cultivation and transplantation of trichogramma dendrolimi pupae using corcyra cephalonica eggs

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

Corcyra cephalonica 계란을 사용한 Trichogramma dendrolimi 번데기의 재배 및 이식

시작하려면 아라비아 껌 가루와 물을 섞은 용액을 9cm x 16cm 카드에 넣으십시오. 그런 다음 약 5, Corcyra cephalonica의 000 계란을 카드에 놓습니다. 알이 부화하는 것을 방지하기 위해 숙주 달걀 카드를 30분 동안 자외선을 조사합니다.그런 다음 비활성화 된 계란이 들어있는 종이를 계란 카드로 자르고 각각 약 300-500 개의 계란이 들어 있습니다. 다음으로, 80-120 Trichogramma dendrolimi 말벌 코호트를 유리관에 도입합니다. 면으로 튜브를 밀봉하십시오.기생을 위해 말벌이 6 시간 동안 숙주 알에 알을 낳을 수 있도록하십시오. 그런 다음 즉시 말벌을 제거하십시오. 기생한 숙주 알을 섭씨 25도에서 약 8일 동안 배양합니다.5일 후, 기생한 숙주 알은 검게 변했다. 숙주 계란 카드를 해부 현미경으로 옮깁니다. 한 쌍의 텅스텐 바늘을 사용하여 숙주 알에서 융모막을 꼼꼼하게 제거하고 내부에서 번데기를 회수합니다.다음으로 1.5 % 한천을 기질로 한 플레이트를 준비합니다. 소독 된 그레이버를 사용하여 한천에 여러 개의 홈을 에칭합니다. 이제 작은 브러시를 사용하여 해부된 숙주 알의 번데기를 한천 기질의 홈 중 하나에 이식합니다.

cultivation-transplantation-trichogramma-dendrolimi-pupae-using

Research

JoVE Journal

Biology

Cultivation and Transplantation of Trichogramma dendrolimi Pupae Using Corcyra cephalonica Eggs

48 조회수. 시작하려면 아라비아 껌 가루와 물을 섞은 용액을 9cm x 16cm 카드에 넣으십시오. 그런 다음 약 5, Corcyra cephalonica의 000 계란을 카드에 놓습니다. 알이 부화하는 것을 방지하기 위해 숙주 달걀 카드를 30분 동안 자외선을 조사합니다.그런 다음 비활성화 된 계란이 들어있는 종이를 계란 카드로 자르고 각각 약 300-500 개의 계란이 들어 있습니다. 다음으로, 80-120 Trichogramma dendroli...

비디오: Corcyra cephalonica 계란을 사용한 Trichogramma dendrolimi 번데기의 재배 및 이식

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.