Variation between geographical populations of Lutzomyia (Nyssomyia) whitmani (Antunes & Coutinho, 1939) sensu lato (Diptera:Psychodidae:Phlebotominae) in Brazil

Phylogenetic analysis of morphometric and biological characters indicated that there are two distinct forms of Lutzomyia whitmani in Brazil: one is present both north and south of the River Amazonas in the State of Pará while the other occurs in northeast Brazil, in the State of Ceará, and further south, including the type locality in State of Bahia. The Amazonian form is reportedly neither strongly anthropophilic nor synanthropic, and it is the vector of Leishmania shawi; whereas the southern form is often collected peridomestically, while biting man, and has been found infected with Le.(V.) braziliensis. The ratio of the length of the genital filaments to that the genital pump was found to be consistently smaller in males of the Amazonian populations. A middle repetitive DNA element was isolated by differentially screening a genomic library made using Amazonian material, and the sequence was diagnostic for this form of Lu. whitmani (being absent or occurring in low copy number in the southern form). The total evidence suggests there are at least two, geographically-isolated forms of Lu. whitmani, which may represent different cryptic species.

Biological variation in Anopheles darlingi root

Behavioural variation in the South American malaria vector Anopheles darlingi is described. At the centre of its distribution, in forest areas close to the city of Manaus, Brazil, it is primarily exophagic and exophilic. Mosquitoes from this area are chromosomally diverse. Towards the northern edge of its distribution (in Guyana and Venezuela) it is more endophagic and less diverse chromosomally. Similarly in the south (in the state of Minas Gerais) it is less polymorphic. In this area, however, it is primarily zoophilic and exophagic. Evidence is presented that female wing size may vary between populations. The possibility that this widely distributed species may be a complex could have important implications for future malaria control schemes.

Mating system and avpr1a promoter variation in primates.

It has been suggested that primate mating and social behaviours may be influenced by variation in promoter region repetitive DNA of the vasopressin receptor 1a gene (avpr1a). We show that male mating behaviour does not covary in a simple way with promoter repetitive DNA in 12 Old World primates. We found that one microsatellite (-553 bp upstream) was present in all species, irrespective of their behaviour. By contrast, two microsatellites (-3956 and -3625 bp upstream) were present only in some species, yet this variation did not correlate with behaviour. These findings agree with a recent comparative analysis of voles and show that the variation in repetitive DNA in the avpr1a promoter region does not generally explain variation in male mating behaviour. Phylogenetic analysis revealed a GAGTA motif that has been independently deleted three times and involved in another larger deletion. Importantly, the presence/absence of this GAGTA motif leads to changes in predicted transcription factor-binding sites. Given the repeated loss of this motif, we speculate that it might be of functional relevance. We suggest that such non-repetitive variation, either in indels or in sequence variation, are likely to be important in explaining interspecific variation in avpr1a expression.

Variation in novel exons (RACEfrags) of the MECP2 gene in Rett syndrome patients and controls.

The study of transcription using genomic tiling arrays has lead to the identification of numerous additional exons. One example is the MECP2 gene on the X chromosome; using 5'RACE and RT-PCR in human tissues and cell lines, we have found more than 70 novel exons (RACEfrags) connecting to at least one annotated exon.. We sequenced all MECP2-connected exons and flanking sequences in 3 groups: 46 patients with the Rett syndrome and without mutations in the currently annotated exons of the MECP2 and CDKL5 genes; 32 patients with the Rett syndrome and identified mutations in the MECP2 gene; 100 control individuals from the same geoethnic group. Approximately 13 kb were sequenced per sample, (2.4 Mb of DNA resequencing). A total of 75 individuals had novel rare variants (mostly private variants) but no statistically significant difference was found among the 3 groups. These results suggest that variants in the newly discovered exons may not contribute to Rett syndrome. Interestingly however, there are about twice more variants in the novel exons than in the flanking sequences (44 vs. 21 for approximately 1.3 Mb sequenced for each class of sequences, p=0.0025). Thus the evolutionary forces that shape these novel exons may be different than those of neighboring sequences.

