Grâce aux progrès expérimentaux permettant d'étudier deux phénotypes qui sont d'un intérêt majeur dans la compréhension des capacités d'adaptation d'A. gambiae s.s. à son environnement. Les différences de résistance à la dessiccation mises en évidence entre les différents caryotypes liés à l'inversion chromosomique 2La et entre les formes moléculaires M et S offrent des pistes intéressantes pour l'identification de facteurs génétiques impliqués dans la divergence écologique au sein de ce complexe d'espèces. / Thanks to progress in sequencing, the genomes of many organisms are known and available. Thus, functional genomics, the elucidation of gene function in sequenced genome, is currently booming. However, there is a gap between our growing knowledge in genetic and the current sparse information on phentoypic data ( "phenotype gap"). All organisms whose genome has been sequenced are facing this problem, including Anopheles gambiae.Anopheles gambiae sensu lato is a complex of sibling species, indistinguishable from a morphological point of view, present on almost the entire African continent. A. gambiae demonstrates an extreme environmental ubiquity and the characterization of phenotypes associated with adaptation to varying environments as well as the identification of genes involved in this adaptation is one of the main research axes in the post-genome area of this major malaria vector.We have studied some phenotypes associated with aridity and temperature in the nominal species of the A. gambiae complex. These two parameters are discriminent in the distribution of molecular forms and chromosomal inversions that characterize this species and may be involved in ecological divergence and speciation. We first measured desiccation resistance of adult mosquitoes of A. gambiae s.s. and we then studied the preferred temperatures of larvae in a choice device set-up (the shuttlebox). We compared the thermoregulation behavior and thermal preferences of a laboratory strain with field larvae of A. gambiae s.s. We also presented preliminary data on the preferred temperatures measured in field larvae of the S and M molecular forms.From a technical point of view, we improved an existing device for testing the survival of mosquitoes in highly desiccated conditions by coupling it with a video surveillance system, which help to increase the accuracy in determining the survival time, to avoid disturbing the system during the experiment and allow to test relatively large numbers of individuals. This study revealed a significant association between the 2La chromosomal inversion and resistance to desiccation in A. gambiae and highlighted the role of body size in the survival of this mosquito in dry environments.We also adapted a new device to study experimentally the thermopreference of A. gambiae s.s. larvae. The results showed that laboratory larvae and field M molecular form larvae had similar thermal preferences, consistent with the values of temperature usually found in natural breeding sites. In addition, the S molecular form larvae from southern Cameroon had preferences similar to those of northern Cameroon, regardless of karyotypes related to chromosomal inversions. In addition, the comparison of data for the M and S molecular forms larvae revealed that there was no significant difference in thermal preferences or in thermoregulatory behavior.Our results have contributed to the development of two experimental devices to study two phenotypes that are of major interest in understanding the adaptation of A. gambiae s.s. to its environment. The differences in desiccation resistance between the different karyotypes associated with the 2La chromosomal inversion and between the M and S molecular forms offer interesting new possibilities for the identification of genetic factors involved in their ecological divergence.

O gene A1 da alfa amilase foi isolado de uma biblioteca genômica em lambda EMBL3, feita com DNA do díptero primitivo Anopheles merus (Díptera, Nematocera, Culicoidea). Ele foi parcialmente seqüenciado, caracterizado pelo padrão da clivagem das enzimas restritivas e clonado no plasmídeo pIBI24 por Pernasetti (1991 ). No presente trabalho relata-se sua expressão em: bactéria (Escherichia coli AD494), células de inseto ( Spodoptera frugiperda) e em levedura (Pichia pastoris), tendo-se observado que somente nas células de Spodoptera frugiperda a enzima é expressa com atividade. A expressão extracelular do gene A1 foi ensaiada em E.coli AD494 usando o vetor pRSETc. Para este fim a seqüência do gene contendo seu próprio peptídeo sinal, foi inserida em fase de leitura (\"frame\") nesse vetor. A expressão sem peptídeo sinal foi executada usando o vetor pAE2, uma construção derivada- do pRSETc. Numa outra construção, o gene A1 foi inserido em fase de leitura após a seqüência do promotor e do peptídeo sinal do gene A2 (alfa amilase de Bacillus stearothermophilus) utilizando-se o vetor pIBI24-Bst. As células de E.coli AD494 transformadas com estes construções expressaram a enzima sem atividade. A amilase A1 não foi secretada nem com seu peptídeo sinal nem com o da amilase A2. A expressão do gene