Dissertations / Theses on the topic 'Insects, vectors, virus diseases'

La Fièvre catarrhale ovine (FCO) est une arbovirose émergente en Europe depuis la fin desannées 90. Elle affecte principalement les ruminants par la piqûre de petits moucheronshématophages, les Culicoides (Diptera : Ceratopogonidae). Pendant l’été 2006, l’introductiondu sérotype 8 de la FCO, dans la région de Maastricht (Pays-Bas) a rapidement diffusé dansles Ardennes, générant de lourdes pertes pour les éleveurs de bovins et d’ovins. Cesévènements interrogent sur la capacité des Culicoides de la région paléarctique à transmettrela FCO. Ils révèlent la nécessité de mieux connaître la biologie de ces diptères.Nous avons développé successivement dans ce travail, trois axes de recherche qui se sontappuyés sur un travail de terrain réalisé principalement au sein de deux élevages situés dansles Ardennes françaises.Dans un premier temps, nous avons réalisé une expérimentation de gorgement de Culicoidesde captures et d’émergences, provenant des Ardennes, sur petits ruminants virémiques pour leBTV8. A l’issue des expérimentations, une femelle gorgée de l’espèce Culicoides obsoletus apondu et a été retrouvée faiblement positive lors de la recherche du génome du virus de laFCO. Les résultats obtenus ainsi que les difficultés rencontrées lors de la réalisation de cetype d’expérimentation sont discutés.Le deuxième travail exposé s’est intéressé au comportement trophique des Culicoides parl’étude de l’origine du repas sanguin de femelles de Culicoides piégées dans des biotopesvariés. A cette fin, nous avons utilisé des marqueurs moléculaires pour amplifier l’ADN devertébré présent dans les estomacs de femelles gorgées. Ces analyses ont permis de mettre enévidence que des espèces appartenant aux complexes Obsoletus, Pulicaris, ou encore,Culicoides dewulfi, avaient un spectre d’hôte large. Certaines d’entre elles peuvent se gorger àla fois sur les ruminants domestiques et sur la faune sauvage. De plus, ce type d’étuderenseigne sur l’écologie des différentes espèces de Culicoides.Enfin, nous présentons les résultats d’une étude faunistique fondée sur des captures avec despièges lumineux, mais aussi, des prélèvements de boue pour la recherche des gîtes larvaires.Les résultats de piégeages entre les deux exploitations ont été comparés, notamment en termesde biodiversité, et sont discutés en regard des différences de pratiques d’élevage entre lesdeux exploitations choisies d’une part, et la mise en évidence des gîtes larvaires d’autre part.De nombreuses espèces de Culicoides ont émergé au laboratoire à partir des prélèvements deboues, qui ont été caractérisés macroscopiquement. Les gîtes larvaires de C. obsoletus, peuconnus jusqu’alors, ont été mis en évidence dans les deux fermes. Ils ont fait l’objet d’un suivisur plusieurs mois.L’ensemble de ces études contribue à la meilleure connaissance des Culicoides présents dansles Ardennes et de leur biologie, elles permettent de rendre compte des espèces qui semblenttrès inféodées à l’élevage de bovins, et celles qui sont plus ubiquistes. Certains travauxprésentés pourraient être poursuivis pour mettre en évidence les espèces ou populations deCulicoides plutôt sylvatiques, et pour mettre en place de nouvelles expérimentations sur lacompétence et la capacité vectorielle des Culicoides
Since the late 90’s, Bluetongue disease (BT) can be considered as an emerging arbovirose inEurope. This disease is mainly transmitted to ruminants by the bites of minute size midges,the Culicoides (Diptera: Ceratopogonidae), also known as biting midges. An outbreak of BTserotype 8 occurred during summer 2006, in the region of Maastricht (Netherlands) andspread quickly to the Ardennes region. The epizooty lead to severe losses in cattle and sheepholdings. These events highlighted the lack of knowledge on the vectorial capacity ofpaleartic Culicoides species, and more generally on their biology.Three approaches are successively treated in this document. They are all based on field workconducted mainly in two holdings located in the Ardennes region.First, an experiment to assess oral susceptibility of Culicoides to Bluetongue virus (BTV) 8was undertaken. Field collected and emerging Culicoides coming from the Ardennes wereengorged on viremic small ruminants. At the end of the experiments, one Culicoides obsoletusfemale was found bloodfed and laid eggs. She was tested for BTV and was found weaklypositive for BTV genome. This result and the difficulties met during the experiment havebeen discussed.The second study focused on the bloodmeal origin of engorged females of Culicoides. Thesewere collected by light traps set in different kinds of environment. Molecular markers wereused in order to amplify the DNA of vertebrates present in the stomach of bloodfed females.Some of the species processed belonging to the Obsoletus or the Pulicaris complex, andCulicoides dewulfi fed on a wide variety of hosts, including domestic ruminants and wildanimals. Moreover, this kind of study brings information on the ecology of different speciesof Culicoides.Finally, a faunistic survey is presented. It was achieved through light trap collections ofmidges and also thanks to the sampling of potential breeding sites. Biodiversity in thecollection of midges captured by light traps between the two holdings were compared.Differences observed are discussed taking into account the differences in breeding practicesbetween the two holdings and the breeding sites investigations. Numerous species ofCulicoides emerged in the laboratory from soil samples which were macroscopicallydescribed. Breeding sites of C. obsoletus, which were not well documentated in the literature,were found in both farms. These were monitored over some months.This work contributes to a better knowledge of the Culicoides present in the Ardennes andtheir biology. It highlights the species which are closely related to the cattle holdingenvironment, and those which are ubiquist. Some of these studies could be continued in orderto highlight the species more related to the forested areas, and to set new experiments onvectorial competence and capacity

Des pullulations d’aleurodes du complexe d'espèces cryptiques de Bemisia tabaci ont été associées à la propagation de deux maladies frappant le manioc en Afrique orientale: la maladie de la mosaïque du manioc (CMD) et, plus récemment (2000), la maladie de la striure brune du manioc (CBSD). Parmi les espèces d’aleurodes de ce complexe, l’espèce SSA2 a été associée à la première épidémie de CMD au cours des années 1990 en Ouganda. Cependant, SSA2 aurait été remplacée par SSA1 dans les années 2000, provoquant une recrudescence de CMD et de CBSD, participant à leur propagation dans plusieurs pays voisins. L’hypothèse défendue à ce jour expliquant la propagation de ces maladies vers le sud et l'ouest de l'Afrique incrimine cette nouvelle espèce considérée comme émergente dans certains de ces pays. Dans ma thèse, j’ai utilisé des données écologiques et des approches moléculaires afin de mieux comprendre les facteurs à l'origine des pullulations de vecteurs en Afrique de l'Est. Nous avons ainsi analysé : i) l’abondance, la diversité et la répartition des espèces sur un transect comprenant : Ouganda, Tanzanie et Malawi, ii) la diversité génétique et la structure des populations actuelles des espèces de B. tabaci, iii) des échantillons des années 90 comparés aux populations actuelles (2017). Cette étude nous a permis d’avoir une image d’une situation plus complexe qu’attendue, en effet, l’espèce SSA1 a été détectée comme à l’origine dans certaines des pullulations observées mais également d’autres espèces, notamment IO et SSA1-SG3 ont aussi montrées cette capacité. Les pullulations observées ne sont donc pas uniquement liées à une seule espèce en Afrique de l’Est. En outre, nous avons pu montrer que la communauté d'espèces et sa diversité génétique diffère d'un pays à l'autre, impliquant des situations épidémiologiques différentes, sans aucun schéma d'invasion détecté entre pays. En outre, l’analyse des anciens échantillons n’a pas montré l’implication d’une nouvelle espèce ou population en 20 ans, toutefois, nous avons observé un changement de dynamique au sein des groupes génétiques représentés au cours du temps
High population of the whitefly, Bemisia tabaci Gennadius, a cryptic species complex had been associated with the vectoring and spread of viruses causing two diseases of cassava in East Africa: the cassava mosaic disease (CMD) and cassava brown streak disease (CBSD). Among the B. tabaci species, sub-Saharan Africa 2 (SSA2) was the vector associated with an epidemic of CMD since the 1990s in Uganda. However, this species is now replaced by the SSA1 and led to development of another epidemic by CBSD since the mid 2000s. The spread of both diseases toward South and West Africa is feared with this new supposed invader. In my thesis I have used ecological data and molecular approaches (mitochondrial and nuclear markers) to better understand the factors driving the presence of the superabundant whitefly populations on cassava in East Africa. We have analyzed: i) species abundance, diversity and distribution (geographic and host plants) along a transect survey over three East African countries: Uganda, Tanzania, Malawi, ii) the genetic diversity and structure of current populations of B. tabaci species, and iii) comparing genetic changes between the old and new populations collected in 1997 and 2017, respectively.This study involving large number of samples provided insights of a more complex picture than expected. SSA1 was found to be the source of the some observed outbreaks although other species, notably IO and sub-group 3 of SSA1 (SSA1-SG3) have also shown this capability. The observed outbreaks are therefore not just related to a single species in East Africa. In addition, we showed that the species community and its genetic diversity differ from one country to another, involving different epidemiological situations, without any clear pattern of invasion detected between the countries. Analysis of old samples did not show the involvement of a new species or the emergence of a new population in 20 years, although the dynamics within the whitefly genetic groups was observed over time. Our results contributed new knowledge on the super abundant populations on cassava in Eastern Africa and help develop targeted control measures for the local populations