Intra- and interindividual variation in flank gland secretions of free-ranging shrews Crocidura russula

Individual differences in Rank gland secretions were examined among males of the monogamous shrew Crocidura russula during the breeding and nonbreeding seasons. Gas chromatography was used to measure intra- and interindividual variation of flank gland secretions of free-ranging shrews from different populations. The number of compounds detected by gas chromatographic analyses was correlated with body mass, flank gland size, and the presence of blood parasites in individual shrews. Very few compounds were detected from the Bank gland area of juvenile males. After they reached sexual maturity, however, the number of compounds detected from the Rank gland secretions increased significantly. At the beginning of the reproductive season 48 different compounds were detected from male flank gland secretions. In the middle of the breeding season 70 compounds were detected, while only 11 compounds were detected during the nonbreeding season. Few compounds were common to all males. There were more volatile compounds in the Bank gland secretions of males in the beginning of the breeding season than later in the breeding season. Males from the same population had fewer differences in the elution profile of compounds than males from different populations indicating that individuals from a distinct population have similar elution profiles of compounds and that each population has its own type of elution profile. No correlations were found between the number of compounds detected by gas chromatography for each male and the male's body mass or flank gland size. Blood parasites (trypanosomes, Trypanosoma crocidurae) were found in only three of 30 males investigated.

Diagnostic interview for genetic studies (DIGS): inter-rater and test-retest reliability of the French version.

The National Institute of Mental Health developed the semi-structured Diagnostic Interview for Genetic Studies (DIGS) for the assessment of major mood and psychotic disorders and their spectrum conditions. The DIGS was translated into French in a collaborative effort of investigators from sites in France and Switzerland. Inter-rater and test-retest reliability of the French version have been established in a clinical sample in Lausanne. Excellent inter-rater reliability was found for schizophrenia, bipolar disorder, major depression, and unipolar schizoaffective disorder while fair inter-rater reliability was demonstrated for bipolar schizoaffective disorder. Using a six-week test-retest interval, reliability for all diagnoses was found to be fair to good with the exception of bipolar schizoaffective disorder. The lower test-retest reliability was the result of a relatively long test-retest interval that favored incomplete symptom recall. In order to increase reliability for lifetime diagnoses in persons not currently affected, best-estimate procedures using additional sources of diagnostic information such as medical records and reports from relatives should supplement DIGS information in family-genetic studies. Within such a procedure, the DIGS appears to be a useful part of data collection for genetic studies on major mood disorders and schizophrenia in French-speaking populations.

Isoenzymes Detect Variation in Populations of Triatoma brasiliensis (Hemiptera: Reduviidae: Triatominae)

Triatoma brasiliensis is one of the most important vectors of Chagas disease in the semiarid zone of the northeast of Brazil. Intraspecific morphological and behavioural variation has been reported for different populations. Results for four distinct populations using eight isoenzymes are reported here. The literature describes three subspecies: T. brasiliensis brasiliensis Neiva, 1911; T. brasiliensis melanica Neiva & Lent, 1941 and T. brasiliensis macromelasoma Galvão, 1956. These subspecies differ mainly in their cuticle colour pattern and were regarded as synonyms by Lent and Wygodzinsky (1979). In order to evaluate whether the chromatic pattern is a morphological variation of different melanic forms within T. brasiliensis or due to interspecific variation, field collections were performed in localities where these three subspecies have been described: Caicó (Rio Grande do Norte), the type-locality for T. b. brasiliensis; Petrolina (Pernambuco) for T. b. macromelasoma and Espinosa (Minas Gerais) for T. b. melanica. A fourth distinct chromatic pattern was found in Juazeiro (Bahia). A total of nine loci were studied. Values of Nei's genetic distance (D) were calculated. T. b. brasiliensis and T. b. macromelasoma are the closest populations with a D=0.295. T. b. melanica had a D ³ 0.537 when compared to the others, a distance in the range of interspecific variation for other triatomine species

Genetic Variation among Natural and Laboratory Colony Populations of Lutzomyia longipalpis (Lutz & Neiva, 1912)(Diptera:Psychodidae) from Colombia