A1 em levedura (Pichia pastoris) foi feita usando o vetor pPic9, que permite a expressão tanto intracelular quanto a secreção da proteína. O gene foi amplificado por PCR usando \"primers forward\" com o intuito de obter o gene com ou sem peptídeo sinal. O gene contendo o peptídeo sinal foi construído com diferentes seqüências tipo Kozak precedendo o códon ATG. Para a expressão extracelular usou-se o peptídeo sinal do fator alfa do pPic9. Os resultados mostraram que a enzima é expressa mas que permanece dentro da célula sem atividade. As mesmas construções do gene A1 feitas para Pichia foram inseridas em Baculovírus para transfectar células de Spodoptera frugiperda. Neste sistema a amilase foi expressa com atividade principalmente no sobrenadante das culturas transformadas com construções usando o gene contendo o seu próprio peptídeo sinal assim com o peptídeo sinal da amilase de Zabrotes subfasciatus. O gene da alfa amilase de Bacillus stearothermophilus foi expresso no sistema Baculovírus-Spodoptera e na levedura Pichia, usando o gene contendo o peptídeo sinal da amilase de Z. subfasciatu. A proteína foi secretada e tinha atividade em ambos os sistemas. / The alpha amylase A1 gene was isolated trom a genomic library of lambda EMBL3 made with DNA from the primitve Díptera Anopheles merus (Díptera, Nematocera, Cuclicoidea). This gene was partialy sequenced, characterized by standard restriction enzyme cleavage, and cloned into the pIBI24 plasmid by Pernasetti (1991). Here we present the expression of this gene in: bacteria (Escherichia coli AD494), insect cell (Spodoptera frugiperda) and yeast (Pichia pastoris). It was shown that only in Spodoptera frugiperda cells the protein display enzymatic activity. The presence of the A1 product in the extracelullar culture media was tested in Escherichia coli AD494 using the vector pRSETc. To this end, the gene sequence containing its own signal peptide was inserted in frame into the vector. Expression without a signal peptide was conducted using the pAE2 vector, a construct derived from pRSETc. In another construct, the A1 gene was inserted in frame after the promoter sequence and the signal peptide of the A2 gene (alpha amylase Bacillus stearothermophilus) using the pIBI24-Bst vector. Although the E.coli AD494 cells transformed with these constructs expressed the enzyme, no enzyme activity was detected. A1 was not secreted, either with its own signal peptide or with that of amylase A2. Expression of the A1 gene in yeast (Pichia pastoris) was conducted using the pPic9 vector, wich allows intracellular expression or secretion of this protein. The gene was amplified by PCR with forward primers, so as to amplyfy the gene sequence with or without the signal peptide. The gene containing the signal peptide was constructed with different Kozak sequences preceeding the ATG codon. For extracellular expression, the signal peptide of the pPic9 alpha factor was used. The results showed that the enzyme is expressed , but remained within the cell and do not display activity. The same constructs of the A1 gene made for P. pastoris were inserted in Baculovirus to infect Spodopera frugiperda cells. In this system, the amylase was successfully expressed and display enzymatic activity, mainly in the extracellular supernatant, using constructs of the gene with own signal peptide or with the signal peptide for amylase of Zabrotes subfasciatus. The gene A2 was expressed in the Baculovirus/Spodoptera system and the yeast P. pastoris using constructs containing the signal peptide of alpha amylase of Z. subfasciatus, the protein was secreted with activity.

Introdução – Anopheles (Nyssorhynchus) lanei foi descrito por Galvão e Amaral em 1938, a partir de espécimes coletados em Campos do Jordão, na Serra da Mantiqueira, Estado de São Paulo, sendo provável espécie endêmica desta região. Linthicum (1988) redescreveu An. lanei baseando-se em um espécime adulto fêmea e duas genitálias masculinas de Campos do Jordão; porém, a ilustração de genitália masculina foi feita a partir de um espécime coletado na cidade de Estância, Estado de Sergipe, Brasil. Evidências morfológicas somadas a diferenças ambientais entre estas localidades indicam que os espécimes analisados por Linthicum não correspondem a An. lanei. Culex (Culex) dolosus foi descrito por Lynch Arribálzaga em 1891 com base em espécimes coletados na Província de Buenos Aires, Argentina. O último trabalho a tratar de aspectos da morfologia de Cx. dolosus é anterior à descrição de Culex eduardoi Casal & García, espécie similar a esta. Dados de literatura apontam para a existência de formas morfológicas diferenciadas em populações de Cx. dolosus, o que parece ser o caso dos espécimes que ocorrem na região de Campos do Jordão. Objetivos – Este estudo visa contribuir para o conhecimento taxonômico de espécies pertencentes aos subgêneros Nyssorhynchus e Culex, que ocorrem na Serra da Mantiqueira, por meio da redescrição de An. lanei e Cx. dolosus, e também da identificação de fauna de Culicidae associada. Métodos – As redescrições foram baseadas na análise de exemplares coletados na região de Campos do Jordão e