Aphids secrete watery saliva into plant tissues from the onset of stylet penetration

Aphid feeding requires the secretion of two types of saliva: gelling saliva (from the principal gland) that forms an intercellular sheath for the penetrating stylet, and watery saliva [from accessory salivary glands (ASGs)] that facilitates intracellular penetration and phloem feeding. Plant viruses can be used as salivary markers to investigate key steps in aphid feeding, and penetration can be monitored electrically using the electrical penetration graph (EPG) approach. We conducted a series of EPG-controlled transmission experiments using Cucurbit aphid-borne yellows virus [CABYV; Polerovirus spec. (Luteoviridae)], which is retained in the ASGs, as a marker for watery saliva secretions. The melon aphid, Aphis gossypii Glover (Hemiptera: Aphididae), was used as a vector and melon seedlings, Cucumis melo L. (Cucurbitaceae), as host plants. Viruliferous aphids were interrupted at various stages during stylet penetration, i.e., during intercellular penetration prior to intracellular puncture and following a potential drop within the first probe. Viruliferous aphids and leaf disc samples obtained from the stylet penetration site were used to detect CABYV by quantitative real-time RT-PCR. Approximately half of the inoculated leaf discs were found to be infected with CABYV after very brief (12.9 +/- 1.9 s) intercellular stylet probes and before intracellular stylet puncture. The number of virus particles ejected during such probes was similar to the number ejected by aphids during longer probes including a single intracellular puncture. Our results therefore suggest that watery saliva is secreted by aphids from the onset of stylet penetration.