Genetic diversity among three field populations of Lutzomyia longipalpis in Colombia was studied using isozyme analysis. Study sites were as much as 598 km apart and included populations separated by the eastern Cordillera of the Andes. Genetic variability among populations, estimated by heterozygosity, was within values typical for insects in general (8.1%). Heterozygosity for field populations were compared with a laboratory colony from Colombia (Melgar colony) and were only slightly lower. These results suggest that establishment and long term maintenance of the Melgar colony has had little effect on the level of isozyme variability it carries. Genetic divergences between populations was evaluated using estimates of genetic distance. Genetic divergence among the three field populations was low (D=0.021), suggesting they represent local populations within a single species. Genetic distance between field populations and the Melgar colony was also low (D=0.016), suggesting that this colony population does not depart significantly from natural populations. Finally, comparisons were made between Colombian populations and colonies from Brazil and Costa Rica. Genetic distance values were high between Colombian and both Brazil and Costa Rica colony populations (D=0.199 and 0.098 respectively) providing additional support for our earlier report that populations from the three countries represent distinct species

Evolutionary Control of Infectious Disease: Prospects for Vectorborne and Waterborne Pathogens

Evolutionary theory may contribute to practical solutions for control of disease by identifying interventions that may cause pathogens to evolve to reduced virulence. Theory predicts, for example, that pathogens transmitted by water or arthropod vectors should evolve to relatively high levels of virulence because such pathogens can gain the evolutionary benefits of relatively high levels of host exploitation while paying little price from host illness. The entrance of Vibrio cholerae into South America in 1991 has generated a natural experiment that allows testing of this idea by determining whether geographic and temporal variations in toxigenicity correspond to variation in the potential for waterborne transmission. Preliminary studies show such correspondences: toxigenicity is negatively associated with access to uncontaminated water in Brazil; and in Chile, where the potential for waterborne transmission is particularly low, toxigenicity of strains declined between 1991 and 1998. In theory vector-proofing of houses should be similarly associated with benignity of vectorborne pathogens, such as the agents of dengue, malaria, and Chagas' disease. These preliminary studies draw attention to the need for definitive prospective experiments to determine whether interventions such as provisioning of uncontaminated water and vector-proofing of houses cause evolutionary reductions in virulence

