microinjecting dsrna into trichogramma dendrolimi pupae and monitoring developmental outcomes

Técnicas avançadas de RNAi em Trichogramma dendrolimi para análise da função gênica

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.

Autor em destaque: Investigando a partenogênese Thelytokous induzida por Wolbachia e o desenvolvimento do kit de ferramentas genéticas por meio da interferência de RNA

Interferência de RNA no parasitoide de ovos, 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.

Microinjeção de dsRNA em pupas de Trichogramma dendrolimi e monitoramento de resultados de desenvolvimento

Para começar, ligue o puxador de agulha de vidro e, em seguida, ajuste o calor para 280, a velocidade para 170, o atraso para 250 e o poste para 30. Biselar a ponta da agulha de vidro capilar. Em seguida, usando uma micro bomba de injeção, carregue aproximadamente dois microlitros de solução de RNA de fita dupla de homologia de cadeia pesada de ferritina na agulha de injeção.Transfira a placa de ágar contendo pupas de Trichogramma dendrolimi para o estágio do microscópio composto. Insira cuidadosamente a agulha de injeção no abdômen de uma pupa em um ângulo de aproximadamente 30 graus e injete gradualmente cinco nanolitros de solução de RNA de fita dupla de homologia de cadeia pesada de ferritina. Incubar aproximadamente 700 pupas por tratamento a 25 graus Celsius por 24 ou 48 horas.Extrair conteúdo de RNA de 100 pupas por replicação. A partir de aproximadamente 50 pupas por tratamento, observar a emergência das vespas. Finalmente, registre a taxa de emergência de vespas e a taxa de deformidade.Pupas de T. dendrolimi injetadas com homologia de cadeia pesada de ferritina de fita dupla exibiram uma taxa de emergência significativamente menor em comparação com aquelas injetadas com GFP de fita dupla ou sem injeção. Em vespas emergidas, 51,85% das vespas T. dendrolimi submetidas à homologia de cadeia pesada de ferritina de fita dupla desenvolveram pequenas asas deformadas. Essa deformação estava ausente em vespas injetadas com GFP de fita dupla ou naquelas sem qualquer injeção.Adicionalmente, pupas de T. dendrolimi injetadas com homologia de cadeia pesada de ferritina de fita dupla mostraram melanismo, indicativo de metabolismo anormal do ferro. A melanina não foi observada em vespas injetadas com GFP de fita dupla ou naquelas sem qualquer injeção.

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

Research

JoVE Journal

Biology

Microinjecting dsRNA into Trichogramma dendrolimi Pupae and Monitoring Developmental Outcomes

37 Views.  Shenyang Agricultural University. To begin, turn on the glass needle puller, then set the heat to 280, the velocity to 170, the delay to 250, and the pole to 30. Bevel the tip of the capillary glass needle. Then using a micro injection pump, load approximately two microliters of ferritin heavy chain homology double-stranded RNA solution into the injection needle.Transfer the agar plate containing Trichogramma dendrolimi pupae onto the compound microscope stage. Carefully insert the injection needle into the abdomen of ...