BACKGROUND
Previous studies of brown planthopper (BPH), Nilaparvata lugens, showed that carrying the plant pathogenic virus, rice ragged stunt virus (RRSV), enhanced the lethality of the entomopathogenic fungus, Metarhizium anisopliae (YTTR). The underlying mechanism for this was not established but a serine protease cascade was hypothesized to be involved.


RESULTS
Two immune response genes, NlKPI and NlVenomase, were identified and shown to be involved. Synthesized dsRNA techniques used in this study to explore gene function revealed that treatment with dsRNA to silence either gene led to a higher BPH mortality from M. anisopliae infection than the dsRNA control treatment. NlKPI and NlVenomase play vital roles in BPH immunity to defend against alien pathogens. And both genes participate in the immune response process of BPHs against co-infection with RRSV and M. anisopliae YTTR by regulating the expression of antimicrobial peptides and phenoloxidase activity.


CONCLUSION
Our study provided new targets for BPH biocontrol and laid a solid foundation for further research on the interaction of "plant virus- insect vector- entomopathogenic fungus". This article is protected by copyright. All rights reserved.

The immune response mechanism of Nilaparvata lugens against a combined infection of rice ragged stunt virus and Metarhizium anisopliae.

The brown planthopper, Nilaparvata lugens (Stål), is a major pest of rice crops, and its control is critical for food security. Pymetrozine has been recommended as an alternative to imidacloprid for controlling N. lugens, but the pest has developed high resistance to it, making its prohibition and restriction urgent. To address this issue, we conducted a study using a mixture of pymetrozine and zhongshengmycin with the effective ratio of 1:40, to evaluate the fitness costs in N. lugens. Our results showed that N. lugens had a relative fitness of 0.03 under this ratio, with significantly reduced longevity, female and male adult periods, total pre-oviposition days, and fecundity. Moreover, the expression levels of the uricase gene (EC1.7.3.3) and farnesyl diphosphate farnesyl transferase gene (EC2.5.1.21) were reduced in N. lugens. These genes are involved in urea metabolism and steroid biosynthesis pathway, respectively, and their suppression can interfere with the normal nutritional function of N. lugens. Our study demonstrates that the combination of chemical insecticides and antimicrobials can delay the development of resistance and improve the efficiency of pest control. This information is valuable for researchers developing management strategies to delay the development of pymetrozine resistance in N. lugens.

Fitness costs of resistance to insecticide pymetrozine combined with antimicrobial zhongshengmycin in Nilaparvata lugens (Stål)

Abstract In this study, two vital genes in the serine protease cascade, NlK-serpin and NlVenomase , were identified. Two genes show differential responses in brown planthopper ( Nilaparvata lugens , BPH) carrying the insect fungal pathogen Metarhizium anisopliae YTTR and/or the plant viral pathogen rice ragged stunt virus (RRSV). RNA interference (RNAi) combined with/without M. anisopliae YTTR were used to explore the genes’ functions. And BPH mortality, the transcriptional response of four antimicrobial peptides (AMPs) genes, and the phenoloxidase (PO) activity were measured to unveil the underlying mechanism. Results showed that the coding sequences (CDSs) of NlK-serpin and NlVenomase contained 291-bp and 1,194-bp nucleotides, respectively. Phylogenetic analysis showed both genes have the closest kinship to Laodelphax striatellus . NlK-serpin is highly expressed in the adult and 3rd -instar stages and NlVenomase is highly expressed in 2nd and 3rd -instar nymphs. Both genes possess high expression in the fat body. Synthesized dsRNAs have strong interference efficiency against the expression of target genes. Ds NlK-serpin injection significantly reduced BPHs survival rates in RRSV-free BPHs. However, when RRSV existed, the differences in survival rates among the three dsRNA treatments were insignificant. DsRNA combined with M. anisopliae YTTR produced a synergistic effect leading to higher BPHs mortality. Results of AMPs expression and PO activity indicated that NlK-serpin and NlVenomase play a vital role in BPH humoral immunity against M. anisopliae YTTR infection. And both genes participate in the process of RRSV-mediated immune responses of BPH to M. anisopliae YTTR infection by regulating AMPs expression and PO activity. Our study provided new targets for BPH control and laid a solid foundation for further research the interaction of “plant virus- insect vector- entomopathogenic fungus”. 

/pdf/rice-ragged-stunt-virus-mediates-immune-responses-in-spxqa5ol.pdf

Rice ragged stunt virus mediates immune responses in Nilaparvata lugens against Metarhizium anisopliae YTTR infection: evidence from two novel genes NlK-serpin and NlVenomase

In insects, serine proteases and serine protease homologs (SPs/SPHs) are involved in a variety of physiological processes including digestion, development, and immunity. Here, we identified 112 SP and 88 SPH genes in the genome of the yellow mealworm, Tenebrio molitor. Based on the features of domain structure, they were divided into "S" group containing single Tryp-SPc or Tryp-SPHc domain, "C" group containing 1-4 CLIP domain (CLIPA-D) and "M" group containing the CBD, CUB, EGF, Fz, Gd, LDLa, PAN, SEA, SR, Sushi, and TSP domains, and have 115, 48, and 37 gene members, respectively. According to the active sites in the catalytic triad, the putative trypsin, chymotrypsin, or elastase-like enzyme specificity of the identified SPs/SPHs were predicted. Phylogenetic and genomic location analyses revealed that gene duplication exists in the large amount of SPs/SPHs. Gene expression profiling using RNA-seq data along with real time reverse transcription-polymerase chain reaction analysis showed that most SP/SPH genes display life stage specific expression patterns, indicating their important roles in development. Many SP/SPH genes are specifically or highly expressed in the gut, salivary gland, fat body, hemocyte, ovary, and testis, suggesting that they participate in digestion, immunity, and reproduction. The findings lay the foundation for further functional characterization of SPs/SPHs in T. molitor.

Identification and expression profiling of serine protease-related genes in Tenebrio molitor.

Nilaparvata lugens proclotting enzymes (NlPCEs) belong to the clip domain serine protease (clip-SP) family, which is a characteristic protease family in arthropods. NlPCE3 was previously reported to regulate egg production and development in female N. lugens, but its role in male N. lugens is unclear. In the present study, qPCR analysis showed that NlPCE3 was expressed in three different tissues (gut, testis and fat body). RNAi revealed that dsNlPCE3 injection made the male vas deferens thinner and reduced the oviposition level of the females that mated with dsNlPCE3-treated males, causing eggs not to hatch. Furthermore, immunofluorescence staining showed that NlPCE3 was widely expressed in the male internal genitalia. However, after dsNlPCE3 injection, expression of NlPCE3 was diffuse in the male internal genitalia, whose peripheral cells seemed degraded. Overall, these results indicate that NlPCE3 is important for reproduction in male N. lugens.

PCE3 Plays a Role in the Reproduction of Male Nilaparvata lugens.

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PCE3 Plays a Role in the Reproduction of Male Nilaparvata lugens.