cultivation and transplantation of trichogramma dendrolimi pupae using corcyra cephalonica eggs

Utveckla RNAi-tekniker inom Trichogramma dendrolimi för analys av genfunktion

Trichogramma spp. are a group of egg parasitoids that have been extensively utilized as highly efficient biological control agents against a wide spectrum of lepidopteran pests in agricultural and forest ecosystems worldwide1,2,3,4. The application of mass-reared Trichogramma provides an environmentally friendly approach for the sustainable management of pests5,6,7. Understanding the molecular biology of Trichogramma wasps provides valuable insights into enhancing the mass-rearing efficiency and field performance of these biological control agents8,9by investigating the methodology of gene regulation and genome editing10.
Since the discovery of double-stranded RNA (dsRNA)-mediated specific genetic interference in Caenorhabditis elegans in 1998, the RNA-interference (RNAi) method has evolved into a vital genetic toolkit for exploring the regulatory mechanisms of organisms by suppressing the expression of target genes11. RNAi experiments have become a standard methodology widely applied to study gene function in numerous insect species12,13. Nevertheless, the manipulation of RNAi presents a formidable challenge in many parasitoid species, particularly among those belonging to the endoparasitic Chalcidoidea family14,15,16. The RNAi method has been documented in at least 13 parasitoid species14,15,16,17,18,19. Among these, the RNAi approach has been comprehensively conducted in Nasonia wasps and is applicable throughout the developmental stages, including embryos, larvae, pupae, and adults14,15,16. It is noteworthy that Nasonia wasps are ectoparasitoids, with their offspring developing in the interstitial space between the host pupa and the puparium, enabling their cultivation in vitro and making them tolerate certain treatments, such as micro-injection. Unlike Nasonia wasps, Trichogramma individuals undergo their entire embryonic, larval, and pupal development inner the host egg. The layer at embryo and larva stages (which may impede dsRNA permeability), vulnerability to damage, and the difficulty in surviving in vitro present formidable obstacles20,21,22. Additionally, the diminutive size of Trichogramma individuals, approximately ~0.5 mm in adult or pupal length, renders them exceedingly intricate to manipulate20,21,22.
In the present study, we outline a comprehensive procedure for conducting RNA interference (RNAi) experiments in Trichogramma denrolimi Matsumura. This procedure encompasses the following procedures: (1) the design and synthesis of double-stranded RNA (dsRNA), (2) microinjection of T. denrolimi pupae, (3) the transplantation and in vitro incubation of these pupae, and (4) the detection of target gene knockdown through RT-qPCR analysis. The target gene selected for the RNAi experiment is the ferritin heavy chain homology (Ferhch). FerHCH, an iron-binding protein, contains a ferroxidase center endowed with antioxidant capabilities, facilitating the oxidation of Fe2+ to Fe3+. It plays an indispensable role in the growth and development of various organisms by maintaining redox equilibrium and iron homeostasis. Depletion of FerHCH can result in the overaccumulation of iron, leading to irreversible tissue damage, and often culminating in significant phenotypic alterations, including growth defects, deformities, and mortality23,24. This study offers a step-by-step guide for conducting RNAi in T. denrolimi, which will be invaluable for investigating the gene functions within the broader context of Trichogramma wasps.

Författare i fokus: Undersöker Wolbachia-inducerad thelytokous partenogenes och utveckling av genetiska verktyg genom RNA-interferens

RNA-interferens i äggparasitoiden, Trichogramma dendrolimi matsumura

Trichogramma wasps are a group of egg parasitoids used to control lepidopteran pests in agriculture. The intracellular bacteria Wolbachia induces thelytokous parthenogenesis in some Trichogramma wasps. We are trying to answer the mechanism of Wolbachia and sex-determination systems in Trichogramma wasps.We have developed the RNA interference method in Trichogramma wasps. This method is a vital genetic toolkit for investigating the gene functions of Trichogramma. The immature stages of Trichogramma offspring develop within the host egg.The body size of Trichogramma is extremely small and is approximately 0.5 millimeter in adult length. The manipulation of genetic tools such as RNA interference and genome editing presents a formidable challenge for these tiny parasitoid wasps. For decades, gene functions within the entire Trichogramma genus have been rarely explored.The present methodology provides a robust model for investigating gene regulation during developmental and physiological activities in Trichogramma wasps.

Odling och transplantation av Trichogramma dendrolimi puppor med hjälp av Corcyra cephalonica-ägg

Börja med att tillsätta en lösning av gummi arabpulver och vatten till ett nio centimeter x 16 centimeter stort kort. Lägg sedan cirka 5 000 ägg av Corcyra cephalonica på kortet. Utsätt värdäggkorten för 30 minuters ultraviolett bestrålning för att förhindra kläckning av ägg.Skär sedan papperet med inaktiverade ägg i äggkort, som var och en innehåller cirka 300 till 500 ägg. Introducera sedan en kohort av 80 till 120 Trichogramma dendrolimi getingar i ett glasrör. Försegla röret med bomull.För parasitisering, låt getingarna deponera sina ägg i värdäggen i sex timmar. Ta sedan omedelbart bort getingarna. Odla de parasiterade värdäggen i cirka åtta dagar vid 25 grader Celsius.Efter fem dagar blev de parasiterade värdäggen svarta. Överför värdäggkortet till ett dissekerande mikroskop. Använd ett par volframnålar för att noggrant ta bort korionen från värdäggen och hämta puppan inifrån.Förbered sedan en tallrik med 1,5 % agar som substrat. Använd en desinficerad graver och etsa flera spår på agar. Använd nu en liten borste och transplantera en puppa från det dissekerade värdägget till en av spåren på agarsubstratet.

Research

JoVE Journal

Biology

Cultivation and Transplantation of Trichogramma dendrolimi Pupae Using Corcyra cephalonica Eggs

RNA Interference in the Egg Parasitoid, Trichogramma dendrolimi Matsumura

The egg parasitoids, Trichogramma spp, are recognized as efficient biological control agents against various lepidopteran pests in agriculture and ...

Microinjecting dsRNA into Trichogramma dendrolimi Pupae and Monitoring Developmental Outcomes

Mikroinjicering av dsRNA i Trichogramma dendrolimi Puppor och övervakning av utvecklingsresultat