No description available.

Wing polyphenism in insects is a type of phenotypic plasticity where environmental factors trigger the development of a set of discrete wing morphologies. In the water strider Gerris buenoi, photoperiods are the main environmental cue that trigger wing morph determination. The genetic mechanisms connecting environmental cues and the determination of wing morph in G. buenoi are not clear. However, recent experimental work suggests that engagement of the Hippo pathway via ecdysone signalling is a promising model for further investigation. In this study, a reverse-transcription quantitative PCR (RT-qPCR) protocol was developed, aimed at elucidating this potential transduction pathway by quantifying gene expression of Fat, Dachsous, Yorkie, EcR, E75 and E74. This was done using melt curve analysis, gel electrophoresis, sequencing of RT-qPCR products and qPCR standard curves. Additionally, wing morph distribution in extreme and intermediate photoperiods were examined. Wing morph proportions were significantly different between adults emerging in the intermediate photoperiods 15.30:8.30 and 15:9 (hours light : hours dark). An effect of sex was observed, with a higher probability of males becoming long-winged compared to females. This has likely evolved as a result of a dispersal-reproduction trade-off. Taken together, this study provided insight for future investigations of periodically induced wing morph determination and its genetic mechanisms in G. buenoi that will contribute to the understanding of phenotypic plasticity.

Wing polyphenism is a type of phenotypic plasticity present in several insect species whereby a genotype have the ability to develop alternative wing morphs when exposed to different environmental cues. One organism demonstrating a clear case of wing polyphenism is the water strider species, Gerris buenoi, which develop long- or short wings depending on exposure to different photoperiods (the time the organism is exposed to light during a 24 h period). The molecular mechanism behind wing polyphenism in insects in general, and in water striders in particular, is largely unknown. From a study on wing polyphenism in the Brown planthopper (Nilaparvata lugens), some candidate genes have been identified and include two insulin receptor genes and the Forkhead transcription factor (FOXO). Since these genes have been demonstrated to affect wing polyphenism in Brown planthopper (BPH) and since G. buenoi contains an additional insulin receptor homolog, the potential role of these genes in regulating wing polyphenism in G. buenoi have in this project been investigated. The functional genetic technique RNA interference (RNAi) was used to evaluate the function of the genes. This method knock down gene expression in the genes mentioned above, one at a time, to investigate if they have a function in wing polyphenism in G. buenoi. DsRNA with specific homology to each target gene was successfully produced. However, when attempting to inject the dsRNA through micro injection all injected liquid leaked out from the body cavity, and the RNAi was therefore not successful. Further optimisation of the injection protocol has to be done to be able to perform RNAi properly in the future. Thereafter, RT-qPCR was used to evaluate whether the insulin receptor genes and FOXO are differentially expressed between the two photoperiods giving rise to the different wing morphs. The differential gene expression experiment showed differences between the mRNA levels of all target genes between G. buenoi being reared in the two different photoperiods. More specific upregulation of the genes FOXO and insulin receptor 2 in short winged G. buenoi were demonstrated. Further, insulin receptor 1-like, was also demonstrated to be upregulated in the short winged morph. Results presented in this project are in line with the previously identified regulation pattern in BPH, still the results need further evaluation. Since gene expression differences were present for all candidate genes between G. buenoi reared in the different photoperiods, theses genes could still be seen as potential candidate genes in wing polyphenism in water striders.

