The cuticular protein gene ApCP7 and ApCP62 are essential for reproduction in Acyrthosiphon pisum, affecting ecdysis and survival.

Cuticle proteins, in conjunction with chitin, compose the insect exoskeleton, and play a key role in the growth, development, and molting of insects. However, the specific functions of most cuticular protein genes in the growth, development, and reproductive processes of the pea aphid (Acyrthosiphon pisum) remain unclear. In this study, we have identified six cuticle protein genes in the pea aphid, namely ApCP7, ApCP10, ApCP19, ApCP19.8-like, ApCP35 and ApCP62. We found that the expression levels of six genes were highly expressed during the adult stage, and except for ApCP10, which is highly expressed in the pea aphid cuticle, other genes were highly expressed in the ovaries. Subsequently, we observed that the survival rate and fecundity of pea aphid were significantly lower than those of the control group after silencing ApCP7 and ApCP62 through RNA interference. Furthermore, when ApCP7 transcript levels were reduced, aphid encountered difficulties in molting, were smaller in body sizes, and exhibited a darker body color. These results indicate that ApCP7 and ApCP62 are involved in the development and reproduction of pea aphid, and could be used as RNAi targets for controlling pea aphid.

GARP: A family of glycine and alanine-rich proteins that helps spider mites feed on plants.

Spider mites (Tetranychidae) are destructive agricultural pests which have evolved strategies to overcome plant defenses, such as the ability to puncture the leaves of their hosts to feed. The expression of many genes with unknown functions is altered during feeding, but little is known about the role of these genes in plant-mite interactions. Here, we identified 3 novel gene families through analysis of genomic and transcriptomic data from 3 spider mite species. These GARP family genes encode glycine and alanine-rich proteins; they are present in mites (Acariformes) but absent in ticks (Parasitiformes) in the subclass Acari, indicating that these genes have undergone a significant expansion in spider mites and thus play important adaptive roles. Transcriptomic analysis revealed that the expression of GARP genes is strongly correlated with feeding and the transfer to new hosts. We used RNA interference to silence GARP1d in the two-spotted spider mite Tetranychus urticae, which inhibited feeding and egg laying and significantly increased mortality when the mites were transferred to soybean shoots; a similar effect was observed after TuVATPase was silenced. However, no changes in mite mortality were observed after TuGARP1d-silenced mites were placed on an artificial diet, which was different from the effect of TuVATPase silencing. Our results indicate that GARP family members play important roles in mite-plant interactions. Additional studies are needed to clarify the mechanisms underlying these interactions.

Biochemical and Molecular Analysis of Field Resistance to Spirodiclofen in Panonychus citri (McGregor).

Spirodiclofen is one of the most widely used acaricides in China. The citrus red mite, Panonychus citri (McGregor) (Acari: Tetranychidae), is one of the most destructive citrus pests worldwide and has developed a high resistance to spirodiclofen. However, the molecular mechanism of spirodiclofen resistance in P. citri is still unknown. In this study, we identified a field spirodiclofen-resistant strain (DL-SC) that showed 712-fold resistance to spirodiclofen by egg bioassay compared to the susceptible strain. Target-site resistance was not detected as non-synonymous mutations were not found by amplification and sequencing of the ACCase gene of resistant and susceptible strains; in addition, the mRNA expression levels of ACCase were similar in both resistant and susceptible strains. The activity of detoxifying enzymes P450s and CCEs in the resistant strain was significantly higher than in the susceptible strain. The transcriptome expression data showed 19 xenobiotic metabolisms genes that were upregulated. Stage-specific expression profiling revealed that the most prominent upregulated gene, CYP385C10, in transcriptome data was significantly higher in resistant strains in all stages. Furthermore, functional analysis by RNAi indicated that the mortality caused by spirodiclofen was significantly increased by silencing the P450 gene CYP385C10. The current results suggest that overexpression of the P450 gene, CYP385C10, may be involved in spirodiclofen resistance in P. citri.

RNA virome screening in diverse but ecologically related citrus pests reveals potential virus-host interactions.

As an evergreen ecosystem, citrus orchards have specialized pest species and stable ecological homeostasis; thus, they provide an ideal model for investigating RNA viromes in diverse but ecologically related species. For this purpose, we collected specialized citrus pests from three classes of invertebrates, Insecta, Arachnida, and Gastropoda and we constructed two kinds of libraries (RNA and small RNA) for the pests by deep sequencing. In total, six virus-derived sequences were identified, including four Picornavirales, one Jingchuvirales and one Nidovirales. The picornavirus-derived small RNAs showed significant small RNA peaks and symmetric distribution patterns along the genome, which suggests these viruses infected the hosts and triggered host antiviral immunity RNA interference. Screening of virus-derived sequences in multiple species of citrus pests (n = 10 per species) showed that Eotetranychus kankitus picorna-like virus and Tetranychus urticae mivirus may be present in multiple pests. Our investigation in citrus pests confirmed that RNA viruses revealed by metagenomics could impact host immunity (e.g. RNAi). An approach with parallel deep sequencing of RNAs and small RNAs is useful not only for viral discoveries but also for understanding virus-host interactions of ecologically related but divergent pest species.

