Genetic variation in the aphid Pemphigus spyrothecae

This project used genetic variation to investigate dispersal, inbreeding and social behaviour in the aphid $\textit{Pemphigus spyrothecac}$. \textit{\textit{P. spyrothecae}} is cyclically parthenogenetic, reproducing sexually on the bark of its primary host, $\textit{Populus nigra}$, and asexually within galls on the leaf petioles. Within the gall, a soldier caste defends and cleans the gall, potentially reducing its own fitness. $\textit{P. spyrothecae}$ collected across the UK from 1997 to 1999 and from mainland Europe and America in 1999 were genotyped using seven variable microsatellite markers that I developed in collaboration with William Amos and Kate Llewellyn. Using population genetic analysis, I showed that $\textit{P. spyrothecae}$ populations were temporally stable over three years, and spatially structured. Populations from trees 5 to 1700 km apart were significantly differentiated, and loosely followed an isolation-by-distance model. There was slight evidence of differentiation between neighbouring trees (5 to 500 m apart), but not between samples taken from within trees (less than 5 m apart). By contrast, $\textit{P. bursarius}$, a closely related species that, unlike $\textit{P. spyrothecae}$, has a secondary host, showed no differentiation between populations 150 km apart, suggesting that population isolation in $\textit{P. spyrothecae}$ may be a consequence of losing its secondary host. Populations within trees were highly inbred, probably due to selfing between sexuales from the same clone. This finding corresponds with the theory that female-biased sex ratios in P. spyrothecae evolved through local mate competition. There was no evidence for a correlation between inbreeding and population density. Genetic variation was also found within galls. Of 633 aphids in one gall, 619 shared one genotype, while the remaining 14 were immigrants from at least nine other clones. One immigrant was found among 49 aphids from four other galls. Such a low level of clonal mixing probably favoured the evolution of soldiers, and may represent an investment in dispersal by the clone as an insurance against its death

Molekularna tipizacija tuniskih klonova vrste Myzus persicae (Hemiptera: Aphididae) pomoću mikrosatelitskih biljega

In order to assess the genetic differentiation among Tunisian clones belonging to the Myzus persicae complex (M. persicae (Sulzer), M. antirrhinii (Macchiati) and M. nicotianea Blackman), the molecular technique of microsatellites was used in this study. These markers offer sensitivity and are useful in population genetic studies of parthenogenetic organisms. Here, nine polymorphic microsatellite loci were amplified to distinguish between six parthenogenetic clones belonging to M. persicae complex collected from two different Tunisian areas. Interestingly, this technique allowed discrimination between five different genotypic classes among the six clones. Furthermore, analysis of genetic relatedness between the genotypic classes revealed that two Tunisian clones did not cluster either in M. persicae or in M. antirrhinii taxa, whereas, the four other Tunisian clones clustered into the M. persicae Sulzer taxa.U cilju utvrđivanja genetičkih razlika između tuniskih klonova kompleksa Myzus persicae (Sulzer), M. antirrhinii (Macchiati) i M. nicotianea (Blackman) upotrijebljena je molekularna mikrosatelitska tehnika. Ovi su biljezi vrlo osjetljivi i korisni u takvim istraživanjima partenogenetskih organizama. Umnoženo je devet polimorfnih lokusa mikrosatelita kako bi se razlikovalo šest partenogenetskih klonova kompleksa M. persicae sabranih u dva različita područja u Tunisu. Zanimljivo je da je ova tehnika omogućila razlikovanje između pet različitih genotipskih razreda tih šest klonova. Nadalje, analize genetičke srodnosti između genotipskih razreda pokazale su da dva tuniska klona nisu u istoj grupi niti s M. persicae, niti s M. antirrhinii, dok se četiri tuniska klona nalaze unutar vrste M. persicae Sulzer

Adaptive suicide : is a kin-selected driver of fatal behaviours likely?

Funding: Perry Foundation and the University of St Andrews.While several manipulated host behaviours are accepted as extended phenotypes of parasites, there remains debate over whether other altered behaviours in hosts following parasitic invasion represent cases of parasite manipulation, host defence or the pathology of infection. One particularly controversial subject is 'suicidal behaviour' in infected hosts. The host-suicide hypothesis proposes that host death benefits hosts doomed to reduced direct fitness by protecting kin from parasitism and therefore increasing inclusive fitness. However, adaptive suicide has been difficult to demonstrate conclusively as a host adaptation in studies on social or clonal insects, for whom high relatedness should enable greater inclusive fitness benefits. Following discussion of empirical and theoretical works from a behavioural ecology perspective, this review finds that the most persuasive evidence for selection of adaptive suicide comes from bacteria. Despite a focus on parasites, driven by the existing literature, the potential for the evolution of adaptive suicidal behaviour in hosts is also considered to apply to cases of infection by pathogens, provided that the disease has a severe effect on direct fitness and that suicidal behaviour can affect pathogen transmission dynamics. Suggestions are made for future research and a broadening of the possible implications for coevolution between parasites and hosts.PostprintPeer reviewe