The risks posed by rapidly evolving RNA viruses to human and animal health are well recognized. Epidemics in managed and wildlife populations can lead to considerable economic and biodiversity losses. Yet, we lack understanding of the ecological and evolutionary factors that promote disease emergence. Host-switching viruses may be a particular threat to species important for human welfare, such as pollinating bees. Both honeybees and wild bumblebees have faced sharp declines in the last decades, with high winter mortality seen in honeybees. Infectious and emerging diseases are considered one of the key drivers of declines, acting in synergy with habitat loss and pesticide use. Here I focus on multihost viruses that pose a risk to wild bumblebees. I first identify the risk factors driving viral spillover and emergence from managed honeybees to wild bumblebees, by synthesising current data and literature. Biological factors (i.e. the nature of RNA viruses and ecology of social bees) play a clear role in increasing the risk of disease emergence, but anthropogenic factors (trade and transportation of commercial honeybees and bumblebees) creates the greatest risk of viral spillover to wild bees. Basic knowledge of the pathogenic effect of many common pollinator viruses on hosts other than A. mellifera is currently lacking, yet vital for understanding the wider impacts of infection at a population level. Here, I provide evidence that a common bumblebee virus, Slow bee paralysis virus (SBPV), reduces the longevity of Bombus terrestris under conditions of nutrition stress. The invasion of Varroa destructor as an ectoparasitic viral vector in European honeybees has dramatically altered viral dynamics in honeybees. I test how this specialist honeybee vector affects multi-host pathogens that can infect and be transmitted by both honeybees and wild bumblebees. I sampled across three host species (A. mellifera, B. terrestris and B. pascuorum) from Varroa-free and Varroa-present locations. Using a combination of molecular and phylogenetic techniques I find that this specialist honeybee vector increases the prevalence of four multi-host viruses (deformed wing virus (type A and B), SBPV and black queen cell virus) in sympatric wild bumblebees. Furthermore, wild bumblebees are currently experiencing a DWV epidemic driven by the presence of virus-vectoring Varroa in A. mellifera. Overall this thesis demonstrates that wild bumblebees are at high risk of viral disease emergence. My research adds to the ever-expanding body of evidence indicating that stronger disease controls on commercial bee operations are crucial to protect our wild bumblebees.

Providence virus (PrV) is a member of the Tetraviridae, a family of small, positive sense, single-stranded RNA viruses, which characteristically infect the midgut tissue of heliothine larvae. PrV is the only known tetravirus that replicates in cultured insect cells. The virus comprises a monopartite genome resembling members of the genus Betatetravirus with the capsid precursor protein undergoing autoproteolytic cleavage at its C-terminus consistent with other tetravirus capsid precursor proteins. Analysis of viral cDNA predicted the presence of three potential overlapping gene products (from 5` to 3`): (1) p130, a protein of unrecognized nucleotide or amino acid homology with a 2A-like processing site at its N-terminus; (2) p104, the replicase ORF, which was found to be phylogenetically related to tombus-and umbraviruses replicases. The presence of a read-through stop signal in the p104 ORF was proposed to produce and amino terminal product with a predicted MW of 40 kDa (p40) and (3) the capsid protein precursor (81 kDa) which has two 2A-like processing sites at its N-terminus. Metabolic radiolabelling of viral translation products in persistently infected MG8 cells and in vitro translation of the individual ORFs were performed in order to analyse the expression of PrV gene products. p130 was translated with no evidence of 2A-like processing. Two products of 40 kDa and 104 kDa were translated from the p104 ORF, indicating that the read-through stop signal was likely to be functional. Finally, the capsid protein precursor ORF produced a major translation product of 68 kDa corresponding to the capsid protein precursor as well a peptide of 15 kDa that was attributed to the activity of the second 2A-like site at the N-terminus of the p81 ORF. The subcellular distribution of viral RNA (vRNA) and p40 in MG8 cells was investigated using immunofluorescence and biochemical fractionation. The results showed that p40/p104 and vRNA accumulated in polarized, punctate structures in some but not all MG8 cells and in some cases, co-localization was observed. This thesis concludes that PrV is a novel tetravirus with significant similarities plant carmolike viruses that should be re-classified at the family level.