Les pucerons sont des hémiptères ravageurs des cultures agronomiques particulièrement adaptés à leur environnement. Acyrthosiphon pisum (le puceron du pois) présente un cycle de vie basé sur l’alternance d’une reproduction sexuée ou asexuée en réponse à la photopériode. Ils présentent ainsi un polyphénisme de reproduction aboutissant à la formation de trois phénotypes distincts : femelles asexuées, femelles sexuées, et mâles. Ces derniers étant obtenus par élimination d’un chromosome X, A. pisum est une espèce hétérogamétique mâle présentant un système chromosomique XX chez les femelles et X0 chez les mâles. Le déséquilibre du nombre de chromosome X entre mâles et femelles engendré par cette hétérogamétie nécessite chez certains organismes d’être corrigé par des mécanismes de compensation de dose. Les polyphénismes et compensation de dose impliquent chez d’autres organismes des régulations transcriptionnelles notamment régulées par l’accessibilité de la chromatine.Ma thèse vise ainsi à étudier le polyphénisme de reproduction et la compensation de dose des pucerons sous l’angle d’analyses bio-informatiques de données d’expression des gènes (RNA-seq) et d’accessibilité de la chromatine (FAIRE-seq) dans le but de caractériser l’impact des mécanismes épigénétiques dans ces deux processus biologiques fondamentaux du cycle de vie des pucerons. Les résultats développés dans ma thèse ont permis de montrer d’une part la présence d’une compensation de dose chez le puceron du pois au niveau transcriptomique, supportée par une accessibilité accrue de la chromatine de l’unique X des / Aphids are hemipterous crops pests that are particularly adapted to their environment. Acyrthosiphon pisum (pea aphid) displays a life cycle based on the alternation of sexual or asexual reproduction in response to photoperiod. They thus exhibit a reproductive polyphenism resulting in the formation of three distinct phenotypes: asexual females, sexual females, and males. The latter being obtained by elimination of an X chromosome, A. pisum is a male heterogametic species with a XX chromosomal system in females and X0 in males. The X chromosome number between males and females caused by this heterogamy requires in some organisms to be corrected by dosage compensation mechanisms. Polyphenisms and dosage compensation both involve in other organisms transcriptional regulations that are notably regulated by the chromatin accessibility regulations. My thesis aims to study the reproductive polyphenism and dosage compensation in aphids in the context of bioinformatic analyzes of gene expressioThe results developed in my thesis have shown, on one hand, the presence of dose compensation in pea aphid at the transcriptomic level, which is supported by increased chromatin accessibility of the males’ single X in somatic cells. On the other hand, specific sites of chromatin opening between sexual and asexual embryos seem to participate in the definition of their reproduction mode by modulating the expression of certain genes and by allowing the fixation of transcription factors. Their analysis shows the involvement of ecdysone as a new hormonal pathway that may trigger sexual reproducti

Alate morphs can benefit aphid populations by facilitating dispersal from deteriorating food sources and by escaping from natural enemies. Wing development, however, imposes constraints on fecundity. The soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), is an important economic pest in the U.S. and Canada. I conducted a series of laboratory and field experiments to determine the environmental factors inducing wing development in this species, and to determine the effects of asexual alate individual production on an A. glycines population under predation. My results reveal that wing induction in A. glycines occurs in response to interactions between crowding and decreased plant quality cues, and that alate aphid production benefits an aphid population under predation by increasing prevalence at a temporary cost to fecundity. My results contribute to the growing knowledge on the production of asexual alate aphids and provide insight into the biology of A. glycines as an agricultural pest. / May 2017

