microinjecting dsrna into trichogramma dendrolimi pupae and monitoring developmental outcomes

유전자 기능 분석을 위한 Trichogramma dendrolimi 의 RNAi 기술 발전

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.

저자 스포트라이트: RNA 간섭을 통한 Wolbachia-Induced Thelytokous partthenogenesis 및 Genetic Toolkit Development 조사

계란 기생충의 RNA 간섭, Trichogramma dendrolimi Matsumura

Trichogramma dendrolimi 번데기에 dsRNA 미세 주입 및 발달 결과 모니터링

시작하려면 유리 바늘 당김기를 켠 다음 열을 280으로, 속도를 170으로, 지연을 250으로, 극을 30으로 설정합니다. 모세관 유리 바늘의 끝을 비스듬히 깎습니다. 그런 다음 마이크로 주입 펌프를 사용하여 약 2마이크로리터의 페리틴 중쇄 상동성 이중 가닥 RNA 용액을 주사 바늘에 로드합니다.Trichogramma dendrolimi 번데기가 함유된 한천 플레이트를 복합 현미경 스테이지로 옮깁니다. 주사 바늘을 번데기의 복부에 약 30도 각도로 조심스럽게 삽입하고 5나노리터의 페리틴 중쇄 상동성 이중 가닥 RNA 용액을 서서히 주입합니다. 치료당 약 700마리의 번데기를 섭씨 25도에서 24시간 또는 48시간 동안 배양합니다.복제당 번데기 100마리에서 RNA 함량을 추출합니다. 치료 당 약 50 마리의 번데기에서 말벌의 출현을 관찰하십시오. 마지막으로 말벌 출현률과 기형율을 기록합니다.이중 가닥 페리틴 중쇄 상동성을 주입한 T.dendrolimi 번데기는 이중 가닥 GFP로 주입되거나 주입되지 않은 것에 비해 현저히 낮은 출현율을 나타냈습니다. 출현한 말벌에서 이중 가닥 페리틴 중쇄 상동성을 받은 T.dendrolimi 말벌의 51.85%가 변형된 작은 날개를 발달시켰습니다. 이 변형은 이중 가닥 GFP가 주입된 말벌 또는 주입이 없는 말벌에서는 나타나지 않았습니다.또한, 이중 가닥 페리틴 중쇄 상동성을 주입한 T.dendrolimi 번데기는 비정상적인 철 대사를 나타내는 멜라니즘을 나타냈습니다. 멜라닌은 이중 가닥 GFP로 주입 된 말벌 또는 주입없이 관찰되지 않았습니다.

microinjecting-dsrna-into-trichogramma-dendrolimi-pupae-monitoring

Research

JoVE Journal

Biology

Microinjecting dsRNA into Trichogramma dendrolimi Pupae and Monitoring Developmental Outcomes

40 조회수. 시작하려면 유리 바늘 당김기를 켠 다음 열을 280으로, 속도를 170으로, 지연을 250으로, 극을 30으로 설정합니다. 모세관 유리 바늘의 끝을 비스듬히 깎습니다. 그런 다음 마이크로 주입 펌프를 사용하여 약 2마이크로리터의 페리틴 중쇄 상동성 이중 가닥 RNA 용액을 주사 바늘에 로드합니다.Trichogramma dendrolimi 번데기가 함유된 한천 플레이트를 복합 현미경 스테이지로 옮깁?...

비디오: Trichogramma dendrolimi 번데기에 dsRNA 미세 주입 및 발달 결과 모니터링

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 forests. The immature stages of Trichogramma offspring develop within the host egg, exhibiting remarkable diminutiveness (approximately 0.5 mm in adult length). RNA-interference (RNAi) methodology has emerged as a crucial tool for elucidating gene functions in numerous organisms. However, manipulating RNAi in certain small parasitoid species, such as Trichogramma, has generally posed significant challenges. In this study, we present an efficient RNAi method in Trichogramma denrolimi. The outlined procedure encompasses the acquisition and isolation of individual T. dendrolimi specimens from host eggs, the design and synthesis of double-stranded RNA (dsRNA), the in vitro transplantation and cultivation of T. dendrolimi pupae, the micro-injection of dsRNA, and the subsequent assessment of target gene knockdown through RT-qPCR analysis. This study furnishes a comprehensive, visually detailed procedure for conducting RNAi experiments in T. dendrolimi, thereby enabling researchers to investigate the gene regulation in this species. Furthermore, this methodology is adaptable for RNAi studies or micro-injections in other Trichogramma species with minor adjustments, rendering it a valuable reference for conducting RNAi experiments in other endoparasitic species.

The manipulation of RNA interference (RNAi) presents a formidable challenge in many parasitoid species with diminutive size, such as Trichogramma wasps. This study delineated an efficient RNAi method in Trichogramma denrolimi. The present methodology provides a robust model for investigating gene regulation in Trichogramma wasps.