Segmental copy number variation shapes tissue transcriptomes

Abstract : Copy number variation (CNV) of DNA segments has recently gained considerable interest as a source of genetic variation likely to play a role in phenotypic diversity and evolution. Much effort has been put into the identification and mapping of regions that vary in copy number among seemingly normal individuals, both in humans and in a number of model organisms, using both bioinformatic and hybridization-based methods. Synteny studies suggest the existence of CNV hotspots in mammalian genomes, often in connection with regions of segmental duplication. CNV alleles can be in equilibrium within a population, but can also arise de novo between generations, illustrating the highly dynamic nature of these regions. A small number of studies have assessed the effect of CNV on single loci, however, at the genome-wide scale, the functional impact of CNV remains poorly studied. We have explored the influence of CNV on gene expression, first using the Williams-Beuren syndrome (WBS) associated deletion as a model, and second at the genome-wide scale in inbred mouse strains. We found that the WBS deletion influences the expression levels not only of the hemizygous genes, but also affects the euploid genes mapping nearby. Consistently, on a genome wide scale we observe that CNV genes are expressed at more variable levels than genes that do not vary in copy number. Likewise, CNVs influence the relative expression levels of genes that map to the flank of the genome rearrangements, thus globally influencing tissue transcriptomes. Further studies are warranted to complete cataloguing and fine mapping of CNV regions, as well as to elucidate the different mechanisms by which CNVs influence gene expression. Résumé : La variation en nombre de copies (copy number variation ou CNV) de segments d'ADN suscite un intérêt en tant que variation génétique susceptible de jouer un r81e dans la diversité phénotypique et l'évolution. Les régions variables en nombre de copies parmi des individus apparemment normaux ont été cartographiées et cataloguées au moyen de puces à ADN et d'analyse bioinformatique. L'étude de la synténie entre plusieurs espèces de mammifères laisse supposer l'existence de régions à haut taux de variation, souvent liées à des duplications segmentaires. Les allèles CNV peuvent être en équilibre au sein d'une population ou peuvent apparaître de novo. Ces faits illustrent la nature hautement dynamique de ces régions. Quelques études se sont penchées sur l'effet de la variation en nombre de copies de loci isolés, cependant l'impact de ce phénomène n'a pas été étudié à l'échelle génomique. Nous avons examiné l'influence des CNV sur l'expression des gènes. Dans un premier temps nous avons utilisé la délétion associée au syndrome de Williams-Beuren (WBS), puis, dans un second temps, nous avons poursuivi notre étude à l'échelle du génome, dans des lignées consanguines de souris. Nous avons établi que la délétion WBS influence l'expression non seulement des gènes hémizygotes, mais également celle des gènes euploïdes voisins. A l'échelle génomique, nous observons des phénomènes concordants. En effet, l'expression des gènes variant en nombre de copies est plus variable que celles des gènes ne variant pas. De plus, à l'instar de la délétion WBS, les CNV influencent l'expression des gènes adjacents, exerçant ainsi un impact global sur les profils d'expression dans les tissus. Résumé pour un large public : De nombreuses maladies ont pour cause un défaut génétique. Parmi les types de mutations, on compte la disparition (délétion) d'une partie de notre génome ou sa duplication. Bien que l'on connaisse les anomalies associées à certaines maladies, les mécanismes moléculaires par lesquels ces réarrangements de notre matériel génétique induisent les maladies sont encore méconnus. C'est pourquoi nous nous sommes intéressés à la régulation des gènes dans les régions susceptibles à délétion ou duplication. Dans ce travail, nous avons démontré que les délétions et les duplications influencent la régulation des gènes situés à proximité, et que ces changements interviennent dans plusieurs organes.

Molecular Basis of Ribotype Variation in the Seventh Pandemic Clone and its O139 Variant of Vibrio cholerae

Ribotyping has been widely used to characterise the seventh pandemic clone including South American and O139 variants which appeared in 1991 and 1992 respectively. To reveal the molecular basis of ribotype variation we analysed the rrn operons and their flanking regions. All but one variation detected by BglI, the most discriminatory enzyme, was found to be due to changes within the rrn operons, resulting from recombination between operons. The recombinants are detected because of the presence of a BglI site in the 16S gene in three of the nine rrn operons and/or changes of intergenic spacer types of which four variants were identified. As the frequency of rrn recombination is high, ribotyping becomes a less useful tool for evolutionary studies and long term monitoring of the pathogenic clones of Vibrio cholerae as variation could undergo precise reversion by the same recombination event.

Mechanisms Mediating Antigenic Variation in Trypanosoma brucei

Antigenic variation in Trypanosoma brucei is a highly sophisticated survival strategy involving switching between the transcription of one of an estimated thousand variant surface glycoprotein (VSG) genes. Switching involves either transcriptional control, resulting in switching between different VSG expression sites; or DNA rearrangement events slotting previously inactive VSG genes into an active VSG expression site. In recent years, considerable progress has been made in techniques allowing us to genetically modify infective bloodstream form trypanosomes. This is allowing us to reengineer VSG expression sites, and look at the effect on the mechanisms subsequently used for antigenic variation. We can now begin a dissection of a highly complicated survival strategy mediated by many different mechanisms operating simultaneously.

Mitochondrial DNA sequence variation within the remnant populations of the endangered numbat (Marsupialia: Myrmecobiidae: Myrmecobius fasciatus).

The numbat has been reduced to two populations in Western Australia. To better understand the effects of range reduction on gene flow and genetic variation, and to address questions crucial for the species' management, we analysed mitochondrial DNA (mtDNA) sequences of free-ranging individuals and museum specimens. The results suggest recent connectivity between the remnant populations, although one of those may have lost significant amounts of genetic diversity during the recent population size reduction. We propose that for management purposes the remnant populations should be treated as a single historical lineage and that, subject to certain caveats, consideration should be given to population augmentation by translocation.

