cultivation and transplantation of trichogramma dendrolimi pupae using corcyra cephalonica eggs

Avancement des techniques ARNi chez Trichogramma dendrolimi pour l’analyse de la fonction des gènes

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.

Pleins feux sur l’auteur : Étude de la parthénogenèse thélytokous induite par Wolbachia et du développement d’une boîte à outils génétique par interférence ARN

Interférence de l’ARN chez le parasitoïde de l’œuf, Trichogramma dendrolimi Matsumura

Culture et transplantation de pupes de Trichogramma dendrolimi à l’aide d’œufs de Corcyra cephalonica

Pour commencer, ajoutez une solution de gomme arabique en poudre et d’eau à une carte de neuf centimètres sur 16 centimètres. Placez ensuite environ 5 000 œufs de Corcyra cephalonica sur la carte. Soumettre les cartes d’œufs de l’hôte à 30 minutes d’irradiation ultraviolette pour éviter l’éclosion des œufs.Ensuite, coupez le papier avec les œufs inactivés en cartes d’œufs, chacune contenant environ 300 à 500 œufs. Ensuite, introduisez une cohorte de 80 à 120 guêpes Trichogramma dendrolimi dans un tube de verre. Scellez le tube avec du coton.Pour le parasitage, laissez les guêpes déposer leurs œufs dans les œufs de l’hôte pendant six heures. Ensuite, retirez rapidement les guêpes. Cultivez les œufs de l’hôte parasité pendant environ huit jours à 25 degrés Celsius.Après cinq jours, les œufs de l’hôte parasité sont devenus noirs. Transférez la carte de l’œuf hôte dans un microscope à dissection. À l’aide d’une paire d’aiguilles en tungstène, retirez méticuleusement le chorion des œufs de l’hôte et récupérez la chrysalide de l’intérieur.Ensuite, préparez une plaque avec 1,5% d’agar comme substrat. À l’aide d’un graveur désinfecté, gravez plusieurs rainures sur la gélose. Maintenant, à l’aide d’un petit pinceau, transplantez une chrysalide de l’œuf hôte disséqué dans l’une des rainures du substrat de gélose.

cultivation-transplantation-trichogramma-dendrolimi-pupae-using

Research

JoVE Journal

Biology

Cultivation and Transplantation of Trichogramma dendrolimi Pupae Using Corcyra cephalonica Eggs

48 vues. Pour commencer, ajoutez une solution de gomme arabique en poudre et d’eau à une carte de neuf centimètres sur 16 centimètres. Placez ensuite environ 5 000 œufs de Corcyra cephalonica sur la carte. Soumettre les cartes d’œufs de l’hôte à 30 minutes d’irradiation ultraviolette pour éviter l’éclosion des œufs.Ensuite, coupez le papier avec les œufs inactivés en cartes d’œufs, chacune contenant environ 300 à 500 œufs. Ensuite, introduisez une cohorte de 80 à 120...

Vidéo: Culture et transplantation de pupes de Trichogramma dendrolimi à l’aide d’œufs de Corcyra cephalonica

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.