implicaram em etapas de quetotaxia das larvas e pupas, biometria dos adultos e formas imaturas e elaboração de ilustrações das estruturas anatômicas da morfologia externa. Resultados – Deste trabalho resultaram redescrições morfológicas detalhadas, tabelas de quetotaxia das formas imaturas e ilustrações das larvas, pupas e estruturas das genitálias masculinas. Considerações finais - Espera-se que os resultados alcançados possam ampliar e dar continuidade a estudos taxonômicos de Culicidae e também contribuir para a correta identificação de espécies de Anopheles e Culex. / Introduction – Anopheles (Nyssorhynchus) lanei was described by Galvão and Amaral in 1938, based on specimens collected in the Campos do Jordão, in Serra da Mantiqueira, State of São Paulo, being a probable endemic species from this region. Linthicum (1988) re-described An. lanei based on one adult female and two male genitalia from Campos do Jordão; however, the illustration of the male genitalia was generated from a specimen collected in Estância municipality, State of Sergipe, Brazil. Morphologic evidences, in addiction to environmental differences between both localities, suggest that the specimens examined by Linthicum (1988) do not belong to An. lanei. Culex (Culex) dolosus was described by Lynch Arribálzaga in 1891 based in exemplars from the Buenos Aires Province, in Argentina. The last work to deal with morphologic aspects of Cx. dolosus precedes the description of Culex eduardoi Casal & García, a similar species to the former. Literature records point to the existence of differentiate morphologic forms of Cx. dolosus, what seems to be the case of the specimens that occur in Campos do Jordão region. Objectives – This study aim to contribute to the knowledge about species from the subgenera Nyssorhynchus and Culex, that occur in the Serra da Mantiqueira, by the re-description of An. lanei and Cx. dolosus and also by the identification of the Culicidae associated fauna. Methods – The redescriptions were based in the analyzes specimens collected in the region of Campos do Jordão and implied in stages of chaetotaxy of the larvae and pupae, biometry of the adults and immature forms, and elaboration of illustrations of the anatomic structures of the external morphology. Results – This work resulted in detailed morphologic redescriptions, chaetotaxy tables of the immature forms and illustrations of the larvae, pupae and male genitalia structures. Final considerations – It is expected that the reached results might expand and continuity taxonomic studies on Culicidae and also contribute to the correct identification of Anopheles and Culex.

Submitted by Cleide Dantas (cleidedantas@ufpa.br) on 2014-02-07T14:12:35Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Tese_ImportanciaAnophelesDarlingi.pdf: 5215794 bytes, checksum: bdef0d9d3cb37a1d41a7c1f3d53e5d79 (MD5) / Approved for entry into archive by Ana Rosa Silva(arosa@ufpa.br) on 2014-02-10T15:12:47Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Tese_ImportanciaAnophelesDarlingi.pdf: 5215794 bytes, checksum: bdef0d9d3cb37a1d41a7c1f3d53e5d79 (MD5) / Made available in DSpace on 2014-02-10T15:12:47Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Tese_ImportanciaAnophelesDarlingi.pdf: 5215794 bytes, checksum: bdef0d9d3cb37a1d41a7c1f3d53e5d79 (MD5) Previous issue date: 2010 / Projeto PIATAM Mar / A malária no município de Macapá é principalmente peri urbana, áreas estas caracterizadas por ressaca, pela presença de fragmentos de floresta e assentamentos desordenados (invasões). O objetivo deste estudo foi verificar a importância dos Anopheles darlingi e Anopheles marajoara na transmissão de malária em Macapá. O estudo foi realizado de outubro de 2007 a setembro de 2008, na comunidade de Lagoa dos Índios, Macapá. Foram coletados 4.601 mosquitos, dos quais 3.029 foram Anopheles marajoara (65,8%), 917 Anopheles darlingi (19,9%), 429 Anopheles braziliensis (9,3%), 208 Anopheles triannulatus (4,5%), 18 Anopheles peryassui (0,4%) e cinco Anopheles nuneztovari (0,1%). Apenas 32,8% dos espécimes foram coletadas no intradomicilio (1.511) e 67,2% no peridomicilio (3.090). O Indice de Picada Homem Hora do An. darlingi no intradomicilio variou entre 0 a 6,5 e no peridomicilio de 0 a 22 picadas homem hora. Já para o An. marajoara a variaçao foi de 0 a 22 no intradomicilio e de 0 a 175,5 no peridomicilio. A analise das exúvias e da dissecção de genitália resultaram na confirmaçao das duas espécies estudadas, An. darlingi e An. marajoara, e que o An. marajoara é a única espécie do complexo albitarsis circulante na área. A abundância dos vetores flutuou associada com o padrão sazonal das chuvas. An. darlingi é mais abundante no final e inicio das chuvas, enquanto o An. marajoara esteve presente em alta densidade durante todo o período de chuvas. Dos 4.601 mosquitos testados, 100 foram positivos para plasmódios humanos pelo método de ELISA, resultando em uma taxa de infecção de 2,17%. Dos 3.029 An. marajoara testados 71 (2,34%) foram positivos e dos 917 An. darlingi, 28 (3,05%). Este estudo demonstrou que as duas espécies estudadas mantêm a transmissão de malária durante todo