Plant viruses transmitted by insects cause tremendous losses in most important crops around the world. The identification of receptors of plant viruses within their insect vectors is a key challenge to understanding the mechanisms of transmission and offers an avenue for future alternative control strategies to limit viral spread. We here report the identification of two cuticular proteins within aphid mouthparts, and we provide experimental support for the role of one of them in the transmission of a noncirculative virus. These two proteins, named Stylin-01 and Stylin-02, belong to the RR-1 cuticular protein subfamily and are highly conserved among aphid species. Using an immunolabeling approach, they were localized in the maxillary stylets of the pea aphid Acyrthosiphon pisum and the green peach aphid Myzus persicae, in the acrostyle, an organ earlier shown to harbor receptors of a noncirculative virus. A peptide motif present at the C termini of both Stylin-01 and Stylin-02 is readily accessible all over the surface of the acrostyle. Competition for in vitro binding to the acrostyle was observed between an antibody targeting this peptide and the helper component protein P2 of Cauliflower mosaic virus. Furthermore, silencing the stylin-01 but not stylin-02 gene through RNA interference decreased the efficiency of Cauliflower mosaic virus transmission by Myzus persicae. These results identify the first cuticular proteins ever reported within arthropod mouthparts and distinguish Stylin-01 as the best candidate receptor for the aphid transmission of noncirculative plant viruses.

Many viruses of agricultural importance are transmitted to host plants via insect vectors. Characterizing virus-vector interactions at the molecular level is essential if we are to fully understand the transmission mechanisms involved and develop new strategies to control viral spread. Hitherto, insect proteins involved in virus transmission have been characterized only poorly. Recent advances in this topic, however, have significantly filled this knowledge gap. Among the vector molecules identified, cuticular proteins have emerged as key molecules for plant virus transmission, regardless of transmission mode or vector considered. Here, we review recent evidence highlighting that the CPR family, and particularly RR-1 proteins, undoubtedly deserves special attention.

Aphids are phloem-feeding insects known as major pests in agriculture able to transmit hundreds of plant viruses. The majority of these viruses, classified as noncirculative, are retained and transported on the inner surface of the cuticle of the needle-like mouthparts while the aphids move from plant to plant. Identification of receptors of viruses within insect vectors is a key challenge because they are promising targets for alternative control strategies. The acrostyle, an organ discovered earlier paving the common food/salivary canal at the tip of aphid maxillary stylets, displays proteins at the cuticle-fluid interface, some of which are receptors of noncirculative viruses. To assess the presence of stylet- and acrostyle-specific proteins and identify putative receptors, we have developed a comprehensive comparative analysis of the proteomes of four cuticular anatomical structures of the pea aphid, stylets, antennas, legs and wings. In addition, we performed a systematic immunolabeling detection of the cuticular proteins identified by mass spectrometry in dissected stylets. We thereby establish the first proteome of stylets of an insect and determine the minimal repertoire of the cuticular proteins composing the acrostyle. Most importantly, we propose a short list of plant virus receptor candidates, among which RR-1 proteins are remarkably predominant.

The acrostyle is a distinct anatomical region present on the cuticle at the inner face of the common food/salivary canal at the tip of aphid maxillary stylets. This conserved structure is of particular interest as it harbors the protein receptors of at least 1 plant virus, Cauliflower mosaic virus, and presumably has other roles in plant–insect interactions. Previously we reported immunolabeling of a highly conserved motif of cuticular proteins from the CPR family (named for the presence of a Rebers and Riddiford consensus) within the acrostyle. Here we report the development of novel tools to further study the proteomic composition of this region and to identify proteins involved in insect‐virus interactions. Using a series of antibodies against cuticular proteins from the RR‐2 subfamily, we identified additional peptides present within the acrostyle. Our results demonstrated that the acrostyle is a complex structure containing multiple domains of cuticular proteins accessible for interaction. In addition, an array of overlapping peptides, which covers the diversity of the majority of the RR‐2 subfamily, was developed as a generic tool to characterize cuticular protein/pathogen interactions. Upon probing this array with Cucumber mosaic virus particles, consensus peptide sequences from hybridizing peptides were identified. Use of these novel tools has extended our knowledge of the proteomic composition of insect maxillary stylets and identified sequences that could be involved in virus binding, thus contributing to further elucidation of the various properties and functions of the acrostyle.

Background: Aedes aegyptimosquito, the principal global vector of arboviral diseases, lays eggs and undergoes larval and pupal development to become adult mosquitoes in fresh water (FW). It has recently been observed to develop in coastal brackish water (BW) habitats of up to 50% sea water, and such salinity tolerance shown to be an inheritable trait. Genomics of salinity tolerance inAe. aegyptihas not been previously studied, but it is of fundamental biological interest and important for controlling arboviral diseases in the context of rising sea levels increasing coastal ground water salinity.Results: BW- and FW-Ae. aegyptiwere compared by RNA-seq analysis on the gut, anal papillae and rest of the carcass in fourth instar larvae (L4), proteomics of cuticles shed when L4 metamorphose into pupae, and transmission electron microscopy of cuticles in L4 and adults. Genes for specific cuticle proteins, signalling proteins, moulting hormone-related proteins, membrane transporters, enzymes involved in cuticle metabolism, and cytochrome P450 showed different mRNA levels in BW and FW L4 tissues. The salinity-tolerantAe. aegyptiwere also characterized by altered L4 cuticle proteomics and changes in cuticle ultrastructure of L4 and adults.Conclusions: The findings provide new information on molecular and ultrastructural changes associated with salinity adaptation in FW mosquitoes. Changes in cuticles of larvae and adults of salinity-tolerantAe. aegyptiare expected to reduce the efficacy of insecticides used for controlling arboviral diseases. Expansion of coastal BW habitats and their neglect for control measures facilitates the spread of salinity-tolerantAe. aegyptiand genes for salinity tolerance. The transmission of arboviral diseases can therefore be amplified in multiple ways by salinity-tolerantAe. aegyptiand requires appropriate mitigating measures. The findings inAe. aegypti have attendant implications for the development of salinity tolerance in other fresh water mosquito vectors and the diseases they transmit.