1.1 The rosy apple aphid
Aphids are parasites of plants and, in an agricultural and horticultural context, are pests that reduce the quality and yield of produce. Sustainable control of UK pests is essential for a competitive UK industry. Of the five common aphids infesting apple, the rosy apple aphid (RAA, Dysaphis plantaginea) is the most serious as it causes severe economic loss to UK growers, through extensive fruit damage.
The current control strategy for this aphid is by spraying insecticides as soon, or even before, the pest is detected in the orchard. This is because the economic damage threshold is very low for this aphid, with few aphids causing extensive fruit deformation. Due to the damaging nature of this pest, the limited number of aphidicides compatible with IPM, and the high risk of resistance to such pesticides (a strain of RAA has already appeared in the south of France that is resistant to many aphidicides, including the main one pirimicarb), it is becoming necessary to focus on developing alternative techniques to control this aphid.
RAA has a host alternating holocyclic life cycle. It emerges from an egg in spring and spends early summer on apple, forming dense colonies and causing fruit and leaf malformation. Around mid-summer winged aphids are produced that fly to a herbaceous secondary host, plantain. The aphid remains on plantain for a few generations then migrates back to the primary host in autumn to sexually reproduce and lay overwintering eggs. It is unclear why some aphids host alternate and yet others, such as D. devecta (also an apple pest) do not. Various cues have been put forward as potential inducers of host alternation in the summer, including a range of abiotic factors (temperature; photoperiod) as well as biotic factors (crowding; host decline; host defence; maternal effects; predator/pathogen build-up). This migration provides two windows of opportunity to disrupt the aphid’s life cycle.
1.2 Plant-aphid interactions
During the course of evolution, plants have evolved a vast array of defence strategies to counteract aphid attack. These adaptations include surveillance genes, toxic secondary metabolites, and defence proteins which affect aphid host preference, longevity, development rate, fecundity and egg viability. Aphids have in response armed themselves with counter-defence strategies to overcome plant defences. These include evading detection, modifying toxin target sites, or by qualitative and or quantitative production of enzymatic detoxification systems.
Breeders have been able to identify some aphid resistance material in the apple (Malus) gene pool and have used it to breed varieties resistant to two apple aphid species (rosy leaf curling aphid and woolly aphid). However, there is only one commercially available apple cultivar that is sold as containing RAA resistance genes; a French dessert–apple called Florina (Rat-Morris, 1999). So far, it has proven impossible to identify sources of natural resistance to RAA that could be used for future commercial apple breeding.
This project attempts to seek out natural RAA resistance in the Malus gene pool and then study how aphids respond physically and genetically to this defence. Understanding the genetic responses will provide valuable data on the potential durability of any defence mechanism by helping make predictions about how the pest’s response might evolve. Information on sustainability would be invaluable when breeding difficult-to-replace perennial plants.
The vast majority of plant-aphid genetic interaction studies have focussed exclusively on the effect of the aphid on the host. (Qubbaj et al., 2005; Zhu-Salzman et al., 2004, 2008; Kehr, 2006; Smith and Boyko, 2007). These show that aphid feeding induces (possibly via elicitors in their saliva) a defence response characterised by a strong cellular salicylic acid rise and induction of pathogenesis-related (PR) proteins. This is similar to defences to pathogens and suggests aphids avoid triggering damage based defences. Chewing insects, on the other hand, have a defence characterised by the jasmonic acid (JA) pathway and release of protease inhibitors. There is, however, substantial variation in the extent of antagonistic and synergistic cross-talk between these phytohormone networks among published host-pest analyses. Thus, for each plant-insect system, assumptions regarding the important molecular events when developing control solutions should be avoided. JA and SA foliar treatments have already been proposed and tested as novel elicitors of plant resistance in pest management compatible with biocontrol by natural, with variable results on aphids (Cooper and Goggin, 2005; Boughton et al., 2006).
1.3 Genomic techniques
Genomic techniques are now becoming accessible to small-scale projects studying non-model organisms. This provides an opportunity to study the molecular background to questions of relevance to horticultural pest management.