Systems biology of plants : from intraspecific genome variation to computational morphodynamics

Recently, the introduction of second generation sequencing and further advance-ments in confocal microscopy have enabled system-level studies for the functional characterization of genes. The degree of complexity intrinsic to these approaches needs the development of bioinformatics methodologies and computational models for extracting meaningful biological knowledge from the enormous amount of experi¬mental data which is continuously generated. This PhD thesis presents several novel bioinformatics methods and computational models to address specific biological questions in Plant Biology by using the plant Arabidopsis thaliana as a model system. First, a spatio-temporal qualitative analysis of quantitative transcript and protein profiles is applied to show the role of the BREVIS RADIX (BRX) protein in the auxin- cytokinin crosstalk for root meristem growth. Core of this PhD work is the functional characterization of the interplay between the BRX protein and the plant hormone auxin in the root meristem by using a computational model based on experimental evidence. Hyphotesis generated by the modelled to the discovery of a differential endocytosis pattern in the root meristem that splits the auxin transcriptional response via the plasma membrane to nucleus partitioning of BRX. This positional information system creates an auxin transcriptional pattern that deviates from the canonical auxin response and is necessary to sustain the expression of a subset of BRX-dependent auxin-responsive genes to drive root meristem growth. In the second part of this PhD thesis, we characterized the genome-wide impact of large scale deletions on four divergent Arabidopsis natural strains, through the integration of Ultra-High Throughput Sequencing data with data from genomic hybridizations on tiling arrays. Analysis of the identified deletions revealed a considerable portion of protein coding genes affected and supported a history of genomic rearrangements shaped by evolution. In the last part of the thesis, we showed that VIP3 gene in Arabidopsis has an evo-lutionary conserved role in the 3' to 5' mRNA degradation machinery, by applying a novel approach for the analysis of mRNA-Seq data from random-primed mRNA. Altogether, this PhD research contains major advancements in the study of natural genomic variation in plants and in the application of computational morphodynamics models for the functional characterization of biological pathways essential for the plant. - Récemment, l'introduction du séquençage de seconde génération et les avancées dans la microscopie confocale ont permis des études à l'échelle des différents systèmes cellulaires pour la caractérisation fonctionnelle de gènes. Le degrés de complexité intrinsèque à ces approches ont requis le développement de méthodologies bioinformatiques et de modèles mathématiques afin d'extraire de la masse de données expérimentale générée, des information biologiques significatives. Ce doctorat présente à la fois des méthodes bioinformatiques originales et des modèles mathématiques pour répondre à certaines questions spécifiques de Biologie Végétale en utilisant la plante Arabidopsis thaliana comme modèle. Premièrement, une analyse qualitative spatio-temporelle de profiles quantitatifs de transcripts et de protéines est utilisée pour montrer le rôle de la protéine BREVIS RADIX (BRX) dans le dialogue entre l'auxine et les cytokinines, des phytohormones, dans la croissance du méristème racinaire. Le noyau de ce travail de thèse est la caractérisation fonctionnelle de l'interaction entre la protéine BRX et la phytohormone auxine dans le méristème de la racine en utilisant des modèles informatiques basés sur des preuves expérimentales. Les hypothèses produites par le modèle ont mené à la découverte d'un schéma différentiel d'endocytose dans le méristème racinaire qui divise la réponse transcriptionnelle à l'auxine par le partitionnement de BRX de la membrane plasmique au noyau de la cellule. Cette information positionnelle crée une réponse transcriptionnelle à l'auxine qui dévie de la réponse canonique à l'auxine et est nécessaire pour soutenir l'expression d'un sous ensemble de gènes répondant à l'auxine et dépendant de BRX pour conduire la croissance du méristème. Dans la seconde partie de cette thèse de doctorat, nous avons caractérisé l'impact sur l'ensemble du génome des délétions à grande échelle sur quatre souches divergentes naturelles d'Arabidopsis, à travers l'intégration du séquençage