A cultura do tomateiro (Solanum lycopersicum L.) é importante mundialmente devido ao alto consumo de seus frutos. Nos últimos anos surgiram nesta cultura no Brasil alguns vírus emergentes com altas taxas de disseminação, como begomovírus e crinivírus, transmitidos pela Bemisia tabaci biótipo B, que podem causar danos à produção do tomateiro. A espécie de begomovírus atualmente mais encontrada no Brasil, em plantios de tomateiro, é o Tomato severe rugose virus (ToSRV). De 2002 a 2004, pesquisadores relataram incidências desse vírus em mais da metade das amostras com sintomas de geminiviroses coletadas em vários estados brasileiros e sua presença continua sendo verificada frequentemente. No ano de 2006, um crinivírus, o Tomato chlorosis virus (ToCV), foi relatado no Brasil, infectando plantas de tomate no Estado de São Paulo e atualmente encontra-se presente em diveros estados brasileiros. Os objetivos desse trabalho foram: determinar os períodos mínimos de acesso à aquisição e à inoculação do ToSRV e do ToCV pela B. tabaci biótipo B; identificar o período de retenção do ToSRV no inseto e a interação do ToSRV e do ToCV na aquisição e na transmissão por esse aleirodídeo. Também foi avaliada a eficiência do inseticida cloridrato de cartape no controle da disseminação primária e secundária do ToSRV pela B. tabaci biótipo B em tomateiros em gaiolas em casa de vegetação. Finalmente avaliou-se a eficiência do aleirodídeo Trialeurodes vaporariorum na transmissão de um isolado brasileiro do ToCV. Os períodos mínimos de acesso à aquisição e à inoculação de ambos os vírus pela B. tabaci biótipo B foram de cinco minutos. O tempo de retenção do ToSRV em B. tabaci biótipo B foi de 25 dias. A eficiência de um único adulto de B. tabaci na transmissão simultânea do ToSRV e do ToCV para tomateiros foi de 44,7%, similar àquela da transmissão isolada do ToRSV (47,4%) e do ToCV (44,7%). A eficiência de T. vaporariorum na transmissão do ToCV foi inferior à da B. tabaci biótipo B. Usando 40 insetos por vaso com duas plantas as eficiências de transmissão foram 57,7% e 100%, respectivamente. O inseticida cloridrato de cartape reduziu a infecção secundária do ToSRV pela B. tabaci biótipo B, mas não foi eficiente para reduzir a infecção primária em tomateiros.
Tomato (Solanum lycopersicum) is one of the leading vegetables grown and consumed in Brazil and in the world, after potato. The importance of tomato is related to its high consumption worldwide and also its nutritive value. Presently the most important virus diseases responsible for yield losses on tomato crops in Brazil are those caused by begomovirus and crinivirus, both transmitted by Bemisia tabaci biotype B. At the moment the prevalent species of begomovirus is Tomato severe rugose virus (ToSRV). From 2002 to 2004, researchers reported incidence of this virus in more than half of the symptomatic tomato samples collected in several Brazilian states. In 2006, a crinivirus, Tomato chlorosis virus (ToCV), was reported for the first time in Brazil, infecting tomato plants in the State of São Paulo and at present the virus occurs in several Brazilian states. The objectives of this study were to determine the minimum acquisition and inoculation access periods of ToSRV and ToCV by B. tabaci biotype B; identify the retention period of ToSRV in the insect; and the interaction of ToSRV and ToCV on the transmission by this aleyrodidae. It was also evaluated the effectiveness of the insecticide cartap hydrochloride in controlling the primary and secondary spread of ToSRV by B. tabaci biotype B on tomato plants in a greenhouse. Finally, it was evaluated the efficiency of Trialeurodes vaporariorum in the transmission of a Brazilian isolate of ToCV. The minimum acquisition and inoculation access periods for both viruses by B. tabaci biotype B were five minutes. The maximum retention time of ToSRV in B. tabaci biotype B was 25 days. The efficiency of a single adult of B. tabaci to simultaneously transmit ToSRV and ToCV to tomato plants was 44.7%, similar to the transmission of ToRSV (47.4%), and ToCV (44.7%) separately. T. vaporariorum was less efficient than B. tabaci on the transmission of ToCV. Using 40 insects per pot with two plants, transmission efficiencies were 57.7% and 100%, respectively. The insecticide cartap hydrochloride reduced secondary infection of ToSRV transmitted by B. tabaci biotype B, but was not effective in reducing the primary infection in tomato.

Potomac Horse Fever (PHF) is a disease of great concern to many horse owners in the Potomac River area of Maryland and Virginia. It is caused by a rickettsia, Ehrlichia risticii. The involvement of an arthropod vector has been suspected because of the seasonal epidemiology of the disease. This research was an attempt to identify and evaluate potential arthropod vectors. A seasonal activity study of biting arthropods attacking horses in endemic areas of Maryland and Virginia identified five potential vectors: (1) Simulium jenningsi (Diptera: Simuliidae), (2) Stomoxys calcitrans (Diptera: Muscidae), (3) Culicoides obsoletus (Diptera: Ceratopogonidae), (4) C. variipennis, and (5) Dermacentor variabilis (Acari: Ixodidae). These five arthropod species were given status as potential vectors because they were collected feeding on horses just prior to and throughout the PHF season. Simulium jenningsi and D. variabilis have the closest seasonal association with the occurrence of PHF as presented in this study. D. variabilis was determined to have the greatest potential due to its reported association with other rickettsial diseases. A series of laboratory and field studies were designed to examine the potential role of D. variabilis in the transmission of E. risticii. We first attempted to transmit E. risticii by feeding adult D. variabilis collected from an endemic farm on susceptible horses. Other laboratory studies included mouse to horse and mouse to mouse transmission attempts using ticks fed on mice inoculated with E. risticii. A serological survey of 105 trapped field rodents (host of immature D. variabilis) on endemic farms in Maryland showed all specimens collected to be negative for PHF antibodies. These studies and others gave no indication of D. variabilis's involvement in the transmission of the disease in nature. The other species mentioned above were not examined.
Ph. D.

Title from PDF of title page (University of Missouri--Columbia, viewed on Feb 19, 2010). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Thesis advisor: Dr. James E. Schoelz. Includes bibliographical references.

Thesis (MSc)--University of Stellenbosch, 2004.
ENGLISH ABSTRACT: Viruses are some of the most important pathogens of grapevines. There are no effective chemical treatments, and no grapevine- or other natural resistance genes have been discovered against grapevine infecting viruses. The primary method of grapevine virus control is prevention by biological indexing and molecular- and serological screening of rootstocks and scions before propagation. Due to the spread of grapevine viruses through insect vectors, and in the case of GRSPaV the absence of serological screening, these methods of virus control are not always effective. In the past several methods, from cross-protection to pathogen derived resistance (PDR), have been applied to induce plant virus resistance, but with inconsistent results. In recent years the application of post-transcriptional gene silencing (PTGS), a naturally occurring plant defense mechanism, to induce targeted virus resistance has achieved great success. The Waterhouse research group has designed plant transformation vectors that facilitate specific virus resistance through PTGS. The primary focus of this study was the production of virus specific transformation vectors for the introduction of grapevine virus resistance. The Waterhouse system has been successfully utilised for the construction of three transformation vectors with the pHannibal vector as backbone. Each vector contains homologous virus coat protein (CP) gene segments, cloned in a complementary conformation upstream and downstream of an intron sequence. The primary vector (pHann-SAScon) contains complementary CP gene segments of both GRSPaV and GLRaV-3 and was designed for the introduction of multiple-virus resistance. For the construction of the primary vector the GRSPaV CP gene was isolated from RSP infected grapevines. A clone of the GLRaV-3 CP gene was acquired. The second vector (pHann- LR3CPsas) contains complementary CP gene segments of GLRaV-3. The third vector (pHann-LR2CPsas) contains complementary CP gene segments of GLRaV-2. The cassette containing the complementary CP gene segments of both GRSPaV and GLRaV-3 was cloned into pART27 (pART27-HSAScon), and used to transform N tabacum cv. Petit Havana (SRI), through A. tumefaciens mediated transformation. Unfortunately potential transformants failed to regenerate on rooting media; hence no molecular tests were performed to confirm transformation. Once successful transformants are generated, infection with a recombinant virus vector (consisting of PYX, the GFP gene as screenable marker and the complementary CP gene segments of both GRSPaV and GLRaV-3) will be used to test for the efficacy of the vectors to induce resistance. A secondary aim was added to this project when a need was identified within the South African viticulture industry for GRSPaV specific antibodies to be used in serological screening. To facilitate future serological detection of GRSPaV, the CP gene was isolated and expressed with a bacterial expression system (pETI4b) within the E. coli BL2I(DE3)pLysS cell line. The expressed protein will be used to generate GRSPaV CP specific antibodies.
AFRIKAANSE OPSOMMING: Virusse is van die belangrikste patogene by wingerd. Daar bestaan geen effektiewe chemiese beheer nie, en geen wingerd- of ander natuurlike weerstandsgene teen wingerdvirusse is al ontdek nie. Die primêre metode van beheer t.o.v. wingerdvirusse is voorkoming deur biologiese indeksering, en molekulêre- en serologiese toetsing van onderstokke en entlote voor verspreiding. As gevolg van die verspreiding van wingerdvirusse deur insekvektore, en in die geval van GRSPa V die tekort aan serologiese toetsing, is dié metodes van virusbeheer nie altyd effektief nie. In die verlede is metodes soos kruis-beskerming en patogeen-afgeleide weerstand (PDR) gebruik om virusweerstand te induseer, maar met inkonsekwente resultate. In onlangse jare is post-transkripsionele geenonderdrukking (PTGS), 'n natuurlike plantbeskermingsmeganisme, met groot sukses toegepas om geteikende virusweerstand te induseer. Die Waterhouse-navorsingsgroep het planttransformasievektore ontwerp wat spesifieke virusweerstand induseer d.m.v. PTGS. Die vervaardiging van virus spesifieke tranformasievektore vir die indusering van wingerdvirusweerstand was die primêre doelwit van hierdie studie. Die Waterhouse-sisteem was gebruik vir die konstruksie van drie transformasievektore, met die pHannibal vektor as basis. Elke vektor bevat homoloë virus kapsiedproteïen (CP) geensegmente, gekloneer in 'n komplementêre vorm stroom-op en stroom-af van 'n intronvolgorde. Die primêre vektor (pHann-SAScon) bevat komplementêre CP geensegmente van beide GRSPaV en GLRaV-3, en was ontwerp vir die indusering van veelvoudige-virusweerstand. Die CP-geen van GRSPa V was vanuit RSP-geïnfekteerde wingerd geïsoleer, vir die konstruksie van die primêre vektor. 'n Kloon van die GLRa V-3 CP-geen was verkry. Die tweede vektor (pHann-LR3CPsas) bevat komplementêre CP geensegmente van GLRaV-3. Die derde vektor (pHann-LR2CPsas) bevat komplementêre CP geensegmente van GLRa V-2. Die kasset bestaande uit die komplementêre CP geensegmente van beide GRSPaV en GLRaV-3, was gekloneer in pART27 (pART27-HSAScon), en gebruik om N tabacum cv. Petit Havana (SRI) te transformeer d.m.v. A. tumefaciens bemiddelde transformasie. Ongelukkig het potensiële transformante nie geregenereer op bewortelingsmedia nie; gevolglik was geen molekulêre toetse gedoen om transformasie te bevestig nie. Na suksesvolle transformante gegenereer is, sal infeksie met 'n rekombinante-virusvektor (bestaande uit PYX, die GFP geen as waarneembare merker en die komplementêre CP geensegmente van beide GRSPa V en GLRa V-3) gebruik word om die effektiwiteit van die vektore as weerstandsinduseerders te toets. 'n Sekondêre doelwit is by die projek gevoeg toe 'n behoefte aan GRSPaV spesifieke teenliggame binne die Suid-Afrikaanse wynbedryf geïdentifiseer is, vir gebruik in serologiese toetsing. Om toekomstige serologiese toetsing van GRSPa V te bemiddel, was die CP-geen geïsoleer en in 'n bakteriële uitdrukkingsisteem (PETI4b) uitgedruk, in die E. coli BL21(DE3)pLysS sellyn. Die uitgedrukte proteïne sal gebruik word vir die vervaardiging van GRSPa V CP spesifieke antiliggame.

