microinjecting dsrna into trichogramma dendrolimi pupae and monitoring developmental outcomes

Gen Fonksiyon Analizi için Trichogramma dendrolimi'de RNAi Tekniklerinin Geliştirilmesi

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.

Yazar Spotlight: RNA Girişimi Yoluyla Wolbachia'nın Neden Olduğu Thelytokous Partenogenez ve Genetik Araç Seti Gelişiminin Araştırılması

Yumurta Parazitoidinde RNA Girişimi, 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.

dsRNA'nın Trichogramma dendrolimi Pupa'ya Mikroenjekte Edilmesi ve Gelişimsel Sonuçların İzlenmesi

Başlamak için cam iğne çekiciyi açın, ardından ısıyı 280'e, hızı 170'e, gecikmeyi 250'ye ve direği 30'a ayarlayın. Kılcal cam iğnenin ucunu eğin. Daha sonra bir mikro enjeksiyon pompası kullanarak, enjeksiyon iğnesine yaklaşık iki mikrolitre ferritin ağır zincirli homoloji çift sarmallı RNA çözeltisi yükleyin.Trichogramma dendrolimi pupaları içeren agar plakasını bileşik mikroskop aşamasına aktarın. Enjeksiyon iğnesini bir pupa'nın karnına yaklaşık 30 derecelik bir açıyla dikkatlice yerleştirin ve kademeli olarak beş nanolitre ferritin ağır zincirli homoloji çift sarmallı RNA çözeltisi enjekte edin. Tedavi başına yaklaşık 700 pupayı 25 santigrat derecede 24 veya 48 saat inkübe edin.Replika başına 100 pupadan RNA içeriğini çıkarın. Tedavi başına yaklaşık 50 pupadan, eşekarısı oluşumunu gözlemleyin. Son olarak, eşekarısı çıkma oranını ve deformite oranını kaydedin.Çift sarmallı ferritin ağır zincir homolojisi enjekte edilen T.dendrolimi pupaları, çift sarmallı GFP ile enjekte edilenlere veya enjeksiyonsuz olanlara kıyasla önemli ölçüde daha düşük bir ortaya çıkma oranı sergiledi. Ortaya çıkan yaban arılarında, çift sarmallı ferritin ağır zincir homolojisine maruz kalan T.dendrolimi yaban arılarının %51.85'inde deforme olmuş küçük kanatlar gelişmiştir. Bu deformasyon, çift sarmallı GFP enjekte edilen veya herhangi bir enjeksiyonu olmayan eşekarısı yoktu.Ek olarak, çift sarmallı ferritin ağır zincir homolojisi enjekte edilen T.dendrolimi pupaları, anormal demir metabolizmasının göstergesi olan melanizm gösterdi. Melanin, çift sarmallı GFP enjekte edilen veya herhangi bir enjeksiyonu olmayan yaban arılarında gözlenmedi.

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

Research

JoVE Journal

Biology

Microinjecting dsRNA into Trichogramma dendrolimi Pupae and Monitoring Developmental Outcomes

37 Görüntüleme.  Shenyang Agricultural University. Başlamak için cam iğne çekiciyi açın, ardından ısıyı 280'e, hızı 170'e, gecikmeyi 250'ye ve direği 30'a ayarlayın. Kılcal cam iğnenin ucunu eğin. Daha sonra bir mikro enjeksiyon pompası kullanarak, enjeksiyon iğnesine yaklaşık iki mikrolitre ferritin ağır zincirli homoloji çift sarmallı RNA çözeltisi yükleyin.Trichogramma dendrolimi pupaları içeren agar plakasını bileşik mikroskop aşamasına aktarın. Enjeksiyon iğnesini bir pupa'nın karnına yaklaş...

Video: dsRNA'nın Trichogramma dendrolimi Pupa'ya Mikroenjekte Edilmesi ve Gelişimsel Sonuçların İzlenmesi

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.

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

Biyoloji

Cultivation and Transplantation of Trichogramma dendrolimi Pupae Using Corcyra cephalonica Eggs

To begin, add a solution of gum Arabic powder and water to a nine centimeter by 16 centimeter card. Then place approximately 5, 000 eggs of Corcyra cephalonica onto the card. Subject the host egg cards to 30 minutes of ultraviolet irradiation to prevent the hatching of eggs.Subsequently, cut the paper with inactivated eggs into egg cards, each containing approximately 300 to 500 eggs. Next, introduce a cohort of 80 to 120 Trichogramma dendrolimi wasps into a glass tube. Seal the tube with cotton.For parasitization, allow the wasps to deposit their eggs into the host eggs for six hours. Then promptly remove the wasps. Cultivate the parasitized host eggs for approximately eight days at 25 degrees Celsius.After five days, the parasitized host eggs turned black. Transfer the host egg card to a dissecting microscope. Using a pair of tungsten needles, meticulously remove the chorion from the host eggs and retrieve the pupa from within.Next, prepare a plate with 1.5%agar as substrate. Using a disinfected graver, etch several grooves on the agar. Now using a small brush, transplant a pupa from the dissected host egg into one of the grooves on the agar substrate.

Trichogramma dendrolimi Pupalarının Corcyra cephalonica Yumurtaları Kullanılarak Yetiştirilmesi ve Nakli

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 a pupa at an approximately 30 degree angle and gradually inject five nanoliters of ferritin heavy chain homology double-stranded RNA solution. Incubate approximately 700 pupae per treatment at 25 degrees Celsius for 24 or 48 hours.Extract RNA content from 100 pupae per replicate. From approximately 50 pupae per treatment, observe the wasps emergence. Finally, record the wasps emergence rate and deformity rate.T.dendrolimi pupae injected with double-stranded ferritin heavy chain homology exhibited a significantly lower emergence rate compared to those injected with double-stranded GFP or without injection. In emerged wasps, 51.85%of T.dendrolimi wasps subjected to double-stranded ferritin heavy chain homology developed deformed small wings. This deformation was absent in wasps injected with double-stranded GFP or those without any injection.Additionally, T.dendrolimi pupae injected with double-stranded ferritin heavy chain homology showed melanism, indicative of abnormal iron metabolism. The melanin was not observed in wasps injected with double-stranded GFP or those without any injection.