A diferenciação morfo-fisiológica entre rainhas e operárias de Apis mellifera decorre da alimentação recebida durante o desenvolvimento larval. Dentre as diferenças morfológicas entre as duas castas encontram-se estruturas especializadas para a coleta de pólen e própolis, localizadas nas pernas metatorácicas das operárias, ausentes nas pernas de rainhas. Os padrões de expressão de Ultrabithorax (Ubx) durante o desenvolvimento de operárias e rainhas estão associados aos padrões de cerdas das pernas de fêmeas adultas. Pernas mesotorácicas de operárias apresentam estruturas descritas como importantes na coleta de pólen, ausentes em rainhas. Por outro lado, as asas não possuem estruturas casta-específicas. No presente trabalho, análises globais de transcrição gênica por hibridação de lâminas de microarrays a partir de RNA total de pernas metatorácicas de operárias e rainhas em três estágios do desenvolvimento mostram 1952 genes diferencialmente expressos. Discos de pernas metatorácicas de larvas no início do quinto estágio larval, quando comparados aos de estágios mais tardios no desenvolvimento, têm alto níveis de transcritos de ativadores de Ubx, o qual está 25 vezes mais expresso em estágios subseqüentes do desenvolvimento. Buscas por motivos de ligação de fatores de transcrição nos promotores dos grupos de genes diferencialmente expressos revelam que os motivos para ligação de Ubx, Zeste e Twist estão super-representados em um dos conjuntos analisados. Dentro deste grupo, estão presentes genes cujos ortólogos em Drosophila são controlados por Ubx, como o caso do gene lola. Análises do processamento do mRNA de Ubx em pernas e asas de ambas as castas mostram que são produzidas três isoformas diferentes quanto à presença de dois microéxons (m1 and m2), que contêm 42 nt and 53 nt, respectivamente. A isoforma IIa-like, que contém o m2, entretanto, parece não ser capaz de produzir uma proteína Hox, já que possui um códon de terminação antes do homeodomínio. O perfil de transcrição diferencial de Ubx entre as castas está associado a apêndices que apresentam diferenças morfológicas, sendo Ubx mais transcrito em pernas meso e metatorácicas de operárias que rainhas. Quando analisadas as porcentagens de expressão de cada isoforma nos apêndices, claramente a isoforma IVa-like, sem microéxons, é a mais transcrita em todos os tecidos. Entretanto, nota-se que nas asas anteriores, onde há menos Ubx, a isoforma IIa-like é proporcionalmente mais transcrita que II nos outros apêndices. Destaca-se uma tendência à inclusão do microéxon m1 (isoforma IIIa-like) ao mRNA de Ubx transcrito em asas posteriores e pernas de rainhas em comparação a operárias, em detrimento da isoforma IVa-like. Análises do uso da região 3UTR em pupas de operárias mostram que há um microssatélite transcrito na porção distal da região 3UTR deUbx. A estrutura secundária predita agrupa separadamente as regiões codificadora e as regiões 3UTR proximal e distal. Análises de seqüenciamento de última geração revelaram que oito dos 51 microRNAs com sítios-alvo preditos na região 3UTR de Ubx estão mais expressos em asas anteriores, e outros dois em asas posteriores. Assim, nossos resultados mostram que o controle da expressão diferencial de Ubx é dada pela ativação desse gene por fatores de transcrição que se ligam ao promotor, controle do splicing alternativo, e expressão de microRNAs diferencial em cada casta e apêndice, controlando, assim, a morfogênese diferencial dos apêndices de fêmeas observada em A. mellifera. / Along with differences in physiological and behavioral characteristics, workers and queens of Apis mellifera also differ in appendage morphology. Some appendage specializations in the hind legs of honeybee workers, which are highly specialized pollinators, deserve special attention. The hind tibia of the worker has an expanded bristle-free region used for carrying pollen and propolis, the corbicula. In queens, this structure is absent. Although these morphological differences have been well characterized, the genetic inputs triggering the development of this alternative morphology have remained unknown. Through microarray analysis, we detected 1,952 genes that are differentially expressed during worker versus queen hind leg development. The gene expression signatures of the two castes have similar patterns of genes controlling development. At the beginning of the last larval instar, Ultrabithorax (Ubx) activators are more strongly expressed than in prepupae and early pupae; at this time Ubx expression is approximately 25 times higher. Within the gene expression signature, we identified a cluster formed by genes in which Ubx, Twist and Zeste binding sites are over-represented. This cluster includes genes for which Drosophila orthologs are known to be bound by Ubx, as in the case of lola. We also tested the extent of Ubx mRNA processing during wing and leg development. Unexpectedly, we found Ubx alternative splicing in both workers and queens; there were two microexons (m1 and m2) encoding 42 nt and 53 nt, respectively, arguing against the hypothesis that alternative splicing occurs exclusively within the Diptera. Inclusion of the m2 exon inserts a stop codon upstream from the exon containing the homeodomain, producing a truncated protein. Moreover, these bee microexons conserve the nucleotides known to be important for alternative splicing in Drosophila. During bee wing development, Ubx mRNA isoforms are transcribed in similar amounts in both castes; however, during leg development, queens produce 60% of the Ubx levels transcribed by workers. Analysis of 3UTR usage during bee development revealed a microsatellite region transcribed within the Ubx 3UTR. The predicted secondary structure locations separated the coding region into three branches and the proximal and the distal 3UTR regions. Deep-sequencing analysis revealed that eight out of 51 miRNAs predicted to target the Ubx mRNA are more highly expressed in worker forewings and two are more expressed in the hindwings. Therefore, we conclude that Ubx differential expression is activated by transcription factors that bind to its promoter, by control of alternative splicing, and moreover by microRNAs differentially expressed according to tissue and caste, resulting in differential morphogenesis of the hind leg in honeybee females.