Very long chain acyl-coA dehydrogenase (VLCAD) is the rate-limiting step in mitochondrial fatty acid oxidation. VLCAD deficient mice and patients’ clinical symptoms stem from not only an energy deficiency but also long-chain metabolite accumulations. VLCAD deficient mice were treated systemically with 1x10 12 vector genomes of rAAV9-VLCAD. Expression was detected in the liver, heart and muscle. Also substantial expression of VLCAD was noted in the brain, where it was expressed across different sections of the brain and in different cell types with different morphologies. Biochemical correction was observed in vector-treated mice beginning two weeks post-injection, as characterized by a significant drop in long chain fatty acyl accumulates in whole blood after an overnight fast. Changes persisted through the termination point around 20 weeks post injection. Magnetic resonance spectroscopy (MRS) and tandem mass spectrometry (MS/MS) revealed normalization of intramuscular lipids in treated animals. Correction was not observed in liver tissue extracts, but cardiac muscle extracts showed significant reduction of long chain metabolites. Disease-specific phenotypes were characterized, including thermoregulation and maintenance of euglycemia after a fasting cold challenge. Internal body temperatures of untreated VLCAD-/- mice dropped below 20°C and the mice became lethargic, requiring euthanasia. In contrast all rAAV9-treated VLCAD-/- mice and the wild-type controls maintained body temperatures. rAAV9-treated VLCAD-/- mice maintained euglycemia, whereas untreated VLCAD-/- mice suffered hypoglycemia following a fasting cold challenge. These promising results suggest rAAV9 gene therapy as a potential treatment for VLCAD deficiency in humans.

GM1 gangliosidosis is a lysosomal storage disorder caused by a deficiency in the catabolizing enzyme β-galactosidase (βgal). This leads to accumulation of GM1-ganglioside (GM1) in the lysosome inducing ER stress and cell death. GM1 gangliosidosis is primarily a disorder of the central nervous system (CNS) with peripheral organ involvement. In this work we report two major findings, 1) systemic treatment of GM1 gangliosidosis with an adenoassociated virus (AAV9) encoding mouse-βgal (mβgal) in a GM1 gangliosidosis mouse model (βGal-/-), and 2) an investigation into an intracranial injection of a therapeutic AAVrh8 encoding mβgal. Systemic treatment of GM1 gangliosidosis with AAV9 resulted in a moderate expression of enzyme in the CNS, reduction of GM1 storage, significant retention of motor function and a significant increase in lifespan. Interestingly, the therapeutic effect was more robust in females. Intracranial injections of AAVrh8 vector expressing high levels of βgal resulted in enzyme spread throughout the brain, significant retention of motor function and a significant increase in lifespan. Histological alterations were also found at the injection site in both βGal-/- and normal animals. We constructed a series of vectors with a range of decreasing enzyme expression levels to investigate the cause for the unanticipated result. Microarrays were performed on the injection site and we showed that a lower expressing AAVrh8-mβgal vector mitigated the negative response. Intracranial injection of this newly developed vector was shown to clear lysosomal storage throughout the CNS of βGal-/- mice. Taken together, these studies indicate that a combined systemic and fine-tuned intracranial approach may be the most effective in clearing lysosomal storage completely in the CNS while providing therapeutic benefit to the periphery.

Hepatitis C virus (HCV) is a major global pathogen estimated to infect over 170 million people worldwide. A recent study has shown that vaccination with adenoviral vectors, based on rare human and simian serotypes encoding the non-structural (NS) proteins of HCV, induces highly potent, multi-specific and durable T cell responses in healthy human volunteers. In this thesis I assess the safety and immunogenicity of these vaccines (ChAd3–NSmut and Ad6-NSmut), for the first time in HCV infected patients. This work also explores whether vaccine-induced T cell responses target in vivo circulating HCV antigens and common naturally occurring epitope variants. Patients with treatment naive chronic genotype 1 HCV infection were vaccinated (i.m.) with ChAd3-NSmut and Ad6-NSmut in a heterologous prime boost schedule, either with or without current IFN and ribavirin (IFN/RBV). Epitope-specific T cell responses were defined by fine mapping using HCV peptides. Circulating viral genomic sequence was determined in vaccinated patients at baseline and at any point of viral relapse. Cross-reactivity of vaccine-induced T cell responses was determined in T cell assays, using peptides corresponding to both circulating host virus and common population HCV epitope variants. An in vitro dendritic cell /T cell priming model was used to identify possible candidates for a cross-reactive vaccine immunogen at the most immunodominant epitope, NS3₁₄₀₆. 33 patients were vaccinated. Vaccination was well tolerated. At the highest vaccine dose (2.5 x 10¹⁰vp) vaccine-induced T cell responses were detectable in 11/20 patients receiving concurrent IFN/RBV and 2/4 patients receiving vaccination alone. In total 14 antigenic targets were identified, 2 of which have not previously been described. However, T cell responses were of lower magnitude and more narrowly focused than those observed in healthy volunteers vaccinated with the same regimen. Analysis of viral sequence showed that in many cases vaccine-induced T cells did not target the circulating virus. At the most immunodominant epitope (NS3₁₄₀₆), T cells induced by vaccination failed to target common circulating genotype 1 HCV variants. An in vitro model suggested that in order to target all genotype 1 sequences at this epitope, it would be necessary to insert both a genotype 1a and 1b version of this epitope into a vaccine immunogen. Vaccination with adenoviral vectors induces T cell responses in patients with chronic HCV infection, however immune responses are attenuated compared with healthy volunteers. Ultimately a successful therapeutic or prophylactic vaccine strategy will rely on inducing responses that target conserved or cross-reactive epitopes.

