The green peach aphid is a pest in agricultural setting throughout the world. Besides its direct damage through removal of carbon (photoassimilates) and nitrogen (free amino acids) from the phloem, M. persicae is also capable of vectoring over one hundred virus species. In a project that was spearheaded by John Ramsey, we have sequenced ~27.000 ESTs from more than 16 cDNA libraries using conventional Sanger sequencing. Among others, these include libraries from three different M. persicae lineages, aphid tissues (e.g. digestive tract and salivary glands), whole aphids on various host plants, and aphids with and without Potato Leaf Roll Virus. Later in the project, we used next-generation sequencing methods (Roche/454 GS-FLX technology), which allowed for the generation of significantly more M. persicae EST sequences. All ESTs were clustered into contigs (~40.000) and are spotted on an Agilent oligoarray, which is available upon request (requests should be directed to Georg Jander; gj32@cornell.edu). Recently we have extended the number of transcripts using Roche 454 sequencing. This project has yielded a vast amount of novel sequences (Ramsey et al., submitted to IMB), many of which are now also represented on the Agilent oligoarray. We intend to use this array to study aphid responses upon induction of plant secondary metabolites, virus infection and other environmental stresses. (This project is funded by the USDA. Collaborators are: Alex C. C. Wilson, Qi Sun, Cecilia Tamborindeguy, Agnese Winfield, Gaynor Malloch, Dawn M. Smith, Brian Fenton, and Stewart M. Gray)

The first aphid genome is sequenced! ftp://ftp.ncbi.nih.gov/genomes/Acyrthosiphon_pisum/ Members of a large aphid community have, over the past years, worked toward completing the sequencing of Acyrthosiphon pisum, the pea aphid (International Aphid Genomics Consortium, 2009, submitted to PLoS Biology). In contrast to M. persicae, this agricultural pest has specialized to feed on a smaller set of plants in the family of the Fabiaceae. I have been involved in several aspects of the gene annotation process. Firstly, an annotation group, spearheaded by Nicole Gerardo (Emory University), looked into the genes related to stress, immunity and defense in aphids. We have found that, compared to other insects, the pea aphid has a highly reduced number of immunity-related genes. A comparison with our data for M. persicae supports this observation (Gerardo et al., submitted to Genome Biology).

We further annotated all genes in the pea aphid that have a possible role in detoxification of xenobiotics, such as plant metabolites, pesticides etc. We hypothesized that generalist species would encounter many different plant secondary metabolites, and thus would have a greater variety of detoxification enzymes. We addressed this question by comparing the genes encoding esterases, cytochrome P450s and glutathione S-transferases between the green peach aphid and the pea aphid. Whereas we find similar number of genes encoding GSTs and esterases in both aphid species, M. persicae seems to have radiated the P450 protein family (Ramsey et al., submitted to IMB). Future research will aim at identification of the role of these detoxification enzymes. The USDA funded this work.

Aphids feed from a single cell type, the sieve element. In order to minimize damage, they penetrate their slender stylets between the host cells until they reached the phloem. A successful stylet penetration requires the action of aphid saliva. Salivary proteins seem involved in 1) protection of the stylet during penetration, 2) facilitating penetration, and 3) suppression of plant defense responses (e.g. clogging up the sieve elements). Because of the importance of aphid saliva for their successful infestation, it seems likely that plants recognize some salivary components, which in turn could elicit defense responses in the host.
From: Tjallingii J Exp Bot. 2006; 57:739-745.
I have shown that Arabidopsis plants are capable of recognizing aphids through their saliva and mount an appropriate defense response at the aphid-feeding site. Further characterization of these salivary elicitors indicates that Arabidopsis recognizes a proteinaceous elicitor in the aphid saliva. Size fractionation suggests the peptide of small protein ranges from 3 to 10 kDa. This work is accepted for publication in Plant, Cell, and Environment. We are currently working, together with Ted Thannhauser and Tara Fish (USDA/Cornell University) toward identification of proteins/peptides in this particular fraction of the aphid saliva. This work is funded by Netherlands Organization for Scientific Research and NSF.

Meanwhile, we have identified many M. persicae genes encoding salivary proteins from sequences obtained from a salivary gland EST library. To study the in vivo activity of these proteins we use a combination of RNAi in aphids and in planta expression. We have selected ~20 full-length cDNAs for expression in plants. Transient expression in tobacco allows for the rapid testing of the function of salivary proteins. Candidates that show reduced or increased susceptibility to M. persicae have been targeted for stable expression in Arabidopsis. Dezi Elzinga a PhD student in that lab and I soon hope to present our initial results.

M. persicae, like many other aphids, relies on E-beta-farnesene (EBF) to warn other members of their colony of eminent danger. Whereas it has been known for decades that EBF is the aphid alarm pheromone, no one has identified a receptor or components of the signal cascade triggered upon EBF treatment. I am rearing two aphid populations, originating from the same genotype, displaying differential responsiveness to EBF. Whereas, one of the colonies is normally responsive, the other colony is unresponsive to EBF due to continuous stimulation with EBF (habituation). Habituation resulted in the complete down-regulation of genes responsive to EBF (as evidenced by micro-array studies comparing the two colonies). Aphids from the habituated colony can revert back to EBF-responsiveness upon removal of the stimulus, but this takes several generations. We are currently investigating if habituation to EBF has deleterious effects on their fitness when aphid parasitoids or predators are present. This work is done in collaboration with Winnie Cheng, a Cornell Undergraduate.

Although gene expression studies can potentially identify genes important for resistance against M. persicae, ultimately changes in the plant’s protein and metabolite profile result in resistance to aphid infestation. Therefore we have undertaken an approach to study plant primary metabolism during aphid infestation. Comparing the metabolite profile of uninfested control and aphid-infested plants shows changes in a limited number of metabolites. We are now studying the role of these metabolites in resistance against M. persicae in detail. (This is a collaborative project with A. Fernie, J. Lunn, R. Feil, and M. Stitt at the Max Planck Institute for Molecular Plant Physiology (Golm, Germany), which is funded by NSF)

Glucosinolates are arguably the most important secondary metabolites for the defense of cruciferous plants against insect herbivores. Upon tissue rupture, myrosinase hydrolyzes glucosinolates, which results in the formation of toxic breakdown products. We use several mutants in Arabidopsis, impaired in the biosynthesis of glucosinolates, to study the effect of glucosinolates on generalist and specialist herbivores. Specialists, such as the small white cabbage butterfly (Pieris rapae) and the Diamondback moth (Plutella xylostella) have evolved ways to detoxify glucosinolates. It has been suggested that both species require glucosinolates as host recognition cues for egg-laying. The availability of mutants is an excellent resource to prove the in vivo role for glucosinolates in attraction of specialists. We find that mutants that are impaired in the biosynthesis of indole glucosinolates receive fewer eggs than wild-type plants (Pieris rapae and Plutella xylostella). This research was performed in collaboration with Ksenia Kriksunov, an Ithaca high school student, and Joel Sun (Cornell University Undergraduate) and Tanisha Robinson (REU Summer Intern - Norfolk State University).