiency of venom extraction and avoid contamination from other tissues caused by dissection.
This method can also be extended to other parasitoid wasps. For example, polyethylene plastic film oocytes containing a mixture of salt ions and amino acids can be used to obtain T. neustadt venom, and artificial wax eggs containing KCl-MgSO4 solution can be used to obtain T. pretiosum venom. In addition to Trichogramma, it has been reported that Anastatus japonicus13, Microplitis croceipes9, and Habrobracon hebetor10 can parasitize artificial hosts. Using the properties of these parasitoid wasps to parasitize artificial hosts, similar venom extraction methods can be developed.
Parasitoid wasp venom is an underexplored source of biological molecules with potential pest control and medical applications. Recently, the potential uses of parasitoid venom in pharmacology and agriculture have been recognized14,15. Pharmacologically, many components in parasitoid venom have broad potential application prospects in optimizing immunotherapy, treating thrombotic disorders, and finding templates for new antibiotics. In agriculture, some components in parasitoid venom can be used as biological control agents to regulate the development, reproduction, and immunity of pests to achieve the purpose of effectively controlling pests15. However, the lack of efficient venom extraction methods often limits research on the venom of parasitoid wasps, especially tiny parasitoid wasps such as Trichogramma. This paper provides an efficient method to extract the venom of Trichogramma, which provides a method for the follow-up study of Trichogramma venom, such as the identification of protein composition and venom function. In addition, this method can also be used as a reference for other parasitoid wasp venom research and provides support for promoting the screening of bioactive molecules from parasitoid venoms for insecticidal or medical applications.
Subscription Required. Please recommend JoVE to your librarian.
Disclosures
The author has nothing to disclose and no competing financial interests.
Acknowledgments
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 "Shuangchuang Doctor" Foundation of Jiangsu Province (Grant No. 202030472), and the Nanjing Agricultural University startup fund (Grant No. 804018).
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 |
References
- Pennacchio, F., Strand, M. R. Evolution of developmental strategies in parasitic hymenoptera. Annual Review of Entomology. 51, 233-258 (2006).
- Yan, Z. C., Ye, X. H., Wang, B. B., Fang, Q., Ye, G. Y. Research advances on composition, function and evolution of venom proteins in parasitoid wasps. Chinese Journal of Biological Control. 33 (1), 1-10 (2017).
- Asgari, S., Rivers, D. B. Venom proteins from endoparasitoid wasps and their role in host-parasite interactions. Annual Review of Entomology. 56, 313-335 (2011).
- Moreau, S. J. M., Guillot, S. Advances and prospects on biosynthesis, structures, and functions of venom proteins from parasitic wasps. Insect Biochemistry and Molecular Biology. 35 (11), 1209-1223 (2005).
- Yan, Z. C., et al. A venom serpin splicing isoform of the endoparasitoid wasp Pteromalus puparum suppresses host prophenoloxidase cascade by forming complexes with host hemolymph proteinases. Journal Biological Chemistry. 292 (3), 1038-1051 (2017).
- Woelke, J. B., et al. Description and biology of two new egg parasitoid species (Hymenoptera: Trichogrammatidae) reared from eggs of Heliconiini butterflies (Lepidoptera: Nymphalidae: Heliconiinae) in Panama. Journal of Natural History. 53 (11-12), 639-657 (2019).
- Zang, L. S., Wang, S., Zhang, F., Desneux, N. Biological control with Trichogramma in China: History, present status, and perspectives. Annual Review of Entomology. 66, 463-484 (2021).
- Nettles, W. C. J., Morrison, R. K., Xie, Z. N., Ball, D., Shenkir, C. A., Vinson, S. B. Synergistic action of potassium chloride and magnesium sulfate on parasitoid wasp oviposition. Science. 218, 4568 (1982).
- Tilden, R. L., Ferkovich, S. M. Kairomonal stimulation of oviposition into an artificial substrate by the endoparasitoid Microplitis croceipes (Hymenoptera)Braconidae). Annals of the Entomological Society of America. 81 (1), 152-156 (1988).
- Xie, Z. N., Li, L., Xie, Y. Q. In vitro culture of Habrobracon hebetor. Chinese Journal of Biological Control. 5 (2), 49-51 (1989).
- Han, S. T., Liu, W. H., Li, L. Y., Chen, Q. X., Zeng, B. K. Breeding Trichogramma ostriniae with artificial eggs. Journal of Environmental Entomology. 21 (1), 9-12 (1999).
- Li, L. Y., Chen, Q. X., Liu, W. H. Oviposition behavior of twelve species of Trichogramma and its influence on the efficiency of rearing them in vitro. Journal of Environmental Entomology. 11 (1), 31-35 (1989).
- Xing, J. Q., Li, L. Y. Rearing of an egg parasite Anastatus japonicus Ashmead in vitro. Acta Entomologica Sinica. 33 (2), 166-173 (1990).
- Moreau, S. J. M. "It stings a bit but it cleans well": Venoms of Hymenoptera and their antimicrobial potential. Journal of Insect Physiology. 59 (2), 186-204 (2013).
- Moreau, S. J. M., Asgari, S. Venom proteins from parasitoid wasps and their biological function. Toxins. 7 (7), 2385-2412 (2015).