The overall objective of this study was to test the hypothesis that insects can vector F. tumidum conidia to infect gorse plants with the aim of developing an alternative approach to mycoherbicide delivery to control weeds. Four potential insect species (Apion ulicis, Cydia ulicetana, Epiphyas postvittana and Sericothrips staphylinus) were assessed for their ability to vector F. tumidum conidia. To achieve this, the external microflora (bacteria and fungi) and the size and location of fungal spores on the cuticle of these insect species were determined. In addition, the ability of the insects to pick up and deposit F. tumidum conidia on agar was studied. Based on the results from these experiments, E. postvittana was selected for more detailed experiments to determine transmission of F. tumidum to infect potted gorse plants. The factors promoting pathogenicity of F. tumidum against gorse and the pathogen loading required to infect and kill the weed were also determined. The external microflora of the four insect species were recovered by washing and plating techniques and identified by morphology and polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and sequencing of internally transcribed spacer (ITS) and 16S rDNA. A culture-independent technique (direct PCR) was also used to assess fungal diversity by direct amplification of ITS sequences from the washings of the insects. All insect species carried Alternaria, Cladosporium, Nectria, Penicillium, Phoma, Pseudozyma spp. and entomopathogens. Ninety four per cent of the 178 cloned amplicons had ITS sequences similarity to Nectria mauritiicola. E. postvittana carried the largest fungal spores (mean surface area of 125.9 ìm2) and the most fungal CFU/insect. About 70% of the fungi isolated from the insects were also present on the host plant (gorse) and the understorey grass. The mean size of fungal spores recovered from the insect species correlated strongly with their body length (R² = 85%). Methylobacterium aquaticum and Pseudomonas lutea were common on all four insect species. Pseudomonas fluorescens was the most abundant bacterial species. In the pathogenicity trials, the effectiveness of F. tumidum in reducing root and shoot biomass of 16 and 8 wk old gorse plants was significantly increased with wounding of the plants. Older plants (32 wk old) which were wounded and inoculated were significantly shorter, more infected and developed more tip dieback (80%) than plants which were not wounded (32%). This indicates that damage caused by phytophagous insect species present on gorse through feeding and oviposition may enhance infection by F. tumidum. Wounding may release nutrients (e.g. Mg and Zn) essential for conidia germination and germ tube elongation and also provide easier access for germ tube penetration. Conidial germination and germ tube length were increased by 50 and 877%, respectively when incubated in 0.2% of gorse extract solution for 24 h compared with incubation in water. Inoculum suspensions amended with 0.2% of gorse extract caused more infection and significantly reduced biomass production of 24 wk old gorse plants than suspensions without gorse extract. A minimum number of about 900 viable conidia/infection site of F. tumidum were required to infect gorse leaves. However, incorporation of amendments (which can injure the leaf cuticle) or provision of nutrients (i.e. gorse extract or glucose) in the formulation might decrease the number of conidia required for lesion formation. Scanning electron micrographs showed that germ tube penetration of gorse tissue was limited to open stomata which partly explain the large number of conidia required for infection. The flowers and leaves were more susceptible to F. tumidum infection than the spines, stems and pods. An experiment to determine the number of infection sites required to cause plant mortality showed that the entire plant needs to be inoculated in order for the pathogen to kill 10 wk old plants as F. tumidum is a non systemic pathogen. The number of infection sites correlated strongly with disease severity (R² = 99.3%). At least 50% of the plant was required to be inoculated to cause a significant reduction in shoot dry weight. F. tumidum, applied as soil inoculant using inoculated wheat grains in three separate experiments, significantly suppressed gorse seedling emergence and biomass production. In experiments to determine the loading capacity of the insect species, E. postvittana, the largest insect species studied, carried significantly more (68) and deposited significantly more (29) F. tumidum conidia than the other species. Each E. postvittana, loaded with 5,000 conidia of F. tumidum, transmitted approximately 310 conidia onto gorse plants but this did not cause any infection or affect plant growth as determined by shoot fresh weight and shoot height. E. postvittana on its own did not cause any significant damage to gorse and did not enhance F. tumidum infection. It also failed to spread the pathogen from infected plants to the healthy ones. There was no evidence of synergism between the two agents and damage caused by the combination of both E. postvittana and F. tumidum was equivalent to that caused by F. tumidum alone. This study has shown that E. postvittana has the greatest capacity to vector F. tumidum since it naturally carried the largest and the most fungal spores (429 CFU/insect). Moreover, it naturally carried Fusarium spp. such as F. lateritium, F. tricinctum and Gibberella pulicaris (anamorph Fusarium sambucinum) and was capable of carrying and depositing most F. tumidum conidia on agar. Coupled with the availability of pheromone for attracting the male insects, E. postvittana may be a suitable insect vector for delivering F. tumidum conidia on gorse using this novel biocontrol strategy. Although it is a polyphagous insect, and may visit non-target plants, F. tumidum is a very specific pathogen of gorse, broom and a few closely related plant species. Hence, using this insect species to vector F. tumidum in a biological control programme, should not pose a significant threat to plants of economic importance. However, successful control of gorse using this "lure-load-infect" concept would depend, to a large extent on the virulence of the pathogen as insects, due to the large size of F. tumidum macroconidia, can carry only a small number of it.

Abstract :Bluetongue (BT) is a vector-borne infectious disease primarily transmitted to even- toed ungulates by the bite of several Culicoides species. The global distribution of BT can be attributed to the ubiquity of its vectors and its rapid spread, likely to the enhancement of human activities (intensification of animal production, trans- port, changing habitat). During the last decades, BT established in Southern Europe and more recently emerged in Northern Europe, causing the death of millions of domestic ruminants. On the same time, a Belgian research project has been set up to develop remote-sensing tools to study the EPidemiology and Space-TIme dynamicS of infectious diseases (EPISTIS). In that general framework, this thesis aimed to study the space-time distribution of the main Culicoides vectors occurring in Italy and Belgium, at two different scales. Firstly, we aimed to clarify the role of several eco-climatic factors on the regional-scale distribution of C. imicola in time, based on weekly samplings achieved throughout Italy from 2001 to 2006 and to develop an easy-to-use and reproducible tool, which could be widely validated on the basis of former vector sampling and freely accessible remote-sensing data. Secondly, we aimed to investigate how Culicoides species were distributed in the fine-scale habitat encountered throughout the agro-ecological landscapes of Belgium, while recent studies have suggested that the landscapes configuration could explain the spatial distribution of BT. In the first part, we showed that an autoregressive model where the observed monthly growth rate is predicted by monthly temperature, allowed predicting >70% of the seasonal variability in C. imicola trap catches. The model predicted the seasonality, the altitudinal gradient, and the low populations’ activity taking place during the winter. Incorporating eco-climatic indices such as the Normalized Difference Vegetation Index into the model did not enhance its predictive power. In the second part, we quantified how Culicoides populations are spatially structured in the neighbourhood of farms, and demonstrated the unexpectedly high level of population found in forest. We also showed how four classes of land use could influence the relative abundances of Culicoides species in the agro-ecological landscapes of Belgium. Although in summer, BT vectors were abundant in each of the four classes investigated, their relative abundances varied strongly as a function of sex, species and environmental conditions, and we quantified these variations. Finally, we also presented a new method to quantify the interference between Onderstepoort light traps, and used it to measure their range of attraction for several of the most common BT vectors species in Northern Europe. The model developed on C. imicola in Italy provided enthusiastic perspectives regarding the regional-scale analyses of its distribution in time, although further improvements are nevertheless required in order to assess the broad scale ecology of BT vectors throughout Europe. Mapping the abundances of C. imicola in Sardinia high- lighted an important lack of reliability attributable to the many land use classes that are currently not sampled in the vector surveillance achieved across Europe. Together with the novelties presented in the second part and the recent findings establishing that BT could circulate among wild hosts in both epidemiological systems (i.e. in Southern and Northern Europe), we call for increasing epidemiological and entomo- logical studies at the interface between farms and the surrounding natural habitats. Last, depicting in time the landscape-scale findings for Northern Europe highlighted how dramatic could be the role played by intensive farming practices to maintain BT within the agro-ecological landscapes studied and to facilitate its circulation between them. Quantifying the amplitude of the risk of disease transmission linked to these practices would require a further complex modeling approach accounting simultaneously for the diel activity of hosts, mainly resulting from the farming activities, the diel activities of different vector species and the landscapes configuration found in contrasted agro-ecological systems.