All genomic profiling techniques (proteomics, lipidomics, metabolomics, transcriptomics) have their advantages and disadvantages (Feder and Walser, 2005).
A number of larger institutions have taken the genomic approach of sequencing all the genes being expressed in an aphid (Hunter et al., 2003; Sabater-Muñoz et al., 2006; Figueroa et al., 2007; Ramsey et al ., 2007) and its obligate bacterial symbiont Buchnera aphidicula (Shigenobu et al., 2000). Although essentially a cataloguing exercise, these sequences now deposited in GenBank are a very power resource to more focused projects, such as this one. Over 4 million sequences now exist in the NCBI trace archive from the pea aphid genome sequencing project. At the time of writing the EST database contains 120000 Acyrthosiphon pisum (pea aphid) sequences and 29000 Myzus persicae (green peach aphid) sequences.
A range of techniques now exist to profile the transcriptome (the actual genes being expressed as mRNA at a given point in time) of an organism to investigate adaptive gene expression changes involved in biological phenomena.
Most recent studies on aphid-plant interactions have used microarray technology (Voelckel et al., 2004; Wilson et al., 2006; Ramsey et al., 2007). Microarrays require prior knowledge of the sequence to be detected, which is then spotted onto a glass surface and probed with mRNA from the organism of interest. If the gene is expressed its mRNA will bind to the matching sequence on the glass. Problems with this technique are lack of sensitivity, nonspecific- and cross-hybridization, plus the need to select genes.
This project has not used microarray, mainly because the technology to undertake the protocol is not available at EMR. Also, the international databases were deplete of aphid sequences in 2005, so a meaningful screen of the RAA genome was impossible.
The method employed in this project is differential display reverse transcription (DDRT)-PCR, a technique pioneered and made by the GenHunter Corporation (www.genhunter.com). The concept of differential display is to use a limited number of short arbitrary primers in combination with anchored oligo-dT primers (to match the poly-A tail at the end of all eukaryotic mRNAs) to systematically amplify and visualize most of the mRNA in a cell. The amplified mRNAs from different samples can then be run side by side on a gel, so that up- and down-regulated genes can be detected. Bands (genes) can then be cut out of the gel and sequenced for identification. The advantage of using differential display for this project is that the technique can be carried out easily in a lab equipped with standard bench apparatus. Also, the technique makes no assumption about which genes would be assayed, so novel genes could be detected. The main pitfall levelled at this technique are the production of false positives. We have tried to avoid false positives by running replicate samples (never less than 3) of each ‘treatment’ and only characterised bands differentially expressed in all the replicates. Also, as many bands as was possible were characterised to produce a broad range of candidate markers.
Now that databases are filling up with aphid data from bulk sequencing projects, the mining of candidate genes is becoming easier. This should allow the development of gene-specific primer sets that can then be tested in modern real-time PCR machines for differential expression. However, the danger is that, like microarrays, assumptions are made as to what genes might be important before the investigation. At the end of this project an attempt was made to mine the GenBank aphid sequences and design degenerate primer sets to target detoxification genes directly in the RAA genome.
Quantitative RT-PCR is often used to support the findings of transcription profiling. Congruence between the methods is often low. False positives and contamination of probes are both cited as possible reasons. For example, RT-PCR primers will bind similar templates produced by alternatively splicing, gene duplications, paralogues genes and alleles. Primers designed close to the more variable non-coding 3’ region may be expected to be less likely to be prone to these ambiguities.
It is also apparent that transcript abundance may not necessarily correlate with protein abundance and activity, as there are many regulatory steps between mRNA abundance and protein activity. Interestingly, results presented here correlate well with genes discovered using a protein profiling assay on aphids (Francis et al., 2006).
Also, having found a gene expressed consistently under a specific regime, does not mean it turns out to be essential. This is because of the extensive redundancy in the phenome of organisms, as organisms can commonly compensate for perturbations in other gene networks.
In short, trancriptomics is a very powerful first step in highlighting candidate genes related to a particular biological system, but the extent of these gene’s impact on a pest’s fitness needs to be analysed further for redundancy.