o ano, ratificando assim a importância das mesmas. / Malaria in the municipality of Macapá is mainly peri urban, which are characterized by flooding area, presence of forest fragments and disorganized settlements. The aim of this study was to determine the importance of Anopheles darlingi e Anopheles marajoara in the malaria transmission in Macapá. The study was carried out from October 2007 to September 2008, in the Lagoa dos Índios community, Macapá. It was collected 4,601 mosquitoes, from which 3,029 were Anopheles marajoara (65,8%), 917 Anopheles darlingi (19,9%), 429 Anopheles braziliensis (9,3%), 208 Anopheles triannulatus (4,5%), 18 Anopheles peryassui (0,4%) and 5 Anopheles nuneztovari (0,1%). Only 32.8% (1.511) of the adult mosquitoes were collected indoors and 67.2% (3.090) outdoors. The index of the bites by man and by hour for An. darlingi in the intradomicile had ranged from 0 to 6.5 and in the peridomicile from 0 a 22. As for the An. marajoara the range was from 0 to 22 in the intradomicile and from 0 to 175.5 in the peridomicile. The analysis of the exuvia and the genitalia dissecation had resulted in the confirmation of the two species, An. darlingi and An. marajoara, and that the An. marajoara is the only albitarsis complex specie that is circulating in the study area. The vectors abundance had flutuated associated with the sazonal pattern of the rain. An. darlingi is the most frequent at the beginning and end of the rain, while An. marajoara was present, in high density, during the rainy season. From the 4,601 tested mosquitoes, 100 were positive for human malaria parasites by the ELISA method with a infection index of 2.17%. The infection index for An. marajoara was 2,34% (71/3,029) and for An. darlingi 3.05% (28/917). This study had demonstrated that the two studied species are responsible for the malaria transmission maintenance during the whole year, thus confirming the importance of both.

Le MALDI-TOF MS (Matrix Assisted, Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry) est une technique protéomique qui est utilisée en routine pour l’identification des microorganismes dans les laboratoires de microbiologie. Ainsi, dans ce travail nous avons évalué le MALDI-TOF MS pour l’identification du repas sanguin des moustiques. Dans la première partie de notre travail, une revue bibliographique a été effectuée sur les différentes méthodes (sérologiques, biologie moléculaire) connues dans les études de préférence trophiques des arthropodes. La deuxième partie fut l’optimisation du MALDI-TOF MS pour l’identification de l’origine du repas sanguin des moustiques. Pour l’optimisation, Anopheles gambiae Giles et Aedes albopictus ont été artificiellement nourris sur le sang de plusieurs hôtes vertébrés en utilisant l’appareil Hemotek durant deux heures sous les conditions standard. Nos résultats ont montré que la comparaison des spectres provenant des moustiques nourris sur le même type de sang révèle une grande reproductibilité des profils protéiques. L’interrogation des MS spectres contre la base de données a révélé une identification correcte de l'origine du repas sanguin pour les spécimens collectés moins de 24 heures après la prise du repas sanguin. Pour les échantillons collectés sur le terrain, le MALDI-TOF MS a permis de détecter dans le repas de sang des moustiques une grande diversité d’hôtes domestiques. En conséquence la technique MALDI-TOF MS serait un outil efficace pour les études de surveillance épidémiologique des maladies vectorielles et l'identification de la préférence trophique de spécimens fraichement gorgés. / MALDI-TOF MS (Matrix Assisted, Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry) is a proteomic technique that routinely used for microorganisms identification in clinical microbiology laboratory. Recently, the MALDI-TOF MS was successfully used as a innovative tool for arthropod identification. Thus, in this work we evaluated the MALDI-TOF MS to identify the blood meal sources from engorged mosquitoes. In the first part of our work, a bibliographical review was carried out on the different methods (serological, molecular biology) known in the trophic preference determination of hematophagous arthropods. The second part was optimization of the MALDI-TOF MS for identifying the origin of the blood meal of mosquitoes. For optimization, the Anopheles gambiae Giles and Aedes albopictus were artificially fed on several vertebrate hosts blood using the Hemotek device for two hours under standard conditions. Our results showed intra-species reproducibility and inter-species specificity of MS spectra from mosquitoes engorged on the same or different vertebrate hosts. The MS spectra querying against the database reveal a correct identification of the the blood meal origin from the specimens collected less than 24 hours post-feeding. For field samples, MALDI-TOF MS allowed to detect the mosquitoes blood meal fed on wide variety of domestic hosts. Consequently the MALDI-TOF MS technique would be an effective tool for epidemiological surveys of vector-borne diseases and the identification of the trophic preference of mosquito freshly engorged.