Résumé :La fièvre catarrhale ovine (FCO), encore appelée maladie de la langue bleue, est une maladie infectieuse des ruminants transmise par la piqûre d’un vecteur de type moucheron appartenant au genre Culicoides (Diptera :Ceratopogonidae). L’ubiquité de ses vecteurs peut expliquer son succès d’installation à l’échelle globale. Par ailleurs, sa rapide expansion a été grandement facilitée par l’importante activité anthropique (élevage, transport, modification de l’habitat) et peut-être même par les changements climatiques globaux. La FCO a été récemment qualifiée de maladie infectieuse émergente en Europe du fait de (i) son récent établissement dans la région, bien au delà de son aire de répartition traditionnelle, (ii) de sa forte capacité de dispersion affectant chaque jour un nombre plus important d’hôtes et enfin (iii) de sa forte virulence. Après avoir détaillé les caractéristiques majeures des deux principaux foyers de FCO rencontrés en Europe depuis 1998, la présente thèse s’est plus particulièrement intéressée à l’étude de la distribution spatio-temporelle de ses principaux vecteurs dans le sud (partie 1) puis dans le nord (partie 2) de l’Europe, à différentes échelles. Dans la première partie, un modèle discret, spatialement et temporellement explicite, a été développé afin de mesurer l’influence de différents facteurs éco-climatiques sur la distribution de Culicoides imicola, principal vecteur de la FCO dans le Bassin Méditerranéen. Les profils mensuels de distribution rencontrés en Sardaigne durant 6 années consécutives ont ainsi pu être reconstitués, principalement sur base de la température. Une cartographie de l’abondance de C. imicola sur le territoire a permis de mettre à jour le manque d’information sur sa distribution en dehors des exploitations agricoles. Dans la deuxième partie du travail, nous nous sommes penchés sur la distribution spatiale des Culicoides tels qu’on peut les rencontrer au sein de différents paysages agro-écologiques de Belgique. Nous avons ainsi pu décrire la structure adoptée par les populations de Culicoides au voisinage des fermes ainsi que quantifier l’importante population présente dans les forêts avoisinantes. Nous avons par ailleurs montré l’influence de différentes catégories d’utilisation du sol sur l’abondance et la composition en espèces. Enfin, nous avons présenté une méthode permettant de quantifier l’interférence entre des pièges lumineux utilisés dans un même paysage pour échantillonner les populations, et l’avons utilisé afin de mesurer leur rayon d’attractivité sur les espèces vectrices les plus communément rencontrées dans le nord de l’Europe. En guise de conclusion générale et conjointement aux récentes découvertes de cas de FCO au sein de la faune sauvage européenne, nous appelons à réaliser un plus grand nombre d’études éco-épidémiologiques à l’interface entre exploitations agricoles et zones (semi-) naturelles avoisinantes. En outres, les résultats présentés dans la seconde partie ont été mis en relation avec le mode de fonctionnement journalier de nos exploitations agricoles. Nous avons ainsi pu déduire le rôle dramatique joué par les pratiques agricoles intensives dans le maintien du virus de la FCO au sein de nos paysages agro-écologiques, ainsi que dans sa circulation d’un paysage à l’autre. Un cadre de modélisation complexe permettant une analyse simultanée de l’activité nycthémérale des hôtes de la FCO et de ses vecteurs Culicoides en fonction de la configuration des paysages agro-écologiques est néanmoins requis afin de quantifier l’amplitude du risque de transmission de la FCO lié aux pratiques agricoles intensives.
Doctorat en Sciences agronomiques et ingénierie biologique
info:eu-repo/semantics/nonPublished

Bibliography: leaves 129-137
viii, 137 leaves, [27] leaves of plates : ill. (some col.) ; 30 cm.
Title page, contents and abstract only. The complete thesis in print form is available from the University Library.
Thesis (Ph.D.)--Dept. of Entomology, Waite research Institute, University of Adelaide, 1986

African horse sickness virus causes a non-contagious, infectious disease of equids. It is epizootic to sub-Saharan Africa and parts of the Middle East. The epizootics caused by the virus have caused widespread devastation amongst equids worldwide. Fortunately no epizootic has lasted more than 5 years outside of sub- Saharan Africa. It is vectored by species of Culicoides midges (Diptera: Ceratopogonidae) and most importantly by the two Avarita species of C. imicola Keiffer and C. bolitinos Meiswinkel. The literature pertaining to the study and research of the virus, the disease and the vectors is reviewed. Models allowing prediction of future possible outbreaks as well as details of control strategies and findings of researchers are presented and discussed. The virus needs a long term reservoir host in which to overwinter and various theories are discussed. Control measures in South Africa are suggested so that outbreaks of the disease can be reduced.
Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermarizburg, 2008.

Peppermint, Mentha piperita 'Black Mitcham' was established as a host for tomato spotted wilt virus-impatiens serotype (TSWV-I). TSWV-I infection of peppermint, initially observed in a research greenhouse (Corvallis, OR), included stunting and downward curling of leaves accompanied by bronzing, and occasionally tip necrosis. Young leaves appeared either symptomless, deformed, or pale even under high nitrogen conditions. Older leaves had sunken, brownish-grey lesions. Bright yellow mottling was observed on newly mature deep green leaves. A begonia isolate of TSWV-I was transmitted to peppermint both mechanically and by western flower thrips, Frankliniella occidentalis (Pergande). Symptoms of TSWV-I infection were similar although only a faint yellow mottling was produced and only under cool temperatures (15°C). ELISA detection of virus distribution throughout the plant indicated infection was systemic. Bulked groups of thrips (5 thrips/sample) also tested positive for TSWV-I using ELISA. Transmission efficiency of 4, 6, 8, and 10 day old thrips adults given acquisition sources during the entire nymphal stage varied from 0-40% for thrips tested in pairs and 0-20% for single thrips (based on a 12 hour access feeding period). Adults 2 days old failed to transmit the virus. Western flower thrips exposed to TSWV-I had reduced survival and reproductive potential and slower development rates than unexposed thrips. Virusexposed thrips were 1.4 times as likely to die than unexposed thrips on a given day. Both individual and population reproductive potentials were significantly lower. Preoviposition period was extended in virus-exposed thrips. Development time from second instar to adult was 15% longer for virus-exposed thrips. This is the first report of altered population parameters in western flower thrips exposed to TSWV-I.
Graduation date: 1992