The Soldiers in Societies: Defense, Regulation, and Evolution

The presence of reproductively altruistic castes is one of the primary traits of the eusocial societies. Adaptation and regulation of the sterile caste, to a certain extent, drives the evolution of eusociality. Depending on adaptive functions of the first evolved sterile caste, eusocial societies can be categorized into the worker-first and soldier-first lineages, respectively. The former is marked by a worker caste as the first evolved altruistic caste, whose primary function is housekeeping, and the latter is highlighted by a sterile soldier caste as the first evolved altruistic caste, whose task is predominantly colony defense. The apparent functional differences between these two fundamentally important castes suggest worker-first and soldier-first eusociality are potentially driven by a suite of distinctively different factors. Current studies of eusocial evolution have been focused largely on the worker-first Hymenoptera, whereas understanding of soldier-first lineages including termites, eusocial aphids, gall-dwelling thrips, and snapping shrimp, is greatly lacking. In this review, we summarize the current state of knowledge on biology, morphology, adaptive functions, and caste regulation of the soldier caste. In addition, we discuss the biological, ecological and genetic factors that might contribute to the evolution of distinct caste systems within eusocial lineages

Ecological patterns in plant defence chemistry and herbivore responses in natural populations of Brassica oleracea

Relationships between two taxonomic kingdoms; plants and herbivorous insects, are hypothesized to be a major zone of interaction for generating current day biodiversity; and coevolutionary processes between these intricately linked organisms are hypothesized to maintain diversity in plant secondary chemistry. These metabolites play a key role in plant defence against herbivory and a high degree of intraspecific variation is observed at multiple ecological scales. However, the nature of selection maintaining variation in plant defence profiles is still a major question in evolutionary biology and ecology, and progress towards a deeper understanding is hampered by a lack of studies that take into account ecological context and the multivariate nature of plant defence phenotypes. In this thesis, I employ sophisticated chemical analysis techniques to identify a suite of glucosinolate secondary chemicals, representing different biosynthetic pathways, in the wild cabbage, Brassica oleracea, in natural populations in the UK. I used model-based cluster analysis to explore patterns of association between individual glucosinolates, predicting that as simultaneous resource allocation to multiple defences is likely to be constrained; negative associations between defensive traits should be observed. However, results revealed positive associations between glucosinolates. Therefore co-expression of multiple defences may not be costly for this species. Using this information in conjunction with herbivore surveys and experiments, I show that this mixture has the potential to shape patterns of herbivore abundance and host plant utilization: species-specific responses to variation in glucosinolate phenotypes are discovered at various ecological scales. Thus there is the potential for differential selection on plant chemotypes though species-specific attractions and aversions. By conducting fine scale experiments with herbivore species, I also found that glucosinolate variation has an impact on the counter-adaptations that some brassica specialists have evolved: in order to optimally defend against their own natural enemies, Brevicoryne brassicae aphids sequestering glucosinolates from their host plants must do so selectively, and must choose plants whose chemical profile best matches this behaviour. These findings show that glucosinolate profiles may be under natural selection by herbivores in wild populations, and that reciprocal evolution between these plants and their specialists may continue to promote diversity in secondary metabolites. Together these results highlight the complexity inherent in plant-insect interactions, the importance of field studies and generate a wealth of testable hypotheses for future work.European Social Fund Riviera Produc

Olfactory learning of plant genotypes by a polyphagous insect predator

Olfactory learning may allow insects to forage optimally by more efficiently finding and using favourable food sources. Although olfactory learning has been shown in bees, insect herbivores and parasitoids, there are fewer examples from polyphagous predators. In this study, olfactory learning by a predatory coccinellid beetle is reported for the first time. In laboratory trials, adults of the aphidophagous ladybird Coccinella septempunctata did not prefer the odour of one aphid-infested barley cultivar over another. However, after feeding on aphids for 24 h on a cultivar, they preferred the odour of that particular cultivar. The mechanism appeared to be associative learning rather than sensitisation. Although inexperienced ladybirds preferred the odour of an aphid-infested barley cultivar over uninfested plants of the same cultivar, after feeding experience on a different cultivar this preference disappeared. This may indicate the acquisition and replacement of olfactory templates. The odour blends of the different aphid-infested barley cultivars varied qualitatively and quantitatively, providing a potential basis for olfactory discrimination by the ladybird. The results show that predatory coccinellids can learn to associate the odour of aphid-infested plants with the presence of prey, and that this olfactory learning ability is sensitive enough to discriminate variability between different genotypes of the same plant

