Functional analysis of Ornithine decarboxylase in manipulating the wing dimorphism in Nilaparvata lugens (Stål) (Hemiptera: Delphacidae).

The brown planthopper (BPH, Nilaparvata lugens), a major insect pest of rice, can make a shift in wing dimorphism to adapt to complex external environments. Our previous study showed that NlODC (Ornithine decarboxylase in N. lugens) was involved in wing dimorphism of the brown planthopper. Here, further experiments were conducted to reveal possible molecular mechanism of NlODC in manipulating the wing dimorphism. We found that the long-winged rate (LWR) of BPH was significantly reduced after RNAi of NlODC or injection of DFMO (D, L-α-Difluoromethylornithine), and LWR of males and females significantly decreased by 21.7% and 34.6%, respectively. Meanwhile, we also examined the contents of three polyamines under DFMO treatment and found that the contents of putrescine and spermidine were significantly lower compared to the control. After 3rd instar nymphs were injected with putrescine and spermidine, LWR was increased significantly in both cases, and putrescine was a little bit more effective, with 5.6% increase in males and 11.4% in females. Three days after injection of dsNlODC, injection of putrescine and spermidine rescued LWR to the normal levels. In the regulation of wing differentiation in BPH, NlODC mutually antagonistic to NlAkt may act through other signaling pathways rather than the classical insulin signaling pathway. This study illuminated a physiological function of an ODC gene involved in wing differentiation in insects, which could be a potential target for pest control.

Comparison of the signaling pathways of wing dimorphism regulated by biotic and abiotic stress in the brown planthopper.

Wing polymorphism is an evolutionary trait that is widely present in various insects and provides a model system for studying the evolutionary significance of insect dispersal. The brown planthopper (BPH, Nilaparvata lugens) can alter its wing morphs under biotic and abiotic stress. However, whether differential signaling pathways are induced by the 2 types of stress remain largely unknown. Here, we screened a number of candidate genes through weighted gene co-expression network analysis (WGCNA) and found that ornithine decarboxylase (NlODC), a key enzyme in the synthesis of polyamines, was associated with wing differentiation in BPH and mainly responded to abiotic stress stimuli. We analyzed the Kyoto Encyclopedia of Genes and Genomes enrichment pathways of differentially expressed genes under the 2 stresses by transcriptomic comparison, and found that biotic stress mainly influenced insulin-related signaling pathways while abiotic stress mainly influenced hormone-related pathways. Moreover, we found that insulin receptor 1 (NlInR1) may regulate wing differentiation of BPH by responding to both biotic and abiotic stress, but NlInR2 only responded to biotic stress. Similarly, the juvenile hormone epoxide hydrolase associated with juvenile hormone degradation and NlODC may regulate wing differentiation mainly through abiotic stress. A model based on the genes and stresses to modulate the wing dimorphism of BPH was proposed. These findings present a comprehensive molecular mechanism for wing polymorphism in BPH induced by biotic and abiotic stress.

Identification of putative abdominal vibration-related genes through transcriptome analyses in the brown planthopper (Nilaparvata lugens).

The sexually mature female brown planthoppers (BPHs) send out abdominal vibration (AV) signals through the rice so that the males can obtain intraspecific, gender, and localization information to prepare for mating. Destroying vibration signals is an alternative biological method for pest control. However, the regulatory mechanism of AV in female BPHs remains elusive, which presents an obstacle to pest control. We observed that before mating female BHPs emitted abdominal vibration signals that disappeared immediately after mating and reappeared after 6 days. Therefore, ovarian and brain samples of female BPHs from Unmated-6h+ (with AV), Mated-6h- (without AV) and Mated-6d+ (with AV) individuals were collected for transcript analyses. By transcriptional sequencing analyses, 33 candidate genes that might involve in the regulation of female AV were obtained. After selecting 4 candidate genes of them for verification by RNA interference (RNAi), it was found that interference of juvenile hormone binding protein (JHBP) could greatly reduce the probability and frequency of AV for female BPHs. In general, this study identified AV-related candidate genes in female BPHs through transcriptome analyses and provided an important basis for future research on pest control in BPHs.

CRISPR/Cas9-mediated knockout of the NlCSAD gene results in darker cuticle pigmentation and a reduction in female fecundity in Nilaparvata lugens (Hemiptera: Delphacidae).

In insects, cuticular pigmentation genes have been exploited as potential visible markers for constructing genetic manipulation systems. Here, we cloned cysteine sulfinic acid decarboxylase (CSAD), an orthologue of melanin metabolism pathway genes, and performed RNAi experiments in the brown planthopper Nilaparvata lugens (Hemiptera: Delphacidae). The results showed that a decrease in the level of transcription of NlCSAD increased melanin deposition in the body compared to the control group, resulting in darker cuticle pigmentation. Female adults treated with dsNlCSAD and mated with wild-type males laid significantly fewer eggs than the dsGFP-treated group, and lower hatchability of the eggs was also observed. In addition, two melanic mutant N. lugens strains (NlCSAD-/+ and NlCSAD-/-) constructed by the CRISPR/Cas9 genome editing system showed darker cuticular melanisation and a reduced oviposition and hatching rate, but the homozygotes had a darker body colour, fewer eggs and lower hatchability than heterozygotes or individuals after RNAi. Thus, we have provided the first evidence that NlCSAD is required for normal body pigmentation in adults and has a role in the fecundity of females and hatchability of eggs in N. lugens via a combination of RNAi and knockout of target genes based on the CRISPR/Cas9 genome editing system. Our results suggest that NlCSAD is a candidate visual reference gene for genetic manipulation of this important crop pest.

An Overview of Embryogenesis: External Morphology and Transcriptome Profiling in the Hemipteran Insect Nilaparvata lugens.

During embryogenesis of insects, the morphological and transcriptional changes are important signatures to obtain a better understanding of insect patterning and evolution. The brown planthopper Nilaparvata lugens is a serious insect pest of rice plants, but its embryogenesis has not uncovered. Here, we described embryonic development process of the pest and found it belongs to an intermediate-germ mode. The RNA-seq data from different times (6, 30, 96, and 150 h, after egg laying) of embryogenesis were then analyzed, and a total of 10,895 genes were determined as differentially expressed genes (DEGs) based on pairwise comparisons. Afterward, 1,898 genes, differentially expressed in at least two comparisons of adjacent embryonic stages were divided into 10 clusters using K means cluster analysis (KMCA). Eight-gene modules were established using a weighted gene co-expression network analysis (WGCNA). Gene expression patterns in the different embryonic stages were identified by combining the functional enrichments of the stage-specific clusters and modules, which displayed the expression level and reprogramming of multiple developmental genes during embryogenesis. The "hub" genes at each embryonic stage with possible crucial roles were identified. Notably, we found a "center" set of genes that were related to overall membrane functions and might play important roles in the embryogenesis process. After parental RNAi of the MSTRG.3372, the hub gene, the embryo was observed as abnormal. Furthermore, some homologous genes in classic embryonic development processes and signaling pathways were also involved in embryogenesis of this insect. An improved comprehensive finding of embryogenesis within the N. lugens reveals better information on genetic and genomic studies of embryonic development and might be a potential target for RNAi-based control of this insect pest.