Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / The hymenopteran endoparasitoid, Cotesia rubecula, employs integrated forms of active and passive immune-suppression in overcoming the defences of its host, Pieris rapae, a cosmopolitan pest of cruciferous crops. The immune-suppressive activity arises from a complex of maternally secreted proteins and polydnavirus (PDV) particles, which are injected in a host larva with the parasitoid egg at oviposition. The PDV associated with C. rubecula (CrBV) is unusual in that only four main viral genes (CrV1-CrV4) are expressed in P. rapae tissues and that expression is transient, remaining at high levels only in the period between four and eight hours postparasitisation (hpp). Previously, CrV1 was characterised and found to inactivate host haemocytes by causing disruption of their cytoskeleton, leading to abrogation of immune-associated processes such as spreading. In this study, a cDNA library was constructed from parasitised P. rapae larvae and screened with total CrBV DNA, leading to isolation of CrV2 and CrV3. The open reading frame of each gene was cloned in a bacterial expression vector and the resultant recombinant proteins were used to produce antibodies against CrV2 and CrV3. CrV2 has an open reading frame of 960 bp (with no introns) and encodes a glycoprotein of = 40 kDa, which is secreted from infected haemocytes and fat body. Comparison of CrV2 deduced amino acid sequence with other known sequences revealed no significant homologies. CrV2 protein was detected in host larvae at 6 hpp, remaining in large amounts for at least a day and was declining by 48 hpp. A putative coiled-coil region at the C-terminus of CrV2 is suspected of involvement in formation of CrV2 trimers that were detected under non-denaturing conditions. CrV2 was visualised within haemocytes in large endosomes at 24 hpp. Although the function of CrV2 remains unclear, it appears to interact with host haemocytes presumably to suppress their immune function. The CrV3 gene contained and intron and was found to encode a C-type lectin (CTL) homologue, which is secreted from infected host haemocytes and fat body into haemolymph. Two CrV3 monomers (of = 14 and 17 kDa) were detected in parasitised larvae with the larger monomer being an N-glycosylated form of the smaller monomer. CrV3 dimers and tetramers were also detected in vivo. Recombinant CrV3 forms larger complexes and was shown to agglutinate ovine blood cells, an activity that was Mn²⁺- and Mg²⁺-dependent but was independent of Ca²⁺. CrV3-mediated hemagglutination was inhibited by EDTA but not biological concentrations of 29 potential ligands tested. Interestingly, CrV3 is similar to invertebrate CTLs associated with humoral defence but not with previously isolated viral lectins. Further, CrV3 homologues were recently detected in bracoviruses from C. ruficrus and C. karyai, indicating that a novel CTL family is expressed by some Cotesia-associated PDVs CrV3 probably interacts with a soluble host haemolymph component associated with host humoral immune defences. CrVl and Crp32 (an immune-suppressive C. rubecula calyx protein) were used to produce recombinant Autographa californica mutiple nucleopolyhedrosis viruses (AcMNPVs), pathogens with putatively enhanced virulence in P. rapae. Bioassays were undertaken to investigate the pathogencity of wild-type AcMNPV iu P. rapae (previously unreported) and the effect of insertion of Crp32. Although the proportion of larval deaths due to wild-type AcMNPV was significant, the slow rate of mortality indicated that P. rapae is only semi-permissive to AcMNPV. Crp32 insertion proved insignificant in terms of the proportion and rate of larval mortality. Given the semi-permissive nature of P. rapae, recombinant AcMNPVs expressing immune-suppressive and appropriate reporter genes may be useful for elucidating mechanisms of insect immunity and more specifically, how CrBV acts to subvert these mechanisms in P. rapae. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1109473 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture and Wine, 2004

Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / The hymenopteran endoparasitoid, Cotesia rubecula, employs integrated forms of active and passive immune-suppression in overcoming the defences of its host, Pieris rapae, a cosmopolitan pest of cruciferous crops. The immune-suppressive activity arises from a complex of maternally secreted proteins and polydnavirus (PDV) particles, which are injected in a host larva with the parasitoid egg at oviposition. The PDV associated with C. rubecula (CrBV) is unusual in that only four main viral genes (CrV1-CrV4) are expressed in P. rapae tissues and that expression is transient, remaining at high levels only in the period between four and eight hours postparasitisation (hpp). Previously, CrV1 was characterised and found to inactivate host haemocytes by causing disruption of their cytoskeleton, leading to abrogation of immune-associated processes such as spreading. In this study, a cDNA library was constructed from parasitised P. rapae larvae and screened with total CrBV DNA, leading to isolation of CrV2 and CrV3. The open reading frame of each gene was cloned in a bacterial expression vector and the resultant recombinant proteins were used to produce antibodies against CrV2 and CrV3. CrV2 