Three novel RNA viruses in the spider mite Tetranychus urticae and their possible interactions with the host RNA interference response.

As a polyphagous herbivore, the two-spotted spider mite Tetranychus urticae is engaged with various plant hosts and interacts with diverse organisms that share the same ecological niche. Thus, T. urticae faces frequent challenges from viral infections. However, the RNA viruses of T. urticae are still unknown. Here, we constructed two libraries (~8 Gb for RNA and ~10 Mb for small RNA) from a strain of T. urticae using deep sequencing, and identified three novel RNA viruses from the families Kitaviridae, Dicistroviridae, and Chuviridae. Among them, the Kitaviridae and Dicistroviridae viruses presented a possible interaction pattern with the host RNA interference pathway.

Expression dynamics of key ecdysteroid and juvenile hormone biosynthesis genes imply a coordinated regulation pattern in the molting process of a spider mite, Tetranychus urticae.

In insects, the ecdysteroid 20-hydroxyecdysone coordinates with juvenile hormone (JH) to regulate the process of molting, development and metamorphosis; however, this interaction is still unclear in the mites. In this study, we investigated the gene related to ecdysteroid and JH biosynthesis pathways, including four ecdysteroid and 11 JH biosynthesis genes. We examined their expression patterns during molting of different developmental stages of the two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), an important agricultural pest that feeds on more than 1100 plant species. The expression of ecdysteroid biosynthesis Halloween genes exhibited a positive zigzag-like pattern, with a peak after 8 h of molting and a drop 8 h after entering each quiescent stage. In contrast, JH biosynthesis genes expression displayed a negative zigzag-like pattern, with a peak at 8 h after entering each quiescent stage and a drop after 8 h of each molting. These opposite patterns imply that ecdysteroid and JH expression is coordinated during the developmental transition. Our data provide an initial perspective on the co-expression of ecdysteroid and JH biosynthesis genes to regulate this important developmental process in the two-spotted spider mite.

Molecular characterization of ecdysis triggering hormone and its receptor in citrus red mite (Panonychus citri).

Neuropeptide ecdysis triggering hormone (ETH) plays crucial roles in invertebrates by activating a G protein-coupled receptor (GPCR), the ecdysis triggering hormone receptor (ETHR), which has been intensively investigated in Hexapoda. However, the molecular characterization of ETH and ETHR in Chelicerata remains unknown. In this study, we identified and characterized the full-length cDNA of ETH and ETHR in citrus red mite Panonychus citri (McGregor). Phylogenetic analysis indicated that PcETHR was closely related to the insect ETHR subtype B. A calcium mobilization-based functional assay showed that PcETH activated the PcETHR in a dose-dependent manner. Furthermore, a sharp upregulation of PcETH before ecdysis was observed by quantitative real-time PCR (qRT-PCR), and it revealed a tight correlation of ETH signaling and the molting process. These are the first insights into the molecular characterization of ETH and its receptor in mites as P. citri. Our work provides basic information of the ETH and ETHR in P. citri and lays a solid foundation for further physiological functions to better understand the ETH signaling system in mites.

Characterization and expression patterns of key ecdysteroid biosynthesis and signaling genes in a spider mite (Panonychus citri).

Ecdysteroids play a crucial role in regulating molting in the phylum of Arthropoda and much is known with members of the subphylum of Hexapoda including the Insecta. However, this is still unclear in key pests as spider mites belonging to the subphylum of Chelicerata that originated earlier in the Cambrian period. In this study, we investigated 14 key genes of ecdysteroid biosynthesis and signaling and their expression over the different developmental stages in the citrus red mite, Panonychus citri (Acari: Stigmaeidae). P. citri is an economically important and widespread pest of citrus crops and it has five developmental stages of egg, larva, protonymph, deutonymph and adult. Typically, the expression of the ecdysteroid-synthesizing Halloween gene Spook (PcSpo) followed a positive zigzag-like pattern with a peak in the first half of each developmental stage and a drop in the second half prior to the molting to the next stage. Similar to PcSpo, PcDib, PcSad, PcRXR2, PcE75 and PcHR38 showed a positive zigzag-like expression pattern, while that of PcE78, PcHR3 and PcFTZ-F1 was opposite that we called a negative zigzag-like pattern. Silencing of the PcSpo gene by RNAi showed that molting was inhibited. Interestingly, we could rescue these RNAi effects by supplementing ponasterone A (PonA) and not by 20E, which is indicative that mites use PonA rather than 20E as ecdysteroid hormone. Modeling of the ecdysteroid receptor (PcEcR) hormone binding cavity also predicted binding of PonA, but showed a steric hindrance for 20E. We believe our data provide insight into the evolution and expression patterns of key ecdysteroid biosynthesis and signaling genes in a distant, non-insect species, and can become a foundation to develop new targets for controlling important agricultural pests such as spider mites.