Viral diseases are a major limiting factor to cowpea production in many countries of Africa. In Uganda, studies indicated that the cowpea aphid-borne mosaic virus (CABMV) is common and a potential threat to cowpea production in the region. There have been no efforts to develop cowpea cultivars with resistance to CABMV in Uganda. This work focused on the development of cultivars resistant to CABMV. Production of cowpea in Uganda is constrained by several factors, including a lack of awareness of diseases among the majority of farmers. A participatory rural appraisal (PRA) was conducted to elicit farmers’ indigenous knowledge of cowpea production and also to gain insight into their understanding of viral diseases affecting cowpea in Uganda. PRA tools such as group discussions, transect walks, problem listing and ranking were used to gather information. Insect pests, diseases, low yielding cultivars and the high cost of pesticides were perceived to be the most important production constraints. Farmers were not aware of the problem of virus diseases, but provided descriptive names of symptoms. Only three cowpea cultivars (Ebelat, Ecirikukwai and Blackcowpea) were produced in the area. Seed size and colour were seen as important traits in new varieties. Information about the occurrence, distribution and identity of cowpea viruses is limited in Uganda. The objective of this study was to identify the important cowpea virus diseases occurring naturally in the major cowpea growing regions of Uganda. Surveys were conducted to determine the incidence and severity of virus symptoms in four districts (Soroti, Kumi, Pallisa and Tororo) in 2004 and 2005. The incidence ranged from 40.5 to 94.4% and severity ranged from 15.0 to 30.6% (for Kumi and Pallisa districts, respectively) during the 2004 surveys. In 2005, the incidence ranged from 55.9 to 85.4% and severity ranged from 4.7 to 14.5% (for Tororo and Soroti districts, respectively). The CABMV, cowpea mild mottle virus (CPMMV), cowpea severe mosaic virus (CPSMV) and cucumber mosaic virus (CMV) were serologically detected by double antibody sandwich enzyme- linked immunosorbent assay (DAS-ELISA). Fifty four improved cowpea genotypes were screened for resistance to CABMV during the first season of 2004 at Serere Agricultural and Animal Production Research Institute in Uganda. Further screening was conducted in the second season of 2004 using 27 genotypes. The genotypes were planted in single rows between the rows of the susceptible cultivar, Ebelat. This was to provide high pressure of aphid vector (Aphis craccivora Koch) and CABMV inoculum. In addition, the test genotypes were artificially inoculated with a CABMV extract on fully expanded primary leaves of fourteen day-old seedlings. The CABMV incidence and severity was assessed. Disease severity was assessed on a 0-60% visual estimation scale where 0 = with no symptoms and 60 = with severe symptoms. Serological analysis was conducted using DAS-ELISA. Five genotypes showed good levels of resistance to CABMV, namely MU-93, IT82D-889, IT82D-516-2, IT85F-2841 and SECOW-2W. These resistant lines were crossed with three susceptible local landraces, namely Ebelat, Ecirikukwai and Blackcowpea in a North Carolina II mating design. The F1, F2 and BC1F1 populations and the parents were evaluated in the field to assess the response to CABMV and to study the inheritance of resistance to CABMV. The general combining ability (GCA) and specific combining ability (SCA) effects were significant, indicating that both additive and non-additive genetic factors are important in determining the control of CABMV in cowpea. The proportions (%) of the sum of squares for crosses attributable to GCA and SCA for CABMV severity were 51.4% for GCA due to females, 8.4% for GCA due to males and 40.2% for the SCA. The narrow-sense heritability estimates, obtained by regressing F1 on mid-parents was 0.87 and 0.84, F2 on F1 progenies 0.49 and 0.48, and F2 progenies on mid-parents 0.63 and 0.79, for AUDPC and final disease severity, respectively. Single gene conditioned resistance in seven populations, but resistance was quantitatively inherited and involved many genes in eight populations. Observation of transgressive segregation and moderate to high heritability suggests a quantitative mode of gene action and the importance of additive effects. The predominance of GCA variance, high heritability estimates and observation of transgressive segregation suggested that resistance could be improved by selection.
Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2007.