Introdução – Anopheles (Nyssorhynchus) lanei foi descrito por Galvão e Amaral em 1938, a partir de espécimes coletados em Campos do Jordão, na Serra da Mantiqueira, Estado de São Paulo, sendo provável espécie endêmica desta região. Linthicum (1988) redescreveu An. lanei baseando-se em um espécime adulto fêmea e duas genitálias masculinas de Campos do Jordão; porém, a ilustração de genitália masculina foi feita a partir de um espécime coletado na cidade de Estância, Estado de Sergipe, Brasil. Evidências morfológicas somadas a diferenças ambientais entre estas localidades indicam que os espécimes analisados por Linthicum não correspondem a An. lanei. Culex (Culex) dolosus foi descrito por Lynch Arribálzaga em 1891 com base em espécimes coletados na Província de Buenos Aires, Argentina. O último trabalho a tratar de aspectos da morfologia de Cx. dolosus é anterior à descrição de Culex eduardoi Casal & García, espécie similar a esta. Dados de literatura apontam para a existência de formas morfológicas diferenciadas em populações de Cx. dolosus, o que parece ser o caso dos espécimes que ocorrem na região de Campos do Jordão. Objetivos – Este estudo visa contribuir para o conhecimento taxonômico de espécies pertencentes aos subgêneros Nyssorhynchus e Culex, que ocorrem na Serra da Mantiqueira, por meio da redescrição de An. lanei e Cx. dolosus, e também da identificação de fauna de Culicidae associada. Métodos – As redescrições foram baseadas na análise de exemplares coletados na região de Campos do Jordão e implicaram em etapas de quetotaxia das larvas e pupas, biometria dos adultos e formas imaturas e elaboração de ilustrações das estruturas anatômicas da morfologia externa. Resultados – Deste trabalho resultaram redescrições morfológicas detalhadas, tabelas de quetotaxia das formas imaturas e ilustrações das larvas, pupas e estruturas das genitálias masculinas. Considerações finais - Espera-se que os resultados alcançados possam ampliar e dar continuidade a estudos taxonômicos de Culicidae e também contribuir para a correta identificação de espécies de Anopheles e Culex. / Introduction – Anopheles (Nyssorhynchus) lanei was described by Galvão and Amaral in 1938, based on specimens collected in the Campos do Jordão, in Serra da Mantiqueira, State of São Paulo, being a probable endemic species from this region. Linthicum (1988) re-described An. lanei based on one adult female and two male genitalia from Campos do Jordão; however, the illustration of the male genitalia was generated from a specimen collected in Estância municipality, State of Sergipe, Brazil. Morphologic evidences, in addiction to environmental differences between both localities, suggest that the specimens examined by Linthicum (1988) do not belong to An. lanei. Culex (Culex) dolosus was described by Lynch Arribálzaga in 1891 based in exemplars from the Buenos Aires Province, in Argentina. The last work to deal with morphologic aspects of Cx. dolosus precedes the description of Culex eduardoi Casal & García, a similar species to the former. Literature records point to the existence of differentiate morphologic forms of Cx. dolosus, what seems to be the case of the specimens that occur in Campos do Jordão region. Objectives – This study aim to contribute to the knowledge about species from the subgenera Nyssorhynchus and Culex, that occur in the Serra da Mantiqueira, by the re-description of An. lanei and Cx. dolosus and also by the identification of the Culicidae associated fauna. Methods – The redescriptions were based in the analyzes specimens collected in the region of Campos do Jordão and implied in stages of chaetotaxy of the larvae and pupae, biometry of the adults and immature forms, and elaboration of illustrations of the anatomic structures of the external morphology. Results – This work resulted in detailed morphologic redescriptions, chaetotaxy tables of the immature forms and illustrations of the larvae, pupae and male genitalia structures. Final considerations – It is expected that the reached results might expand and continuity taxonomic studies on Culicidae and also contribute to the correct identification of Anopheles and Culex.

