RNAi of yellow-y, required for normal cuticle pigmentation, impairs courtship behavior and oviposition in the German cockroach (Blattella germanica).

The insect cuticle plays a key role in maintaining the insect's physiological function and behavior. Herein, the yellow-y protein is required to produce black melanin, and is expressed in a pattern that correlates with the distribution of this pigment. However, yellow-y can also have other functions, for instance, in insect behavior, but not much is known. In this study, we have studied the yellow-y gene in one important model and pest species, namely the German cockroach (Blattella germanica), which is to our knowledge the first time reported. In essence, we identified the yellow-y gene (BgY-y) and characterized its function by using RNA interference (RNAi). Silencing of BgY-y gene led to different developmental abnormalities (body weight and wings) in both genders. Specifically, there was an abundant decrease in melanin, turning the body color in pale yellow and the cuticle softer and more transparent. Interestingly, we also observed that the knockdown of BgY-y impaired the male cockroaches to display a weaker response to female-emitted contact sex pheromones, and also that the oviposition ability was weakened in the RNAi females. This study comprehensively analyzed the biological functions of the yellow-y gene in German cockroaches from the perspectives of development, body color, courtship behavior and oviposition, and as a consequence, this may opens new avenues to explore it as a novel pest control gene.

The melanin pigment gene black mediates body pigmentation and courtship behaviour in the German cockroach Blattella germanica.

Genes involved in melanin production directly impact insect pigmentation and can affect diverse physiology and behaviours. The role these genes have on sex behaviour, however, is unclear. In the present study, the crucial melanin pigment gene black was functionally characterised in an urban pest, the German cockroach, Blattella germanica. RNAi knockdown of B. germanica black (Bgblack) had no effect on survival, but did result in black pigmentation of the thoraxes, abdomens, heads, wings, legs, antennae, and cerci due to cuticular accumulation of melanin. Sex-specific variation in the pigmentation pattern was apparent, with females exhibiting darker coloration on the abdomen and thorax than males. Bgblack knockdown also resulted in wing deformation and negatively impacted the contact sex pheromone-based courtship behaviour of males. This study provides evidence for black function in multiple aspects of B. germanica biology and opens new avenues of exploration for novel pest control strategies.

Nanoparticle-delivered RNAi-based pesticide target screening for the rice pest white-backed planthopper and risk assessment for a natural predator.

Vacuolar-type (H+)-ATPase (vATPase) is a conserved multi-subunit eukaryotic enzyme composed of 14 subunits that form a functional complex consisting of an ATP-hydrolytic domain (V1) and a proton-translocation domain (V0). ATP hydrolysis and subsequent H+ translocation rely heavily on a fully assembled V1/V0 complex. Since vATPase is crucial for insect survival, it is a viable molecular target for pest control. However, detailed functional analyses of the 14 subunits and their suitability for pest control have not been fully explored in a single insect species. In this study, we identified 22 vATPase subunit transcripts that correspond to 13 subunits (A1, A2, B, C, D, E, F, G, H, a1, a2, c and d) in the white-backed planthopper (WBPH), Sogatella furcifera, a major hemipteran pest of rice. RNAi screens using microinjection and spray-based methods revealed that the SfVHA-F, SfVHA-a2 and SfVHA-c2 subunits are critical. Furthermore, star polymer (SPc) nanoparticles were utilized to conduct spray-induced and nanoparticle-delivered gene silencing (SI-NDGS) to evaluate the pest control efficacy of RNAi targeting the SfVHA-F, SfVHA-a2 and SfVHA-c2 transcripts. Target mRNA levels and vATPase enzymatic activity were both reduced. Honeydew excreta was likewise reduced in WBPH treated with dsRNAs targeting SfVHA-F, SfVHA-a2 and SfVHA-c2. To assess the environmental safety of the nanoparticle-wrapped dsRNAs, Cyrtorhinus lividipennis Reuter, a major natural enemy of planthoppers, was also sprayed with dsRNAs targeting SfVHA-F, SfVHA-a2 and SfVHA-c2. Post-spray effects of dsSfVHA-a2 and dsSfVHA-c2 on C. lividipennis were innocuous. This study identifies SfVHA-a2 and SfVHA-c2 as promising targets for biorational control of WBPH and lays the foundation for developing environment-friendly RNAi biopesticides.

RNA Interference-Screening of Potentially Lethal Gene Targets in the White-Backed Planthopper Sogatella furcifera via a Spray-Induced and Nanocarrier-Delivered Gene Silencing System.