à ultra-haut-débit avec l'hybridation génomique sur puces ADN. L'analyse des délétions identifiées a révélé qu'une proportion considérable de gènes codant était affectée, supportant l'idée d'un historique de réarrangement génomique modelé durant l'évolution. Dans la dernière partie de cette thèse, nous avons montré que le gène VÏP3 dans Arabidopsis a conservé un rôle évolutif dans la machinerie de dégradation des ARNm dans le sens 3' à 5', en appliquant une nouvelle approche pour l'analyse des données de séquençage d'ARNm issue de transcripts amplifiés aléatoirement. Dans son ensemble, cette recherche de doctorat contient des avancées majeures dans l'étude des variations génomiques naturelles des plantes et dans l'application de modèles morphodynamiques informatiques pour la caractérisation de réseaux biologiques essentiels à la plante. - Le développement des plantes est écrit dans leurs codes génétiques. Pour comprendre comment les plantes sont capables de s'adapter aux changements environnementaux, il est essentiel d'étudier comment leurs gènes gouvernent leur formation. Plus nous essayons de comprendre le fonctionnement d'une plante, plus nous réalisons la complexité des mécanismes biologiques, à tel point que l'utilisation d'outils et de modèles mathématiques devient indispensable. Dans ce travail, avec l'utilisation de la plante modèle Arabidopsis thalicinci nous avons résolu des problèmes biologiques spécifiques à travers le développement et l'application de méthodes informatiques concrètes. Dans un premier temps, nous avons investigué comment le gène BREVIS RADIX (BRX) régule le développement de la racine en contrôlant la réponse à deux hormones : l'auxine et la cytokinine. Nous avons employé une analyse statistique sur des mesures quantitatives de transcripts et de produits de gènes afin de démontrer que BRX joue un rôle antagonisant dans le dialogue entre ces deux hormones. Lorsque ce-dialogue moléculaire est perturbé, la racine primaire voit sa longueur dramatiquement réduite. Pour comprendre comment BRX répond à l'auxine, nous avons développé un modèle informatique basé sur des résultats expérimentaux. Les simulations successives ont mené à la découverte d'un signal positionnel qui contrôle la réponse de la racine à l'auxine par la régulation du mouvement intracellulaire de BRX. Dans la seconde partie de cette thèse, nous avons analysé le génome entier de quatre souches naturelles d'Arabidopsis et nous avons trouvé qu'une grande partie de leurs gènes étaient manquant par rapport à la souche de référence. Ce résultat indique que l'historique des modifications génomiques conduites par l'évolution détermine une disponibilité différentielle des gènes fonctionnels dans ces plantes. Dans la dernière partie de ce travail, nous avons analysé les données du transcriptome de la plante où le gène VIP3 était non fonctionnel. Ceci nous a permis de découvrir le rôle double de VIP3 dans la régulation de l'initiation de la transcription et dans la dégradation des transcripts. Ce rôle double n'avait jusqu'alors été démontrée que chez l'homme. Ce travail de doctorat supporte le développement et l'application de méthodologies informatiques comme outils inestimables pour résoudre la complexité des problèmes biologiques dans la recherche végétale. L'intégration de la biologie végétale et l'informatique est devenue de plus en plus importante pour l'avancée de nos connaissances sur le fonctionnement et le développement des plantes.

Annual variation in the biochemical composition of newly hatched larvae of Maja brachydactyla in captivity

Quality of newly hatched larvae (NHL) of Maja brachydactyla in captivity has been characterized throughout the year to evaluate their availability for mass production. Spawning took place every month and NHL were collected and analyzed to estimate individual dry weight (DW) and proximate biochemical composition (protein, carbohydrate and lipids). Lipid class, fatty acid composition, amino acid profile, mineral and vitamins A, E and C contents were analyzed seasonally. NHL obtained throughout the year are a potential source for aquaculture purposes, since the increment in the relative protein and lipid (especially phospholipids and n-3 PUFA) content might compensate the decrease in DW of larvae hatched from broodstock kept during one year in captivity. However, the decrease in vitamins A and E as well as in certain essential amino acids (Lys, Val, and His) and trace elements (Cu and Fe) of NHL at the end of the year might be indicative of a nutritional deficiency in broodstock diets.