Το βακτήριο Wolbachia είναι ένα ενδοκυττάριο και μητρικά κληρονομούμενο συμβιωτικό βακτήριο. Ανήκει στην ομοταξία των Alphaproteobacteria και την τάξη των Rickettsiales. Αποτελεί ίσως τον πιο διαδεδομένο ενδοκυττάριο συμβιωτικό οργανισμό στον πλανήτη, καθώς έχει εντοπιστεί μέχρι στιγμής σε πληθώρα αρθροπόδων και νηματωδών της φιλαρίασης. Πρόσφατες μελέτες εκτιμούν ότι πάνω από το 40% των ειδών αρθροπόδων είναι μολυσμένα με το βακτήριο Wolbachia. Το συμβιωτικό αυτό βακτήριο επηρεάζει τις βιολογικές λειτουργίες και ιδιότητες των ξενιστών του και είναι υπεύθυνο για μια σειρά αναπαραγωγικών ανωμαλιών, όπως η κυτταροπλασματική ασυμβατότητα, η παρθενογένεση, η θανάτωση των αρσενικών εμβρύων και η θηλυκοποίηση. Τα μοναδικά αυτά βιολογικά χαρακτηριστικά του βακτηρίου Wolbachia προσελκύουν όλο και περισσότερο το ενδιαφέρον διαφόρων ερευνητών τόσο για το ρόλο του βακτηρίου σε εξελικτικές διαδικασίες (κυρίως ειδογένεση) όσο και για τη χρησιμοποίησή του σε περιβαλλοντικά φιλικές εφαρμογές καταπολέμησης οργανισμών που είναι επιβλαβείς στους τομείς του γεωργικού και δασικού περιβάλλοντος, και της υγείας. Τα είδη του γένους Glossina (Diptera: Glossinidae), γνωστά και ως μύγες τσε-τσε, αποτελούν ξενιστές του βακτηρίου Wolbachia. Η μύγα τσε-τσε είναι ο σημαντικότερος φορέας των παθογόνων τρυπανοσωμάτων στην τροπική Αφρική, τα οποία προκαλούν την ασθένεια του ύπνου (sleeping sickness) στον άνθρωπο και την αντίστοιχη τρυπανοσωμίαση, γνωστή ως nagana, στα ζώα. Η χρησιμοποίηση του βακτηρίου Wolbachia σε μεθόδους βιολογικής καταπολέμησης της μύγας τσε-τσε προαπαιτεί την πλήρη γνώση της γενετικής του ταυτότητας και των αλληλεπιδράσεων του με το ξενιστή. Προς την κατεύθυνση αυτή, και στα πλαίσια της παρούσας διατριβής, πραγματοποιήθηκε η ανίχνευση του συμβιωτικού βακτηρίου Wolbachia σε περισσότερα από 5300 άτομα από φυσικούς και εργαστηριακούς πληθυσμούς 11 διαφορετικών ειδών μύγας τσε-τσε από 13 Αφρικανικές χώρες. Τα αποτελέσματα έδειξαν τεράστια απόκλιση της παρουσίας του βακτηρίου τόσο μεταξύ ειδών όσο και μεταξύ πληθυσμών του ίδιου είδους. Επίσης, πραγματοποιήθηκε ο γενετικός χαρακτηρισμός των στελεχών Wolbachia από συνολικά 29 αντιπροσωπευτικά δείγματα διαφόρων πληθυσμών και ειδών μύγας τσε-τσε, ενώ σε αρκετά από αυτά παρατηρήθηκαν πολλαπλά στελέχη του βακτηρίου. Διαπιστώθηκε εντυπωσιακή γενετική ποικιλότητα στελεχών Wolbachia που απαντούν στα διάφορα είδη μύγας τσε-τσε καθώς και ασυμφωνία μεταξύ των φυλογενειών των στελεχών Wolbachia και των μυγών τσε-τσε ξενιστών της, γεγονός που σημαίνει οριζόντια μετακίνηση του συμβιωτικού βακτηρίου κατά την εξέλιξη. Επιπρόσθετα, εντοπίστηκαν για πρώτη φορά εκτεταμένα γεγονότα οριζόντιας μεταφοράς βακτηριακών γονιδίων στο γονιδίωμα τριών ειδών μύγας τσε-τσε: στο Glossina morsitans morsitans, Glossina pallidipes και Glossina austeni. Από εξελικτικής σκοπιάς, κρίσιμα ερωτήματα προκύπτουν από τα παραπάνω ευρήματα, και πιο συγκεκριμένα σχετικά με: την προέλευση-μηχανισμό αυτών των γεγονότων οριζόντιας μεταφοράς, τον χρονικό προσδιορισμό τους, τον πιθανό ρόλο τους σε διαδικασίες ειδογένεσης και την επιλεκτική εμφάνισή τους σε ορισμένα μόνο είδη Glossina π.χ. στo υποείδos Glossina morsitans centralis που είναι πολύ συγγενικό του Glossina morsitans morsitans δεν παρατηρήθηκε το φαινόμενο. Εξίσου σημαντική και επιβεβλημένη κρίνεται η διεξοδική διερεύνηση του ενδεχομένου τα βακτηριακά γονίδια που ενσωματώθηκαν στο ευκαρυωτικό γονιδίωμα της μύγας τσε-τσε να ευθύνονται για την έκφραση νέων λειτουργιών-ιδιοτήτων (ή να μεταβάλλουν τις ήδη υπάρχουσες), ιδίως μάλιστα εάν αυτές συνδέονται με την αποδοτικότητα μετάδοσης της νόσου της τρυπανοσωμίασης μέσω του φορέα της, δηλαδή της μύγας τσε-τσε. Τέλος, διαπιστώθηκε πιθανή αρνητική συσχέτιση της παρουσίας του βακτηρίου Wolbachia με τον παθογόνο ιό Salivary Gland hypertrophy Virus (SGHV), γεγονός που συζητείται στα πλαίσια βιολογικών εφαρμογών καταπολέμησης του εντόμου-φορέα και της τρυπανοσωμίασης. Παράλληλα, μεγάλο ενδιαφέρον παρουσιάζει η προοπτική χρησιμοποίησης του βακτηρίου Wolbachia για τη βιολογική καταπολέμηση εντόμων αγροτικής ή /και περιβαλλοντικής σημασίας, όπως είναι οι αφίδες και η καρπόκαψα καστανιάς. Το γεγονός αυτό προϋποθέτει την ανίχνευση και τη γενετική ταυτοποίηση του βακτηρίου σε φυσικούς πληθυσμούς εντόμων. Στα πλαίσια της παρούσας διατριβής πραγματοποιήθηκε ανίχνευση και χαρακτηρισμός του βακτηρίου Wolbachia σε 78 συνολικά άτομα από 22 είδη αφίδων, από 26 φυσικούς πληθυσμούς από την Ελλάδα. Από αυτούς τους 26 πληθυσμούς, μόλις οι 4 βρέθηκαν να είναι μολυσμένοι με το βακτήριο Wolbachia και συγκεκριμένα πληθυσμοί των ειδών: Aphis fabae, Aphis hederae, Metopolophium dirhodum και Baizongia pistaciae. Τα αποτελέσματα αυτά δείχνουν για πρώτη φορά ότι η παρουσία του βακτηρίου Wolbachia στις αφίδες είναι πιθανά πιο διαδεδομένη από ότι προέκυπτε από προηγούμενες μελέτες. Επίσης, μελετήθηκε η ανίχνευση και ο χαρακτηρισμός του βακτηρίου Wolbachia στα είδη Cydia splendana, Cydia fagiglandana και Pammene fasciana. Το βακτήριο Wolbachia ανιχνεύθηκε για πρώτη φορά στα συγκεκριμένα είδη και μάλιστα διαπιστώθηκε ότι η συχνότητα εμφάνισής του ποικίλει τόσο μεταξύ των δύο ειδών Cydia όσο και μεταξύ των πληθυσμών του κάθε είδους. Στο είδος Pammene fasciana, το βακτήριο ανιχνεύθηκε σε όλα τα άτομα που μελετήθηκαν. Τα αποτελέσματα της παρούσας διατριβής συζητούνται από τη σκοπιά τόσο της οικολογικής και εξελικτικής σημασίας τους όσο και της προοπτικής χρησιμοποίησης του συμβιωτικού βακτηρίου Wolbachia για τον πληθυσμιακό έλεγχο επιβλαβών εντόμων όπως οι μύγες τσε-τσε, οι αφίδες και η καρπόκαψα καστανιάς.
Wolbachia is an intracellular and maternally inherited symbiotic bacterium that belongs to the class of Alphaproteobacteria and the order of Rickettsiales. It is the most ubiquitous intracellular symbiotic organism of the planet, since it has been estimated that over 40% of insect species, in addition to filarial nematodes, crustaceans, and arachnids are infected with Wolbachia. In arthropods Wolbachia affects the biological functions and properties of its hosts and it is responsible for a number of reproductive abnormalities, such as cytoplasmic incompatibility (CI), thelytokous parthenogenesis, feminization of genetic males and male killing. These unique biological characteristics of Wolbachia are attracting the interest of various researchers for: (a) decyphering the role of Wolbachia in evolutionary processes (mainly speciation), and (b) for its use in environmentally friendly applications for the control of agricultural pests and disease vectors. The species of genus Glossina (Diptera: Glossinidae) known as tsetse flies, have been found to be infected with Wolbachia. Tsetse flies are the sole vectors of pathogenic trypanosomes in tropical Africa, causing the “sleeping sickness” in humans and the “nagana” in animals. The potential use of Wolbachia for the control of tsetse flies, prerequisite a thorough knowledge of its genetic identity and the interactions with the host. To further characterize the prevalence of Wolbachia in tsetse flies an extensive screen of more than 5300 specimens from natural and laboratory populations of 11 different Glossina species originating from 13 African countries was carried out. Our results indicated a huge divergence in the prevalence of Wolbachia, both among the species and among populations of the same species. Further characterization by MLST and wsp genotyping was carried out for the Wolbachia strains of 29 representative populations and species of tsetse flies. An impressive genetic diversity of Wolbachia strains in tsetse flies was revealed. Interestingly, disconcordance between the phylogeny of Wolbachia and that of the tsetse flies was observed, suggesting horizontal transmission of Wolbachia during the evolution. Moreover, extended horizontal gene transfer events were detected for first time in Glossina morsitans morsitans, Glossina pallidipes και Glossina austeni. These results raise critical questions concerning: (a) the origin/mechanism of these horizontal gene transfer events, (b) their temporal determination, (c) their potential role as agents of speciation and (d) their selective appearance in only some Glossina species e.g in the subspecies Glossina morsitans centralis which is closely related with Glossina morsitans morsitans the phenomenon was not observed. Equally important will be to examine if genes from the chromosomal insertions were potentially expressed and examine if these genes are associated with the vectorial capacity of tsetse flies for the trypanosoma transmission. Finally, a negative correlation between the presence of Wolbachia with the Salivary Gland Hypertrophy Virus (SGHV) was identified. This is further discussed in the context of biological applications for control of tsetse fly-vector and trypanosomiasis. Finally in this thesis, the detection and characterization of Wolbachia in 78 specimens of 22 aphids species, from 26 natural populations, from Greece was examined. Only 4 out of 26 populations were found to be infected with Wolbachia, and specifically the species: Aphis fabae, Aphis hederae, Metopolophium dirhodum και Baizongia pistaciae. These results indicated that the presence of Wolbachia in aphids is probably more prevalent than it was derived from previous studies. Also, detection and characterization of Wolbachia in the Cydia splendana, Cydia fagiglandana and Pammene fasciana was carried out. Wolbachia was detected for first time in these species, and it was found that the prevalence of Wolbachia varies between the two species of Cydia and among populations of each species, with the infection in Pammene fasciana being fixed. At the end the ecological and evolutionary importance of Wolbachia, together with the use of the bacterium for the population control of harmful insects like tsetse flies, aphids and moths is further discussed.