Au Vietnam, malgré les succès du gouvernement dans la lutte contre le paludisme, cette maladie persiste en zones forestières, le long des frontières internationales, en particulier avec le Cambodge, et très peu de données sont disponibles sur la filariose lymphatique de Bancroft (FLB). Aucun vaccin n'étant actuellement disponible, la lutte antivectorielle ciblant les Anopheles, moustiques qui peuvent être vecteurs d'agents du paludisme et de la FLB, reste un des éléments clés pour mieux contrôler ces deux maladies. Toutefois, une meilleure connaissance des vecteurs présents en zones endémiques et l'étude de leur capacité vectorielle s'imposaient compte tenu du manque d'information sur la transmission de ces 2 maladies dans la région centre-sud du Vietnam. La capacité vectorielle étant fortement liée à la flore bactérienne de l'estomac des moustiques, sa biodiversité a été estimée afin de mieux appréhender les familles bactériennes présentes chez les moustiques de terrain, en particulier la présence d'Enterobacteriaceae qui peut avoir une influence sur la résistance des anophèles au développement de Plasmodium, agent du paludisme. Ce champ d'investigation n'avait encore jamais été étudié chez les vecteurs du paludisme au Vietnam.Cette thèse porte sur 2 objectifs principaux : (1) l'évaluation de la capacité vectorielle des espèces d'Anopheles dans la co-transmission de Plasmodium spp. et de Wuchereria bancrofti (agent de la FLB) dans la Province de Dak Nong et de Binh Phuoc du centre-sud Vietnam (à la frontière avec le Cambodge) et (2) l'estimation de la biodiversité de la flore bactérienne de l'estomac de populations d'Anopheles du centre-sud Vietnam et l'évaluation de l'influence de certaines bactéries sur la capacité vectorielle des espèces d'Anopheles. L'identification morphologique et moléculaire des spécimens d'Anopheles collectés dans les sites d'étude nous a permis d'appréhender et de mieux connaître la faune anophélienne du centre-sud Vietnam avec la présence de 24 taxa, dont des vecteurs primaires et secondaires. Les espèces dominantes sont Anopheles dirus (48,2%), An. maculatus (19,1%) et An. minimus (9,8%), trois vecteurs majeurs du paludisme et de la FLB. Pour la première fois au Vietnam, la présence d'An. scanloni, une des 8 espèces du Complexe Dirus et vecteur du paludisme en Thaïlande, a été montrée et confirmée après son identification par techniques moléculaires et séquençage. La relation taxonomique entre An. crawfordi et An. dangi, une espèce nommée de façon informelle au Vietnam en 1987, est clarifiée grâce à une étude phylogénétique qui nous a permis d'établir qu'An. dangi est un variant morphologique d'An. crawfordi.Le taux d'infection parasitaire des anophèles a été analysé grâce à des approches PCR, en temps réel et conventionnelle, afin de déterminer la capacité vectorielle des espèces collectées. Parmi les 765 spécimens d'Anopheles étudiés, 2 spécimens dont 1 An. dirus et 1 An. pampanai, ont été trouvés infectés par P. vivax. Les taux d'infection sont de 0,26% sur l'ensemble de l'échantillon, de 0,41% pour la Province de Binh Phuoc et de 0,28% chez An. dirus et 20% chez An. pampanai. Aucun moustique n'a été trouvé infecté ni par P. falciparum, ni par P. knowlesi et ni par Wuchereria bancrofti. La flore bactérienne de l'estomac des anophèles a été analysée après application de 2 méthodes d'identification basées à la fois sur la culture et la PCR du gène 16S sur 200 abdomens d'anophèles de 11 espèces différentes. Les résultats montrent la présence de 116 genres bactériens, dont 18 genres communs, classés taxonomiquement dans 7 phyla dont Acidobacteria, Actinobacteria, Bacteroidetes, Firmicutes, Planctomycetes, Proteobacteria et Synergistetes. Le genre dominant de Dak Nong est Acinetobacter et celui de Binh Phuoc est Staphyloccocus. Acinetobacter est aussi dominant dans la communauté bactérienne de tous les spécimens d'Anopheles étudiés. / In Vietnam, despite the success of the Government in controlling malaria, the disease persists in forest areas along the international borders, especially with Cambodia, and few data are available on the Bancroftian lymphatic filariasis (BLF). No vaccine is currently available to protect from these two diseases, then vector control of Anopheles mosquitoes, that may transmit both malarial and BLF agents, remains one of the key element to control these two diseases. As there is a real lack of information on the transmission of these two diseases in south-central region of Vietnam, it was necessary to apprehend the vectors occurring in endemic areas and to study their vectorial capacity. As bacterial flora in Anopheles midgut may have a strong influence on the vectorial capacity of the mosquito, its biodiversity was estimated in order to better understand bacterial families present in mosquitoes from the field, in particular the presence of Enterobacteriaceae that can have an influence on the development of Plasmodium, agent of malaria. This field of investigation of bacteria has never studied in malaria vectors of Vietnam.This thesis focuses on two main objectives: (1) to evaluate the vectorial capacity