has an open reading frame of 960 bp (with no introns) and encodes a glycoprotein of = 40 kDa, which is secreted from infected haemocytes and fat body. Comparison of CrV2 deduced amino acid sequence with other known sequences revealed no significant homologies. CrV2 protein was detected in host larvae at 6 hpp, remaining in large amounts for at least a day and was declining by 48 hpp. A putative coiled-coil region at the C-terminus of CrV2 is suspected of involvement in formation of CrV2 trimers that were detected under non-denaturing conditions. CrV2 was visualised within haemocytes in large endosomes at 24 hpp. Although the function of CrV2 remains unclear, it appears to interact with host haemocytes presumably to suppress their immune function. The CrV3 gene contained and intron and was found to encode a C-type lectin (CTL) homologue, which is secreted from infected host haemocytes and fat body into haemolymph. Two CrV3 monomers (of = 14 and 17 kDa) were detected in parasitised larvae with the larger monomer being an N-glycosylated form of the smaller monomer. CrV3 dimers and tetramers were also detected in vivo. Recombinant CrV3 forms larger complexes and was shown to agglutinate ovine blood cells, an activity that was Mn²⁺- and Mg²⁺-dependent but was independent of Ca²⁺. CrV3-mediated hemagglutination was inhibited by EDTA but not biological concentrations of 29 potential ligands tested. Interestingly, CrV3 is similar to invertebrate CTLs associated with humoral defence but not with previously isolated viral lectins. Further, CrV3 homologues were recently detected in bracoviruses from C. ruficrus and C. karyai, indicating that a novel CTL family is expressed by some Cotesia-associated PDVs CrV3 probably interacts with a soluble host haemolymph component associated with host humoral immune defences. CrVl and Crp32 (an immune-suppressive C. rubecula calyx protein) were used to produce recombinant Autographa californica mutiple nucleopolyhedrosis viruses (AcMNPVs), pathogens with putatively enhanced virulence in P. rapae. Bioassays were undertaken to investigate the pathogencity of wild-type AcMNPV iu P. rapae (previously unreported) and the effect of insertion of Crp32. Although the proportion of larval deaths due to wild-type AcMNPV was significant, the slow rate of mortality indicated that P. rapae is only semi-permissive to AcMNPV. Crp32 insertion proved insignificant in terms of the proportion and rate of larval mortality. Given the semi-permissive nature of P. rapae, recombinant AcMNPVs expressing immune-suppressive and appropriate reporter genes may be useful for elucidating mechanisms of insect immunity and more specifically, how CrBV acts to subvert these mechanisms in P. rapae. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1109473 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture and Wine, 2004

Ao longo do processo evolutivo, parasitoides desenvolveram inúmeras estratégias para a colonização de seus hospedeiros, resultando em mecanismos específicos de regulação. Endoparasitoides cenobiontes depositam seus ovos no hospedeiro e manipulam sua fisiologia e suas repostas imunológicas, a fim de tornar o hospedeiro um ambiente seguro e nutricionalmente adequado para o desenvolvimento do imaturo. A associação de parasitoides das famílias Braconidae e Ichneumonidae com vírus da família Polydnaviridae e sua injeção no hospedeiro configuram-se como um dos principais mecanismos de regulação utilizados durante o parasitismo, devido sua ação sobre a expressão de genes do hospedeiro. O conjunto diverso de fatores maternos e larvais apresentados por parasitoides pode apresentar potencial biotecnológico no controle de pragas, que busca por novos nichos de exploração. Apesar disso, a especificidade das interações hospedeiro - parasitoide limita a exploração de eventos que utilizam tais fatores de virulência, baseada na necessidade de táticas que apresentem ação sobre maior número de espécies-praga. Dessa forma, a presente tese teve por objetivo identificar fatores de virulência expressos durante a interação Cotesia flavipes Cameron (Hymenoptera: Braconidae) - Diatraea saccharalis (F.) (Lepidoptera: Crambidae), buscando a identificação de fatores com potencial biotecnológico, e identificar vias metabólicas do hospedeiro alvos de regulação, buscando por genes-alvos de controle. Além disso, essa tese buscou avaliar o potencial biotecnológico de um fator de virulência (CfHTIF) pertencente ao vírus simbionte de C. flavipes e de um fator de virulência larval (TnQuit) expresso por teratócitos associados a Toxoneuron nigriceps (Viereck) (Hymenoptera: Braconidae) contra hospedeiros incomuns. O parasitismo e a injeção de fatores maternos por C. flavipes resultou na regulação de inúmeras vias metabólicas de D. saccharalis, principalmente relacionadas ao controle do sistema endócrino e do sistema imunológico e identificou a expressão de genes virais e larvais associados a C. flavipes. Plantas transformadas com o fator de virulência TnQuit apresentaram efeitos na sobrevivência e no desenvolvimento dos hospedeiros não-permissivos de hábito mastigador, Chrysodeixis includens (Walker) (Lepidoptera: Noctuidae), Spodoptera albula (Walker) (Lepidoptera: Noctuidae) e Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), minador, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae), e