RNA interference (RNAi) is a widespread post-transcriptional silencing mechanism that targets homologous mRNA sequences for specific degradation. An RNAi-based pest management strategy is target-specific and considered a sustainable biopesticide. However, the specific genes targeted and the efficiency of the delivery methods can vary widely across species. In this study, a spray-induced and nanocarrier-delivered gene silencing (SI-NDGS) system that incorporated gene-specific dsRNAs targeting conserved genes was used to evaluate phenotypic effects in white-backed planthopper (WBPH). At 2 days postspraying, transcript levels for all target genes were significantly reduced and knockdown of two gene orthologs, hsc70-3 and PP-α, resulted in an elevated mortality (>60%) and impaired ecdysis. These results highlight the utility of the SI-NDGS system for identifying genes involved in WBPH growth and development that could be potentially exploitable as high mortality target genes to develop an alternative method for WBPH control.

Efficient DIPA-CRISPR-mediated knockout of an eye pigment gene in the white-backed planthopper, Sogatella furcifera.

Although CRISPR/Cas9 has been widely used in insect gene editing, the need for the microinjection of preblastoderm embryos can preclude the technique being used in insect species with eggs that are small, have hard shells, and/or are difficult to collect and maintain outside of their normal environment. Such is the case with Sogatella furcifera, the white-backed planthopper (WBPH), a significant pest of Oryza sativa (rice) that oviposits inside rice stems. Egg extraction from the stem runs the risk of mechanical damage and hatching is heavily influenced by the micro-environment of the rice stem. To bypass these issues, we targeted embryos prior to oviposition via direct parental (DIPA)-CRISPR, in which Cas9 and single-guide RNAs (sgRNAs) for the WBPH eye pigment gene tryptophan 2,3-dioxygenase were injected into the hemocoel of adult females. Females at varying numbers of days posteclosion were evaluated to determine at what stage their oocyte might be most capable of taking up the gene-editing components. An evaluation of the offspring indicated that the highest G0 gene-edited efficacy (56.7%) occurred in females injected 2 d posteclosion, and that those mutations were heritably transmitted to the G1 generation. This study demonstrates the potential utility of DIPA-CRISPR for future gene-editing studies in non-model insect species and can facilitate the development of novel pest management applications.

Efficient nanoparticle-based CRISPR-Cas13d induced mRNA disruption of an eye pigmentation gene in the white-backed planthopper, Sogatella furcifera.

The discovery of the clustered regularly interspaced short palindromic repeat (CRISPR) system has driven gene manipulation technology to a new era with applications reported in organisms that span the tree of life. The utility of CRISPR-mediated editing was further expanded to mRNA following identification of the RNA-targeting Cas13 family of smaller endonuclease proteins. Application of this family to insect research, however, has been more limited. In this study, the smallest Cas13 family member, Cas13d, and guide RNAs (gRNAs) were complexed with a versatile nanomaterial (star polycation, SPc) to generate a proof-of-concept RNA-editing platform capable of disrupting mRNA expression of the eye pigmentation gene tryptophan 2,3-dioxygenase (SfTO) in white-backed planthoppers (WBPHs). The resulting red-eye phenotype was present in 19.76% (with SPc) and 22.99% (without SPc) of the treatment groups and was comparable to the red-eye phenotype generated following conventional RNA interference knockdown (22.22%). Furthermore, the Cas13/gRNA phenotype manifested more quickly than RNA interference. Consistent with the expected Cas13d mechanism, SfTO transcript levels were significantly reduced. Taken together, the results indicate that the SPc-CRISPR-Cas13d/gRNA complex negatively impacted expression of the target gene. These findings confirm the utility of this novel mRNA disruption system in insects and lay the foundation for further development of these tools in the implementation of green agricultural pest management tactics.

Two Antenna-Enriched Carboxylesterases Mediate Olfactory Responses and Degradation of Ester Volatiles in the German Cockroach Blattella germanica.

Insects have evolved an extremely sensitive olfactory system that is essential for a series of physiological and behavioral activities. Some carboxylesterases (CCEs) comprise a major subfamily of odorant-degrading enzymes (ODEs) playing a crucial role in odorant signal inactivation to maintain the odorant receptor sensitivity. In this study, 93 CCEs were annotated in the genome of the German cockroach Blattella germanica, a serious urban pest. Phylogenetic and digital tissue expression pattern analyses identified two antenna-enriched CCEs, BgerCCE021e3 and BgerCCE021d1, as candidate ODEs. RNA interference (RNAi)-mediated knockdown of BgerCCE021e3 and BgerCCE021d1 resulted in partial anosmia with experimental insects exhibiting reduced attraction to ester volatile resources and slower olfactory responses than controls. Furthermore, enzymatic conversion of geranyl acetate by crude male antennal extracts from BgerCCE021e3 and BgerCCE021d1 RNAi insects was also significantly reduced. Our results provide evidence for CCE function in German cockroach olfaction and provide a basis for further exploring behavioral inhibitors that target olfactory-related CCEs.

Functional characterization of five developmental signaling network genes in the white-backed planthopper: Potential application for pest management.