of Anopheles species in the co-transmission of Plasmodium spp. and Wuchereria bancrofti (BLF agent) in the Provinces of Dak Nong and Binh Phuoc in south-central Vietnam (near the border with Cambodia), and (2) to estimate the biodiversity of the bacterial flora in the midgut of Anopheles populations of south-central Vietnam and evaluate the influence of certain bacteria on the vectorial capacity of Anopheles species.Morphological and molecular identification of Anopheles specimens collected in the study sites allowed us to apprehend and better understand the Anopheles fauna in south-central Vietnam composed of 24 taxa, including primary and secondary vectors. The dominant species were Anopheles dirus (48.2%), An. maculatus (19.1%) and An. minimus (9.8%), three major vectors of malaria and the BLF. For the first time, An. scanloni, one of the 8 species of Dirus Complex and malaria vector in Thailand, was collected in Vietnam and confirmed by molecular and sequencing techniques. The relationship between An. crawfordi and An. dangi, a species informally named in Vietnam in 1987, was clarified through a phylogenetic study that allows us to establish that An. dangi is a morphological variation of An. crawfordi.The parasites infection rate of Anopheles was investigated using both real-time PCR and conventional PCR to determine the vectorial capacity of the collected species. Of the 765 Anopheles specimens, 2 individuals, such as 1 An. dirus and 1 An. pampanai, were found infected by P. vivax. Then, the infection rates were of 0.26% on the total sample, 0.41% in Binh Phuoc, 0.28% for An. dirus, and 20% for An. pampanai. No mosquito was found infected by either P. falciparum, P. knowlesi or Wuchereria bancrofti.The bacterial flora in Anopheles midguts was analyzed using 2 identification methods based on culture and on 16S PCR-TTGE processed on 200 abdomens of 11 different Anopheles species. The results obtained showed the presence of 116 bacterial genera, including 18 common genera, belonging to 7 phyla such as Acidobacteria, Actinobacteria, Bacteroidetes, Firmicutes, Planctomycetes, Proteobacteria, and Synergistetes. The dominant genus in Dak Nong was Acinetobacter and Staphyloccocus in Binh Phuoc. Acinetobacter was dominant in the bacterial community of all studied Anopheles. The genus Enterobacter, which can influence the development of the Plasmodium, represented a prevalence of 1.7% of the microbiome of our specimens compared to 39.2% for the predominant genus Acinetobacter. This latter genus was significantly associated to Anopheles infected with Plasmodium vivax.

Philosophiae Doctor - PhD / The discovery of microRNAs (miRNAs) is one of the most exciting scientific breakthroughs in the last decade. miRNAs are short RNA molecules that do not encode proteins but instead, regulate gene expression. Over the past several years, thousands of miRNAs have been identified in various insect genomes through cloning and sequencing, and even by computational prediction. However, information concerning possible roles of miRNAs in mosquitoes is limited. Within this context, we report here the first systematic analysis of these tiny RNAs and their target mRNAs in one of the principal African malaria vectors, Anopheles funestus s.s. Firstly, to extend the known repertoire of miRNAs expressed in this insect, the small RNAs from the four developmental stages (egg, larvae, pupae and the adult females), were sequenced using next generation sequencing technology. A total of 98 miRNAs were identified, which included 65 known Anopheles miRNAs, 25 miRNAs conserved in other insects and 8 novel miRNAs that had not been reported in any species. We further characterized new variants for miR-2 and miR-927 and stem-loop precursors for miR-286 and miR-2944. The analysis showed that many miRNAs have stage-specific expression, and co-transcribed and co-regulated during development. Secondly, for a better understanding of the molecular details of the miRNAs function, we identified the target genes for the Anopheles miRNAs using a novel approach that identifies overlap genes among three target prediction tools followed by filtering genes based on functional enrichment of GO terms and KEGG pathways. We found that most of the miRNAs are metabolic regulators. Moreover, the results suggest implication of some miRNAs not only in the development but also in insect-parasite interaction. Finally, we developed the InsecTar database (http://insectar.sanbi.ac.za) for miRNA targets in the three mosquito species; Anopheles gambiae, Aedes aegypti, and Culex quinquefasciatus, which incorporates prediction and the functional analysis of these target genes. The proposed database will undoubtedly assist to explore the roles of these regulatory molecules in insects. This type of analysis is a key step towards improving our understanding of the complexity and regulationmode of miRNAs in mosquitoes. Moreover, this study opens the door for exploration of miRNA in regulation of critical physiological functions specific to vector arthropods which may lead to novel approaches to combat mosquito-borne infectious diseases.