sugador, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), enquanto que o gene viral CfHTIF interferiu nas eficiências de conversão do alimento ingerido e digerido de S. albula, mas não de S. frugiperda. / Parasitoids developed many strategies for colonization of their hosts during the evolution of associations with their hosts, which resulted in the development of specific mechanisms of host regulation. Koinobiont endoparasitoids deposit their eggs into the host and manipulate the host´s physiology and immune responses in order to turn the host a safe and nutritionally suitable environment for parasitoid immature development. The association of parasitoids belonging to Braconidae and Ichneumonidae with virus of the family Polydnaviridae, which are injected in the host, constitutes one of the main mechanisms of regulation used during the parasitization due to viral regulation of the host gene expression. The diverse set of maternal and larval-derived factors produced by parasitoids may have biotechnological potential for pest control. Nevertheless, the specificity of the host - parasitoid interactions are thought to limit the exploration of events that use such virulence factors, as a consequence on the need of successful tactics for a large number of pest species. Thus, we aimed to identify the virulence factors expressed during the interaction Cotesia flavipes Cameron (Hymenoptera: Braconidae) - Diatraea saccharalis (F.) (Lepidoptera: Crambidae) to identify factors with biotechnological potential and to identify targets of regulation that could aid on the development of additional control tactics. In addition, we evaluated the biotechnological potential of a virulence factor (CfHTIF) belonging to the symbiont virus of C. flavipes and a larval virulence factor (TnQuit) expressed by teratocytes associated to Toxoneuron nigriceps (Viereck) (Hymenoptera: Braconidae) against non-host species. Parasitization and pseudoparasitization of D. saccharalis larvae by C. flavipes resulted in the regulation of several metabolic pathways, particularly those related to the endocrine and immune systems. We also characterized the transcriptome of C. flavipes and of associated bracovirus in parasitized and pseudoparasitized hosts. Plants transformed with the TnQuit virulence factor affected survival and development of the non-permissive hosts Chrysodeixis includens (Walker) (Lepidoptera: Noctuidae), Spodoptera albula (Walker) (Lepidoptera: Noctuidae) and Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) and the number of developing adults of Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae). CfHTIF-transgenic plants affected the efficiency of conversion of the digested and ingested food of S. albula larvae, but not of S. frugiperda larvae.

L'hyménoptère Cotesia congregata (Microgastrinae ; Braconidae) pond ses oeufs à l'intérieur de son hôte, la chenille du lépidoptère Manduca sexta (Noctuidae ; Sphingidae) et introduit des particules virales de bracovirus contenant 30 cercles d'ADN double brin. Les gènes viraux portés par ces cercles codent pour une série de protéines qui sont produites dans les tissus de la chenille parasitée. Ces protéines virales jouent un rôle indispensable à la réussite parasitaire. En effet, l'expression des gènes viraux entraîne de nombreuses altérations de la physiologie de l'hôte, notamment un contournement de l'immunité de la chenille qui permet le développement des larves du parasite. D'autre part, le développement de l'hôte est bloqué à un stade pré-pupal. Les travaux portant sur la caractérisation des génomes de bracovirus ont beaucoup progressé et plusieurs familles de gènes ont été découvertes. Une synthèse des connaissances actuelles sur l'immunité des insectes et les gènes de bracovirus potentiellement impliqués dans le contrôle de l'immunité et du développement des lépidoptères est présentée en introduction.<br />Au cours de ma thèse, le séquençage et l'analyse du génome du bracovirus de Cotesia congregata ont été réalisés (Espagne et al 2004). L'existence de plusieurs familles multigéniques a été mise en évidence, notamment la famille des protéines tyrosines phosphatases (PTP) composée de 27 gènes (Provost et al 2004), la famille des cystatines composée de 3 gènes (Espagne et al soumis) et enfin celle des protéines à motif ankyrine composée de 6 gènes (Pennacchio et al en préparation). La caractérisation détaillée de la famille des PTP a été effectuée. La technique d'électrophorèse en champs inversés (FIGE) a permis la localisation physique de ces gènes sur l'ensemble du génome viral, et leur expression a été analysée dans une série de tissus de l'hôte parasité grâce à une méthode de PCR multiplex. Enfin, des tests d'activité biochimique de PTP de bracovirus produites in vitro grâce à un système d'expression en baculovirus.<br />Les gènes des familles décrites sont exprimés dans l'hôte parasité et les protéines possèdent, en général, la fonction biochimique prédite grâce aux domaines conservés qu'elles contiennent. Ceci suggère que ces protéines virales jouent un rôle actif dans les modifications de la physiologie de l'hôte induite par le parasitisme. La caractérisation des gènes viraux exprimés dans l'hôte est une étape indispensable vers l'identification du rôle individuel de chaque protéine dans le contrôle de la physiologie des chenilles parasitées.