Molecular evidence of intraclonal variation and implications for adaptational traits of grape phylloxera populations (Daktulosphaira vitifoliae, Fitch)

Grape phylloxera (Daktulosphaira vitifoliae Fitch; Homoptera: Phylloxeridae) is an economical important insect pest of grapevine (Vitis spp.) worldwide. The insect was introduced with contaminated plant material from North America in the 1850s and spread rapidly across all European viticultural regions. In the 19th century, nearly three-forths of the ungrafted and highly susceptible European grape species were destroyed by the insect pest. European viticulture did not recover until the development of grafting, combining European Vitis vinifera varieties with resistant rootstocks, bred from American Vitis species. Grape phylloxera is still present in viticulture. Today, grape phylloxera populations mainly persist in abandoned vineyards and rootstock nurseries. Grape phylloxera populations seem to be variable in terms of genotypic composition and host adaptability. The lifecycle described by Fitch (1854) and others in the 19th century does not seem to match actual conditions anymore. This thesis aimed at redefining the genetic structure of European grape phylloxera populations by employing genetic markers. It was shown, that the insect has turned away from its classical holocycle and now mainly reproduces asexually, as already demonstrated for Australian grape phylloxera populations. Despite asexual reproduction, all examined populations revealed a high grade of genotypic diversity. The reports on the emergence of new and more aggressive strains raised the question, how a population composed of asexually reproducing organisms would change and adapt to such an extent. Using a multilocus marker system, eight single founder lineages were genetically monitored over at least 15 generations. All lineages revealed a high grade of intraclonal variation. Sequencing of polymorphic fragments showed, that the genetic variation was not due to contaminating plant or bacterial DNA, but was due to variation within the insect genome. Furthermore, mutations occured already in early generations and were not observed to accumulate in later generations. Mutations were rather generated constantly and only few mutation specific markers were identified to be stable over all following generations. The here documentated genetic variation reveals the great adaptational potential of this insect pest. The adaptability of single founder lineages was further assessed by measuring physiological parameters in single isolation chambers in the greenhouse. Parameters as the number of surviving individuals per generation, the number of eggs or the number of ovarioles per generation exposed differences in performance among the lineages and also within the lineages a high grade of intraclonal variation. A direct correlation of specific multilocus markers and particularly adapted individuals or lineages was not possible in this assay. Two markers, though, were observed to occure in several lineages which performed well on the new host plant. These markers may be a first step to the development of adaptation-related markers and need to be tested on further populations and host plants. When analysing intraclonal variation, the question of putative contaminating factors within the system arises. Symbiotic bacteria occuring in nearly all aphid species certainly are the first to be suspected as a source of genetic variation among single individuals tested. Endosymbiotic bacteria, as Buchnera aphidicola in other aphid species, influencing nutritional condition and fitness of the insect population, were not identified in D. vitifoliae. A bacterium, closely related to Pantoea agglomerans, however, was identified in several grape phylloxera populations, using universal 16S rDNA primers and later specifically developed markers, which were also employed for in situ hybridization. The bacterium was localized in the salivary pump of D. vitifoliae. PCR analysis of in vitro reared populations revealed that the bacterium is present in root- and leaf-feeding parthenogenetic populations of grape phylloxera and, moreover, seems to be transmitted from generation to generation. In other insect species, this bacterium has been demonstrated to produce antifungal and antibacterial substances, which were also found in first in vitro tests with grape phylloxera associated bacteria. The insect may benefit from the antagonistic potential of these bacteria. P. agglomerans may be a further participant in the certainly complex interaction of grape phylloxera and grapevine. This thesis represents a broad approach to elucidate the development of grape phylloxera populations in Europe. Using new molecular marker systems, it has become possible to gain more information on the genetic structure of the insect and its adaptational potential. The predominant clonal reproduction mode of the insect confronts grapevine breeders and pest management