Dans les savanes d'Afrique de l'Ouest, les moustiques Anopheles coluzzii et An. gambiae, qui transmettent le paludisme, pullulent en saison des pluies et disparaissent en saison sèche. Les mécanismes permettant le maintien des populations d'Anophèles d'une année sur l'autre restent inconnus et difficiles à mettre en évidence sur le terrain. Au cours de ce travail, nous avons étudié, par des approches comparatives et expérimentales, la plasticité physiologique et morphologique des femelles An. coluzzii et An. gambiae exprimée en réponse aux conditions déshydratantes de la saison sèche. Des analyses métabolomiques, protéomiques, morphométriques et respirométriques, nous ont permis de mettre en évidence une grande variabilité des profils exprimés entre les espèces et entre les différentes populations d'An. coluzzii. / In dry savannahs of West Africa, the malarial mosquitoes Anopheles coluzzii and An. gambiae swarm during the rainy season but almost disappear at the onset of the dry season. Interestingly, the ecological and physiological bases enhancing the survival of these insects during the desiccating conditions of the dry season remain unknown, and hard to understand with field studies. In the present work, we conducted experimental and comparative studies to analyse the physiological and morphological plasticity of female An. coluzzii and An. gambiae mosquitoes in response to the environmental conditions of the dry season. Metabolomic, proteomic, morphometric and gas exchange analyses highlighted a wide range of responses to the dry season conditions in these species, and enables distinguishing the two considered species.

Les militaires français sont engagés depuis 2002 dans des opérations de lutte contre l’orpaillage illégal en Guyane. Malgré un programme de prévention du paludisme dimensionné et organisé, ces opérations se sont accompagnées d’une augmentation de l’incidence du paludisme. Cette thèse a pour objectif d’identifier les déterminants humains, vectoriels et parasitaires du paludisme en Guyane. La première partie de cette thèse permet de mieux décrire le faciès épidémiologique du paludisme en forêt guyanaise. Nous avons clairement établi le lien entre paludisme et orpaillage illégal. Une étude, menée en milieu orpailleur a démontré l’existence de foyers de paludisme hyperendémiques, avec une prédominance de P. falciparum et un risque d’émergence de résistance aux traitements combinés à base d’artémisinine. Les investigations entomologiques ont permis de confirmer le rôle d’Anopheles darlingi comme vecteur majeur du paludisme en forêt mais aussi l’existence d’une activité de piqûre diurne sous la canopée. Elles ont permis en outre de mieux identifier les autres vecteurs secondaires en forêt : An. nuneztovari et An. triannulatus, mais surtout An. ininii et An. marajoara. En seconde partie, une étude de cohorte en population militaire a permis d’estimer l’exposition au paludisme par l’utilisation d’outils sérologiques. La distribution des preuves sérologiques d’une infection palustre était en faveur d’une exposition plus fréquente à P. falciparum. Les taux d’incidence des infections étaient de 40 pour 100 personnes années que ce soit pour P. falciparum ou P. vivax. Le seul facteur protecteur était l’observance parfaite de la chimioprophylaxie par doxycycline. / Since 2002, French armed forces are involved in operations against illegal gold mining in French Guiana. Despite a properly dimensioned prevention program against malaria conducted, a drastic increase of malaria incidence has been observed among military personnel. The first part of this thesis aimed to describe the determinants of malaria in French Guiana forest, including human, vector and parasite features. We have established the link between malaria among military personnel and illegal gold mining sites. A study conducted among gold miners’ population has shown hyperendemic malaria foci in the rain forest, mainly due to P. falciparum, and highlighted a real risk for emergence of artemisinin resistance. Entomologic investigations supported that Anopheles darlingi is the main vector for malaria in rain forest. In addition, we have demonstrated the existence of daytime biting activity by An. darlingi in the forest, which might play a key role in malaria epidemic outbreaks among military personnel. Other sylvatic vectors were identified, as An. nuneztovari and An. triannulatus, but especially An. ininii and An. marajoara. The second part of the thesis, focused on a prospective cohort study conducted among French military population, highlighted real malaria exposure using serological tools. Serological evidences of Plasmodium infection (SEI) were more frequent for P. falciparum than P. vivax, in agreement with our findings in illegal gold miners’ population. SEI incidence rates were high, around 40 per 100 person-years for P. falciparum and P. vivax. Only complete compliance to malaria chemoprophylaxis using daily 100 mg doxycycline protected against malaria infection.