A diferenciação morfo-fisiológica entre rainhas e operárias de Apis mellifera decorre da alimentação recebida durante o desenvolvimento larval. Dentre as diferenças morfológicas entre as duas castas encontram-se estruturas especializadas para a coleta de pólen e própolis, localizadas nas pernas metatorácicas das operárias, ausentes nas pernas de rainhas. Os padrões de expressão de Ultrabithorax (Ubx) durante o desenvolvimento de operárias e rainhas estão associados aos padrões de cerdas das pernas de fêmeas adultas. Pernas mesotorácicas de operárias apresentam estruturas descritas como importantes na coleta de pólen, ausentes em rainhas. Por outro lado, as asas não possuem estruturas casta-específicas. No presente trabalho, análises globais de transcrição gênica por hibridação de lâminas de microarrays a partir de RNA total de pernas metatorácicas de operárias e rainhas em três estágios do desenvolvimento mostram 1952 genes diferencialmente expressos. Discos de pernas metatorácicas de larvas no início do quinto estágio larval, quando comparados aos de estágios mais tardios no desenvolvimento, têm alto níveis de transcritos de ativadores de Ubx, o qual está 25 vezes mais expresso em estágios subseqüentes do desenvolvimento. Buscas por motivos de ligação de fatores de transcrição nos promotores dos grupos de genes diferencialmente expressos revelam que os motivos para ligação de Ubx, Zeste e Twist estão super-representados em um dos conjuntos analisados. Dentro deste grupo, estão presentes genes cujos ortólogos em Drosophila são controlados por Ubx, como o caso do gene lola. Análises do processamento do mRNA de Ubx em pernas e asas de ambas as castas mostram que são produzidas três isoformas diferentes quanto à presença de dois microéxons (m1 and m2), que contêm 42 nt and 53 nt, respectivamente. A isoforma IIa-like, que contém o m2, entretanto, parece não ser capaz de produzir uma proteína Hox, já que possui um códon de terminação antes do homeodomínio. O perfil de transcrição diferencial de Ubx entre as castas está associado a apêndices que apresentam diferenças morfológicas, sendo Ubx mais transcrito em pernas meso e metatorácicas de operárias que rainhas. Quando analisadas as porcentagens de expressão de cada isoforma nos apêndices, claramente a isoforma IVa-like, sem microéxons, é a mais transcrita em todos os tecidos. Entretanto, nota-se que nas asas anteriores, onde há menos Ubx, a isoforma IIa-like é proporcionalmente mais transcrita que II nos outros apêndices. Destaca-se uma tendência à inclusão do microéxon m1 (isoforma IIIa-like) ao mRNA de Ubx transcrito em asas posteriores e pernas de rainhas em comparação a operárias, em detrimento da isoforma IVa-like. Análises do uso da região 3UTR em pupas de operárias mostram que há um microssatélite transcrito na porção distal da região 3UTR deUbx. A estrutura secundária predita agrupa separadamente as regiões codificadora e as regiões 3UTR proximal e distal. Análises de seqüenciamento de última geração revelaram que oito dos 51 microRNAs com sítios-alvo preditos na região 3UTR de Ubx estão mais expressos em asas anteriores, e outros dois em asas posteriores. Assim, nossos resultados mostram que o controle da expressão diferencial de Ubx é dada pela ativação desse gene por fatores de transcrição que se ligam ao promotor, controle do splicing alternativo, e expressão de microRNAs diferencial em cada casta e apêndice, controlando, assim, a morfogênese diferencial dos apêndices de fêmeas observada em A. mellifera. / Along with differences in physiological and behavioral characteristics, workers and queens of Apis mellifera also differ in appendage morphology. Some appendage specializations in the hind legs of honeybee workers, which are highly specialized pollinators, deserve special attention. The hind tibia of the worker has an expanded bristle-free region used for carrying pollen and propolis, the corbicula. In queens, this structure is absent. Although these morphological differences have been well characterized, the genetic inputs triggering the development of this alternative morphology have remained unknown. Through microarray analysis, we detected 1,952 genes that are differentially expressed during worker versus queen hind leg development. The gene expression signatures of the two castes have similar patterns of genes controlling development. At the beginning of the last larval instar, Ultrabithorax (Ubx) activators are more strongly expressed than in prepupae and early pupae; at this time Ubx expression is approximately 25 times higher. Within the gene expression signature, we identified a cluster formed by genes in which Ubx, Twist and Zeste binding sites are over-represented. This cluster includes genes for which Drosophila orthologs are known to be bound by Ubx, as in the case of lola. We also tested the extent of Ubx mRNA processing during wing and leg development. Unexpectedly, we found Ubx alternative splicing in both workers and queens; there were two microexons (m1 and m2) encoding 42 nt and 53 nt, respectively, arguing against the hypothesis that alternative splicing occurs exclusively within the Diptera. Inclusion of the m2 exon inserts a stop codon upstream from the exon containing the homeodomain, producing a truncated protein. Moreover, these bee microexons conserve the nucleotides known to be important for alternative splicing in Drosophila. During bee wing development, Ubx mRNA isoforms are transcribed in similar amounts in both castes; however, during leg development, queens produce 60% of the Ubx levels transcribed by workers. Analysis of 3UTR usage during bee development revealed a microsatellite region transcribed within the Ubx 3UTR. The predicted secondary structure locations separated the coding region into three branches and the proximal and the distal 3UTR regions. Deep-sequencing analysis revealed that eight out of 51 miRNAs predicted to target the Ubx mRNA are more highly expressed in worker forewings and two are more expressed in the hindwings. Therefore, we conclude that Ubx differential expression is activated by transcription factors that bind to its promoter, by control of alternative splicing, and moreover by microRNAs differentially expressed according to tissue and caste, resulting in differential morphogenesis of the hind leg in honeybee females.