with the task to continously develop new resistant rootstocks and to keep up with new pest management systems.Die Reblaus (Daktulosphaira vitifoliae Fitch; Homoptera: Phylloxeridae) ist ein wirtschaftlich bedeutender Schädling in allen weltweiten Weinbauregionen. Das aus Nordamerika stammende Insekt wurde Mitte des 19. Jahrhunderts nach Europa verschleppt und breitete sich rasant in allen europäischen Weinbaugebieten aus. Erst die Einführung von Pfropfreben - die Verbindung europäischer Edelreiser mit amerikanischen, wurzelresistenten Unterlagen - machte es möglich, die Rebflächen sicher wieder aufzureben. Die Reblaus ist trotzdem auch heute noch im Weinbau präsent. Sie vermehrt sich insbesondere an ausgetriebenen Unterlagen (sogenannten Drieschen) und in Unterlagsschnittgärten. Ausserdem wurde in den letzten Jahrzehnten häufiger vom Befall bisher als resistent eingestufter Unterlagen neuen und aggressiveren Reblausstämmen berichtet. Reblauspopulationen scheinen in Hinsicht auf ihre Wirtsanpassungsfähigkeit und ihre genetische Zusammensetzung sehr variabel zu sein. Der im 19. Jahrhundert beschriebene Lebenszyklus entspricht sicher nicht mehr den aktuellen Gegebenheiten. Ein wichtiges Ziel dieser Arbeit war daher, die genetische Struktur europäischer Reblauspopulationen mit Hilfe genetischer Marker neu zu definieren. Es zeigte sich, wie auch schon in anderen Weinbaugebieten, z.B. Australien nachgewiesen, dass sich die Reblaus vorwiegend asexuell vermehrt und nicht mehr den klassischen holozyklischen Lebenszyklus aufweist. Trotz der klonalen Vermehrungsweise konnte in den untersuchten Populationen eine sehr grosse genotypische Diversität nachgewiesen werden. Die Verbreitung neuer und agressiverer Stämme warf die Frage auf, wie sich eine aus klonalen Organismen bestehende Population derart schnell verändern und anpassen kann. Mit Hilfe eines Multilocus-Markersystems wurden klonale Linien über mindestens 15 Generationen genetisch analysiert. Alle Linien zeigten einen hohen Grad an intraklonaler genetischer Variation. Die Sequenzierungen polymorpher Fragmente zeigten, dass die genetischen Unterschiede innerhalb eines Klons nicht von kontaminierender bakterieller oder pflanzlicher DNS herrührten, sondern insekteneigene, genetische Variationen darstellten. Weiterhin wurde festgestellt, dass sich die bereits in frühen Generationen auftretenden Mutationen zum grossen Teil nicht bis in spätere Generationen akkumulierten. Vielmehr wurden kontinuierlich neue Mutationen generiert und nur einige wenige etablierten sich fest in den folgenden Generationen. Die hier zum ersten Mal auf diese Weise dokumentierte genetische Variabilität klonaler Reblauslinien macht das Adaptationspotential dieses Schädlings deutlich. Die Anpassungsfähigkeit einzelner klonaler Linien wurde zusätzlich mit Hilfe von einfach messbaren physiologischen Parametern im Gewächshaus ermittelt. An Parametern, wie der Anzahl überlebender Individuen pro Generation, der Anzahl Eier oder auch der Anzahl Ovariolen konnte eine intraklonale Variation bei klonalen Linien nachgewiesen werden. Die Identifikation zweier häufig auftretender Marker ist ein erster Schritt zur Entwicklung adaptationsspezifischer Marker, die dann an weiteren Populationen getestet werden sollen. Bei der Analyse intraklonaler Variation liegt die Vermutung nahe, dass symbiontische Bakterien, wie sie bei fast allen Aphiden vorkommen, eine wichtige Kontaminationsquelle sein könnten. Endosymbiontische Bakterien, wie Buchnera aphidicola bei anderen Blattlausarten, welche direkten Einfluss auf Ernährung und Fitness der Insektenpopulationen haben, konnten bei der Reblaus nicht identifiziert werden. Jedoch wurde ein dem Bakterium Pantoea agglomerans nah verwandtes Bakterium in allen untersuchten Reblauspopulationen nachgewiesen. Das Bakterium wurde in der Speichelpumpe von D. vitifoliae lokalisiert. PCR Analysen von in vitro Populationen zeigten, dass das Bakterium nicht nur in Blattpopulationen, sondern auch bei Wurzelläusen vorkommt und von Generation zu Generation übertragen wird. In anderen Insektenarten konnte eine antifungale und antibakterieller Funktion bereits nachgewiesen werden. Erste in vitro Tests bestätigten dies auch für das hier identifizierte Bakterium. Es ist vorstellbar, dass die Reblaus vom antagonistischen Potential dieses Bakteriums profitiert. P. agglomerans könnte somit ein weiterer interessanter Faktor in der komplexen Rebe-Reblaus Interaktion sein. Die vorliegende Arbeit stellt einen Ansatz zur Aufklärung des Verhaltens klonaler Reblauspopulationen in Europa dar. Mit Hilfe neuer molekularer Methoden ist es möglich geworden, neue Informationen zur genetischen Struktur dieses Schadinsekts zu gewinnen und seine Anpassungsfähigkeit zu beobachten. Die klonale Vermehrungsweise stellt den Rebschutz vor die Aufgabe, das Verhalten dieses sich rasant entwickelnden Schädlings genau zu beobachten und die Entwicklung resistenter Unterlagen und neuer Pflanzenschutzmassnahmen voranzutreiben