BACKGROUND: The white-backed planthopper (WBPH, Sogatella furcifera) is a major rice pest that exhibits condition dependent wing dimorphisms - a macropterous (long wing) form and a brachypterous (short wing) form. Although, the gene cascade that regulates wing development and dimorphic differentiation has been largely defined, the utility of these genes as targets for pest control has yet to be fully explored. RESULTS: Five genes typically associated with the developmental signaling network, armadillo (arm), apterous A (apA), scalloped (sd), dachs (d), and yorkie (yki) were identified from the WBPH genome and their roles in wing development assessed following RNA interference (RNAi)-mediated knockdown. At 5 days-post injection, transcript levels for all five targets were substantially decreased compared with the dsGFP control group. Among the treatment groups, those injected with dsSfarm had the most pronounced effects on transcript reduction, mortality (95 ± 3%), and incidence (45 ± 3%) of wing deformities, whereas those injected with dsSfyki had the lowest incidence (6.7 ± 4%). To assess the utility of topical RNAi for Sfarm, we used a spray-based approach that complexed a large-scale, bacteria-based double-stranded RNA (dsRNA) expression pipeline with star polycation (SPc) nanoparticles. Rice seedlings infested with third and fourth instar nymphs were sprayed with SPc-dsRNA formulations and RNAi phenotypic effects were assessed over time. At 2 days post-spray, Sfarm transcript levels decreased by 86 ± 9.5% compared with dsGFP groups, and the subsequent incidences of mortality and wing defects were elevated in the treatment group. CONCLUSIONS: This study characterized five genes in the WBPH developmental signaling cascade, assessed their impact on survival and wing development via RNAi, and developed a nanoparticle-dsRNA spray approach for potential field control of WBPH. © 2023 Society of Chemical Industry.

Functional characterization of tyrosine melanin genes in the white-backed planthopper and utilization of a spray-based nanoparticle-wrapped dsRNA technique for pest control.

As a significant pest of rice the white-backed planthopper (WBPH) Sogatella furcifera is a focus of pest management. However, traditional chemical-based control methods risk the development of pesticide resistance as well as severe ecological repercussions. Although nanoparticle-encapsulated dsRNAs provide a promising alternative method for sustainable pest management, gene targets specific to WBPH have yet to be optimized. Genes in the tyrosine-melanin pathway impact epidermal melanization and sclerotization, two processes essential for insect development and metabolism, have been proposed as good candidate targets for pest management. Seven genes (aaNAT, black, DDC, ebony, tan, TH, and yellow-y) in this group were identified from WBPH genome and functionally characterized by using RNAi for their impact on WBPH body color, development, and mortality. Knockdown of SfDDC, Sfblack, SfaaNAT, and Sftan caused cuticles to turn black, whereas Sfyellow-y and Sfebony knockdown resulted in yellow coloration. SfTH knockdown resulted in pale-colored bodies and high mortality. Additionally, an Escherichia coli expression system for large-scale dsRNA production was coupled with star polycation nanoparticles to develop a sprayable RNAi method targeting SfTH that induced high WBPH mortality rates on rice seedlings. These findings lay the groundwork for the development of large-scale dsRNA nanoparticle sprays as a WBPH control method.

Functional characterization of developmentally critical genes in the white-backed planthopper: Efficacy of nanoparticle-based dsRNA sprays for pest control.

BACKGROUND: Epidermal growth factor receptor (EGFR), zinc finger homeodomain-2 (zfh-2), Abdominal-A (Abd-A), and Abdominal-B (Abd-B) regulate the growth and development of the insect abdomen. However, their potential roles in pest control have not been fully assessed. The development of insecticide resistance to multiple chemistries in the white-backed planthopper (WBPH), a major pest of rice, has prompted interest in novel pest control approaches that are ecologically friendly. Although pest management approaches based on double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) have potential, their susceptibility to degradation limits large-scale field applications. These limitations, however, can be overcome with nanoparticle-dsRNA complexes that have greater environmental stability and improved cellular uptake. RESULTS: In this study, at 5 days post-injection, transcripts for the four gene targets were reduced relative to controls and all of the experimental groups exhibited significant phenotypic defects and increased mortality. To evaluate the potential of these gene targets for field applications, a nanocarrier-dsRNA spray delivery system was assessed for RNAi efficacy. At 11 days post-spray, significant phenotypic defects and increased mortality were observed in all experimental groups. CONCLUSION: Taken together, the results confirm the suitability of the target genes (SfEGFR, Sfzfh-2, SfAbd-A, and SfAbd-B) for pest management and demonstrate the efficacy of the nanocarrier spray system for inducing RNAi-mediated knockdown. As such, the study lays the foundation for the further development and optimization of this technology for large-scale field applications. © 2022 Society of Chemical Industry.