Development of a Stage- and Species-Specific RNAi System for Molecular Insights in Trichogramma Wasps

Abstract

Trichogramma wasps, egg parasitoids widely used to control lepidopteran pests, have long eluded in-depth molecular mechanistic studies due to their minute size and genetic tool scarcity. While previous RNAi efforts were restricted to T. dendrolimi, we developed the first cross-species RNAi system for both T. dendrolimi and the previously intractable T. ostriniae. Temporal expression profiling identified white and laccase 2 as stage-specific RNAi targets, peaking during prepupal/pupal stages, which were tested across species and developmental stages using microinjection and soaking dsRNA delivery methods. Survival analysis prioritized soaking for T. dendrolimi prepupae/pupae, while microinjection was essential for T. ostriniae to bypass prepupal mortality during soaking. Concentration-dependent RNAi targeting the white gene achieved 85.61% transcript reduction in T. dendrolimi via soaking and 89.36% in T. ostriniae via microinjection at 2000 ng/μL, correlating with 64.06% and 32.09% white-eyed pupae, causing a significant reduction in eye pigments. For the laccase 2 gene, soaking at 2000 ng/μL induced 88.35% transcript reduction in T. dendrolimi and 73.31% in T. ostriniae, leading to incomplete cuticle tanning and sclerotization. This study resolves the long-standing challenge of genetic manipulation in Trichogramma wasps, providing a universally applicable framework to decipher parasitoid-host interactions at the molecular scale, which is useful for sustainable pest management strategies.

Keywords: RNA interference; Trichogramma wasps; microinjection; soaking; species-specific optimization.

Figure 1

Spatial expression patterns and developmental timelines of white and laccase 2 genes in T. dendrolimi (Td) and T. ostriniae (To). (A) Developmental expression profile of the white gene in T. dendrolimi analyzed by RT-qPCR. (B) Developmental expression profile of the laccase 2 gene in T. dendrolimi analyzed by RT-qPCR. (C) Developmental expression profile of the white gene in T. ostriniae analyzed by RT-qPCR. (D) Developmental expression profile of the laccase 2 gene in T. ostriniae analyzed by RT-qPCR. (E) Reference developmental stages and native phenotypes of T. dendrolimi from the 5th to 12th day post-parasitism (from prepupa to adult). (F) Reference developmental stages and native phenotypes of T. ostriniae from the 4th to 10th day post-parasitism (from prepupa to adult). The lowercase letters represent the significant difference of the relative gene expression at different developmental stages (p < 0.05, Duncan).

Figure 2

Effects of soaking and microinjection treatments on the survival rate in T. dendrolimi (Td) and T. ostriniae (To). (A) Survival rate of T. dendrolimi (from the 5th to 11th day post-parasitism) treated via soaking. (B) Survival rate of T. dendrolimi (from the 5th to 11th day post-parasitism) treated via microinjection. (C) Survival rate of T. ostriniae (from the 4th to 9th day post-parasitism) treated via soaking. (D) Survival rate of T. ostriniae (from the 4th to 9th day post-parasitism) treated via microinjection.

Figure 3

RNAi-mediated knockdown of white and laccase 2 genes in T. dendrolimi (Td) and T. ostriniae (To) under varying dsRNA soaking durations. (A) The expression of the white gene in T. dendrolimi after soaking with 500 ng/μL or 2000 ng/μL dswhite, compared to H₂O and dsGFP controls. (B) The expression of the laccase 2 gene in T. dendrolimi after soaking with 500 ng/μL or 2000 ng/μL dslaccase 2, compared to H₂O and dsGFP controls. (C) The expression of the laccase 2 gene in T. ostriniae after soaking with 500 ng/μL or 2000 ng/μL dslaccase 2, compared to H₂O and dsGFP controls. The lowercase letters represent the significant difference of the relative gene expression after different treatments (p < 0.05, Duncan).

Figure 4

Gene expression and phenotypic changes following RNAi-mediated knockdown of white in T. dendrolimi (Td) and T. ostriniae (To) via soaking and microinjection. (A) Relative expression of the white gene in T. dendrolimi pupae (8th day post-parasitism) following soaking with varying dswhite concentrations. (B) Proportion of eye color phenotypes in T. dendrolimi pupae (8th day post-parasitism) after white knockdown via soaking. (C) Relative expression of the white gene in T. dendrolimi pupae (8th day post-parasitism) following microinjection with varying dswhite concentrations. (D) Proportion of eye color phenotypes in T. dendrolimi pupae (8th day post-parasitism) after white knockdown via microinjection. (E) Relative expression of the white gene in T. ostriniae pupae (7th day post-parasitism) following microinjection with varying dswhite concentrations. (F) Proportion of eye color phenotypes in T. ostriniae pupae (7th day post-parasitism) after white knockdown via microinjection. Phenotype classification in T. dendrolimi ((G), 8th day post-parasitism) and T. ostriniae ((H), 7th day post-parasitism): four-tier ommochrome deposition scale: Class I (fully pigmented, red), Class II (partial pigmentation, semi-red), Class III (low pigmentation, light red), and Class IV (unpigmented, white). The lowercase letters represent the significant difference of the relative gene expression after different treatments (p < 0.05, Duncan).

Figure 5

RNAi-mediated knockdown of laccase 2 in T. dendrolimi (Td) and T. ostriniae (To) via soaking and microinjection. (A) Relative expression of the laccase 2 gene in T. dendrolimi pupa (9th day post-parasitism) following soaking with varying dsRNA concentrations. (B) Relative expression of laccase 2 in T. dendrolimi pupa (9th day post-parasitism) following microinjection with varying dsRNA concentrations. (C) Relative expression of laccase 2 in T. ostriniae pupa (7th day post-parasitism) following soaking with varying dsRNA concentrations. (D) Relative expression of laccase 2 in T. ostriniae pupa (7th day post-parasitism) following microinjection with varying dsRNA concentrations. (E) Phenotypic comparison of T. dendrolimi pupae (9th–11th day post-parasitism) treated with dsGFP (left) and dslaccase 2 (right). (F) Phenotypic comparison of T. ostriniae pupae (7th–9th day post-parasitism) treated with dsGFP (left) and dslaccase 2 (right). The lowercase letters represent the significant difference of the relative gene expression after different treatments (p < 0.05, Duncan).