Parasitoids: metapopulation ecology and genetics, mating behaviour, and aphid host resistance

Das Verstehen von multitrophischen Interaktionen zwischen Parasitoiden und Blattläusen ist von theoretischem und praktischem Interesse. In dieser Arbeit wurden folgende Aspekte untersucht: 1) Metapopulationsdynamik und Genetik eines spezialisierten Parasitoiden und dessen Blattlaus-Wirtes, 2) Paarungsverhalten von Parasitoiden am Schlupfort und deren Beeinflussung durch Interaktionen mit Ameisen, und 3) Resistenzentwicklung von Blattläuse und deren Auswirkung auf die Fitness von Parasitoiden. Lysiphlebus hirticornis ist ein solitärer Parasitoid der die Blattlaus Metopeurum fuscoviride parasitiert, die ihrerseits auf Rheinfarn, Tanacetum vulgare, spezialisiert ist. T. vulgare kommt typischerweise in einer sehr lückenhaften Verteilung vor. Populationen von L. hirticornis folgen einer klassischen Metapopulationsdynamik, geprägt durch Prozesse des Aussterbens und der Wiederbesiedlung. Dieses tritrophische System wird verwendet um i) die genetische Differenzierung von L. hirticornis auf einer kleinen räumlichen Skala zu überprüfen und daraus das Ausbreitungspotenzial abzuleiten, ii) die Veränderung von Allelhäufigkeiten während einer Saison zu bestimmen und um iii) zu analysieren, wie das System unter hohem Parasitoidendruck fortbestehen kann. Dazu wurden L. hirticornis infizierte Blattlausmumien alle zwei Wochen zwischen Juni und Oktober 2007 an 11 verschiedenen Standorten von unterschiedlichen T. vulgare Pflanzen im Feld gesammelt. Die Standorte lagen in Distanzen von 217m bis 15000m voneinander entfernt. Die Phänologie der Pflanzen und die Populationsdynamik von M. fuscoviride und L. hirticornis wurden jeweils aufgenommen. Die DNA, von im Labor geschlüpften Parasitoiden, wurde zur Bestimmung des Genotyps extrahiert. Neun polymorphische Mikrosatelliten-Marker konnten von L. hirticornis isoliert werden und zwei weitere wurden in einem Cross-Amplification-Screening von bereits bekannten Markern zweier anderer Lysiphlebus Arten gefunden. Insgesamt wurden 11 polymorphische Mikrosatelliten für die populationsgenetische Arbeit verwendet. L. hirticornis Blattlausmumien konnten im Feld an allen Sandorten gefunden werden. Auf einigen Pflanzen-Geneten wurden ausschließlich parasitierte Blattläuse gefunden, wohingegen Blattlauskolonien anderer Geneten gänzlich von Parasitierungen verschont blieben. Die Bestimmung der Genotypen zeigt, dass sich die Anzahl der Allele der verschiedenen Standorte, unabhängig von der Stichprobengröße unterscheidet. Parasitoiden-Populationen verschiedener Standorte grenzten sich genetisch signifikant voneinander ab. Ein hoher Prozentsatz der Allel-Variation konnte durch den Pflanzen-Geneten erklärt werden, was darauf hindeutet dass die Einheit der Differenzierung eher der Genet als der Standort sein könnte. Diese Resultate bestätigen, dass L. hirticornis nur eine geringe Ausbreitung und einen hohen Grad an Inzucht aufweist. Eine räumliche Isolierung der Populationen konnte nicht nachgewiesen werden. Während der Saison nahm die Allelvielfalt ab und die Heterozygotie war im Sommer zunehmend und gegen das Ende der Saison abnehmend. Die Differenzierung der Parasitoiden-Populationen nahm tendenziell mit jener der Blattläuse zu, wobei die Blattlaus-Populationen jeweils stärker differenziert waren. Dies deutet darauf hin, dass der Differenzierungsprozess der Parasitoiden dem der Blattläuse hinterherhinkt. Somit könnte in Kombination mit Variationen in der Phänologie der Pflanzen und Heterogenität der Allele das gemeinsame Fortbestehen von Parasitoiden und Blattläusen erklärt werden