Les changements climatiques altèrent la phénologie des organismes, c’est-à-dire la succession dans le temps des éléments de leur cycle de vie. L’expression de la diapause chez les insectes, soit l’arrêt de développement permettant de survivre aux dégradations saisonnières de l’environnement biotique et abiotique, est en particulier affectée. Cette thèse aborde différents facteurs environnementaux agissant sur les stratégies saisonnières des parasitoïdes de pucerons du genre Aphidius. Dans les agro-systèmes céréaliers de l’ouest de la France, les communautés d’hôtes et de parasitoïdes ont changé rapidement au cours de la dernière décennie. Ainsi, plusieurs espèces de parasitoïdes n’entrent plus qu’en faible proportion en diapause, dû à l’augmentation des températures, à la baisse du nombre de jours de gel et à la présence accrue de leurs hôtes pendant l’hiver ; ces pressions de sélection agissent sur les seuils d’entrée diapause. Les parasitoïdes présentent des ajustements plastiques de leur réponse aux stimuli environnementaux qui induisent la diapause. Ils y entrent en plus forte proportion s’ils se développent dans des morphes de pucerons sexués (annonciateurs de ressources limitées) que dans des hôtes asexués, ce qui souligne la co-évolution de leurs cycles de vie. Cette plasticité s’avère également transgénérationnelle puisque la compétition entre femelles induit la diapause estivale chez leurs descendants. De plus, l’incidence de la diapause hivernale augmente après plusieurs générations de parasitoïdes soumises aux mêmes conditions. Les adaptations locales des parasitoïdes aux nouvelles conditions environnementales mènent à la perte d’expression de la stratégie de diapause – qui comporte des coûts écologiques et physiologiques – au profit de stratégies hivernales d’activité à l’état adulte. La modification de l’activité-densité des espèces au sein des communautés de pucerons-(hyper)parasitoïdes, due aux changements de stratégie d’hivernation, pourrait bouleverser l’équilibre des réseaux trophiques et altérer positivement ou négativement l’efficacité du service écosystémique de lutte biologique. / Climate changes alter the phenology of organisms, i.e. the succession over time of the elements of their life-cycle. Diapause expression in insects – that is, the developmental arrest allowing survival to seasonal degradations in the biotic and abiotic environment – is particularly affected. This thesis addresses the existence of different environmental factors acting on the seasonal strategies of Aphidius aphid parasitoids. In cereal agro-systems of western France, host and parasitoid communities have changed rapidly over the past decade. Hence, some parasitoid species enter diapause at low levels due to temperature increase, decrease in frost events and the presence of their hosts during winter, which impose selection on diapause induction thresholds. Parasitoids show plastic adjustments of their response to environmental stimuli inducing diapause. They enter diapause at higher proportion if they develop in sexual morphs of aphids (which inform for upcoming limited resources) than in asexual hosts, which underlines the co-evolution of their life-cycles. Transgenerational plasticity is also involved in these responses since maternal competition induces summer diapause in their offspring. Moreover, winter diapause incidence increases after some parasitoid generations experience the same environmental conditions. Local adaptations of parasitoids to new environmental conditions lead to a loss of diapause expression – which involves ecological and physiological costs – to the benefit of adult overwintering strategies. Modifications in species activity-density within aphids-(hyper)parasitoids communities, due to changes in overwintering strategies, could affect food-webs’ stability and alter positively or negatively the efficiency of biological pest control.

In this laboratory study, the role of juvenile hormone (JH) and ecdysone in regulating wing polyphenism was investigated in the non-model organism Gerris buenoi. Topical application of the JH analog methoprene elicited reduced pronotum, wing defects, and nymphal-adult intermediates but no changes to wing morph. Similarly, while microinjection of the ecdysone derivative 20-hydroxyecdysone (20E) elicited aberrant phenotypes there was again no influence on the wing morph. Using data from a transcriptomics experiment, RNAi knockdown of the differentially expressed 20E induced receptor gene, Hr4, caused high mortality rates (> 90 %) which resulted in a sample size too small to draw any inferences of Hr4’s involvement in G. buenoi wing polyphenism. My results indicate that both JH and ecdysone are involved in several developmental processes including wing development, but they do not seem to be important for determining wing polyphenism. However, several factors are important to consider in future research which means that the potential role of JH and ecdysone in G. buenoi wing polyphenism should not be dismissed at this stage.

No description available.