Acyrthosiphon pisum: Who do you think y ou are?
Molecular characterisa.on and the dynamics of natural enemy resistance in pea aphid popula.ons Rebecca M. Cornwell
The James HuAon Ins.tute, Dundee and The University of Dundee Email: rebecca.cornwell@huAon.ac.uk
Introduc>on
Aphids are important in arable systems as herbivores, vectors for disease and as prey for parasites and parasitoids. The A. pisum (pea aphid) lifecycle is usually holocyclic with at least one genera.on of parthenogenesis (asexual reproduc.on) interspersed by a single genera.on of sexual reproduc.on over winter. During asexual reproduc.on, female aphids give birth to live nymphs containing the developing embryos of their daughters within their ovarioles that in turn harbour the developing embryos of their daughters. The evolu.onary advantage this life cycle is gene.c recombina.on leading to increased genotypic varia.on during sexual reproduc.on combined with maximum reproduc.ve output due to telescopic genera.ons during asexual reproduc.on. These factors lead to rapid popula.on growth under suitable environmental condi.ons with consequences for the life traits and popula.on dynamics of their natural enemies. The incidence of secondary faculta.ve endosymbio.c bacteria in pea aphid popula.ons may seriously impact aphid host and parasitoid wasp popula.on dynamics through increased natural enemy resistance. This affects the severity of aphid damage to crops and the success of biological control programmes. Resistance to parasi.sm is thought to be dependent on the presence of the Acyrthosiphon pisum secondary endosymbiont bacteriophage (APSE) in the Hamiltonella defensa endosymbiont1 but harbouring secondary endosymbionts may lead to fitness trade-‐offs in aphids2.
Right: Parasitoid wasp aAacking pea aphid (Image by A. Wild).
References 1Oliver et al (2009) Bacteriophages encode factors required for protec.on in a symbio.c mutualism. Science 325: 992-‐994; 2Gwynn et al (2005) Resistance is costly: trade-‐offs between immunity, fecundity and survival in the pea aphid. Proc. R. Soc. B., 272: 1803-‐1808; 3Caillaud et al (2004) Microsatellite DNA markers for the pea aphid Acyrthosiphon pisum. Molecular Ecology Notes 4: 446–448; 4Clarke, H. V. (2013) The role of secondary endosymbionts of Macrosiphum euphorbiae in the dynamics of mul.-‐trophic interac.ons. PhD Thesis. Acknowledgements This work is funded by The James HuAon Ins.tute and EPSRC. Special thanks go to Dr Ali Karley, Hannah Clarke and Professor Steve Hubbard for their help with this part of my research.
Methods
Sixteen pea aphid lines are held in culture on pre-‐flowering cu;ngs of the broad bean plant, Vicia faba, at The James HuAon Ins.tute. These lines have been collected from a variety of host plants from different geographical loca.ons in the UK over a number of years. The pea aphids are kept under summer condi.ons so that all offspring are clones with their microbacterial endosymbionts inherited by ver.cal maternal transmission. LeA: The pea aphid cultures Right: An asexually reproducing adult female pea aphid giving birth viviparously to parthenogene.c offspring. DNA extrac.on DNA was extracted from all aphid lines and samples from each culture frozen for future study and reference. Screening for endosymbio.c bacteria and the APSE bacteriophage Polymerase chain reac.on (PCR) produces high numbers of copies of a pre-‐selected region of a DNA molecule. DNA from all aphid lines was used as the target for amplifica.on of gene fragments in PCR assays using published primers to screen for the presence or absence of the obligate endosymbiont Buchnera, the secondary endosymbionts Serra0a symbio0ca, Hamiltonella defensa, Regiella insec0cola, Ricke<sia, Spiroplasma, PAXS and Ricke<siella and genes within the APSE bacteriophage including the P35 gene responsible for injec.ng phage DNA into symbiont host cells. Genotyping Published primers3 for polymorphic microsatellite loci in A. lo0 and A. pisum using were tested using pea aphid DNA from clonal lines known to vary in their collec.on year and loca.on. Five sets of primers were used to characterise the pea aphid lines by genotype using fluorescent primers and capillary electrophoresis. Product sizes were scored using GeneMapperTM soYware.
Key Results
The secondary endosymbionts Serra0a symbio0ca, Hamiltonella defensa, Regiella insec0cola, PAXS, Spiroplasma and Rickes<siella were detected in some lines. The Acyrthosiphon pisum secondary endosymbiont bacteriophage (APSE) was found in all lines harbouring H. defensa. Two lines harboured no secondary endosymbiont; eight lines harboured varying mul.ple endosymbiont infec.ons. Right: Results of a PCR amplifica.on separated by electrophoresis and visualised under ultraviolet light. Ini.al analysis of microsatellite data suggests sufficient varia.on at the five loci tested to discriminate between pea aphid clonal lines by genotype. There is more genotypic varia.on between pea aphids lines than potato aphid lines4 held at The James HuAon Ins.tute. This is due to field environmental condi.ons in the UK triggering sexual reproduc.on in pea aphids in winter whereas potato aphids in the UK reproduce asexually throughout the year.
Future Work
Colla.on of endosymbiont status, APSE status and genotype will allow for selec.on of suitable pea aphid lines in cage and field experiments to look at the costs and benefits of harbouring infec.on with respect to natural enemies in pea aphids. A key ques.on to be inves.gated, using a combina.on of experimental work and mathema.cal modelling, is whether it is beAer for an aphid to evolve its own genotypic resistance to parasitoids or whether it is beAer to recruit a microbe with the fitness costs which that entails.