De novo transcriptome assembly for a non-model species, the blood-sucking bug Triatoma brasiliensis, a vector of Chagas disease

High throughput sequencing (HTS) provides new research opportunities for work on non-model organisms, such as differential expression studies between populations exposed to different environmental conditions. However, such transcriptomic studies first require the production of a reference assembly. The choice of sampling procedure, sequencing strategy and assembly workflow is crucial. To develop a reliable reference transcriptome for Triatoma brasiliensis, the major Chagas disease vector in Northeastern Brazil, different de novo assembly protocols were generated using various datasets and software. Both 454 and Illumina sequencing technologies were applied on RNA extracted from antennae and mouthparts from single or pooled individuals. The 454 library yielded 278 Mb. Fifteen Illumina libraries were constructed and yielded nearly 360 million RNA-seq single reads and 46 million RNA-seq paired-end reads for nearly 45 Gb. For the 454 reads, we used three assemblers, Newbler, CAP3 and/or MIRA and for the Illumina reads, the Trinity assembler. Ten assembly workflows were compared using these programs separately or in combination. To compare the assemblies obtained, quantitative and qualitative criteria were used, including contig length, N50, contig number and the percentage of chimeric contigs. Completeness of the assemblies was estimated using the CEGMA pipeline. The best assembly (57,657 contigs, completeness of 80 %, < 1 % chimeric contigs) was a hybrid assembly leading to recommend the use of (1) a single individual with large representation of biological tissues, (2) merging both long reads and short paired-end Illumina reads, (3) several assemblers in order to combine the specific advantages of each.

Genetic variation and inference of demographic histories in non-model species

Both long-term environmental changes such as those driven by the glacial cycles and more recent anthropogenic impacts have had major effects on the past demography in wild organisms. Within species, these changes are reflected in the amount and distribution of neutral genetic variation. In this thesis, mitochondrial and microsatellite DNA was analysed to investigate how environmental and anthropogenic factors have affected genetic diversity and structure in four ecologically different animal species. Paper I describes the post-glacial recolonisation history of the speckled-wood butterfly (Pararge aegeria) in Northern Europe. A decrease in genetic diversity with latitude and a marked population structure were uncovered, consistent with a hypothesis of repeated founder events during the postglacial recolonisation. Moreover, Approximate Bayesian Computation analyses indicate that the univoltine populations in Scandinavia and Finland originate from recolonisations along two routes, one on each side of the Baltic. Paper II aimed to investigate how past sea-level rises affected the population history of the convict surgeonfish (Acanthurus triostegus) in the Indo-Pacific. Assessment of the species’ demographic history suggested a population expansion that occurred approximately at the end of the last glaciation. Moreover, the results demonstrated an overall lack of phylogeographic structure, probably due to the high dispersal rates associated with the species’ pelagic larval stage. Populations at the species’ eastern range margin were significantly differentiated from other populations, which likely is a consequence of their geographic isolation. In Paper III, we assessed the effect of human impact on the genetic variation of European moose (Alces alces) in Sweden. Genetic analyses revealed a spatial structure with two genetic clusters, one in northern and one in southern Sweden, which were separated by a narrow transition zone. Moreover, demographic inference suggested a recent population bottleneck. The inferred timing of this bottleneck coincided with a known reduction in population size in the 19th and early 20th century due to high hunting pressure. In Paper IV, we examined the effect of an indirect but well-described human impact, via environmental toxic chemicals (PCBs), on the genetic variation of Eurasian otters (Lutra lutra) in Sweden. Genetic clustering assignment revealed differentiation between otters in northern and southern Sweden, but also in the Stockholm region. ABC analyses indicated a decrease in effective population size in both northern and southern Sweden. Moreover, comparative analyses of historical and contemporary samples demonstrated a more severe decline in genetic diversity in southern Sweden compared to northern Sweden, in agreement with the levels of PCBs found.

Assembly and annotation of a non-model gastropod (Nerita melanotragus) transcriptome: a comparison of De novo assemblers

Background The sequencing, de novo assembly and annotation of transcriptome datasets generated with next generation sequencing (NGS) has enabled biologists to answer genomic questions in non-model species with unprecedented ease. Reliable and accurate de novo assembly and annotation of transcriptomes, however, is a critically important step for transcriptome assemblies generated from short read sequences. Typical benchmarks for assembly and annotation reliability have been performed with model species. To address the reliability and accuracy of de novo transcriptome assembly in non-model species, we generated an RNAseq dataset for an intertidal gastropod mollusc species, Nerita melanotragus, and compared the assembly produced by four different de novo transcriptome assemblers; Velvet, Oases, Geneious and Trinity, for a number of quality metrics and redundancy. Results Transcriptome sequencing on the Ion Torrent PGM™ produced 1,883,624 raw reads with a mean length of 133 base pairs (bp). Both the Trinity and Oases de novo assemblers produced the best assemblies based on all quality metrics including fewer contigs, increased N50 and average contig length and contigs of greater length. Overall the BLAST and annotation success of our assemblies was not high with only 15-19% of contigs assigned a putative function. Conclusions We believe that any improvement in annotation success of gastropod species will require more gastropod genome sequences, but in particular an increase in mollusc protein sequences in public databases. Overall, this paper demonstrates that reliable and accurate de novo transcriptome assemblies can be generated from short read sequencers with the right assembly algorithms. Keywords: Nerita melanotragus; De novo assembly; Transcriptome; Heat shock protein; Ion torrent

Imaging in vivo Neuronal Transport in Genetic Model Organisms Using Microfluidic Devices

Microfluidic devices have been developed for imaging behavior and various cellular processes in Caenorhabditis elegans, but not subcellular processes requiring high spatial resolution. In neurons, essential processes such as axonal, dendritic, intraflagellar and other long-distance transport can be studied by acquiring fast time-lapse images of green fluorescent protein (GFP)-tagged moving cargo. We have achieved two important goals in such in vivo studies namely, imaging several transport processes in unanesthetized intact animals and imaging very early developmental stages. We describe a microfluidic device for immobilizing C. elegans and Drosophila larvae that allows imaging without anesthetics or dissection. We observed that for certain neuronal cargoes in C. elegans, anesthetics have significant and sometimes unexpected effects on the flux. Further, imaging the transport of certain cargo in early developmental stages was possible only in the microfluidic device. Using our device we observed an increase in anterograde synaptic vesicle transport during development corresponding with synaptic growth. We also imaged Q neuroblast divisions and mitochondrial transport during early developmental stages of C. elegans and Drosophila, respectively. Our simple microfluidic device offers a useful means to image high-resolution subcellular processes in C. elegans and Drosophila and can be readily adapted to other transparent or translucent organisms.

Salmonella enterica serovars Typhimurium and Typhi as model organisms Revealing paradigm of host-pathogen interactions

The lifestyle of intracellular pathogens has always questioned the skill of a microbiologist in the context of finding the permanent cure to the diseases caused by them. The best tool utilized by these pathogens is their ability to reside inside the host cell, which enables them to easily bypass the humoral immunity of the host, such as the complement system. They further escape from the intracellular immunity, such as lysosome and inflammasome, mostly by forming a protective vacuole-bound niche derived from the host itself. Some of the most dreadful diseases are caused by these vacuolar pathogens, for example, tuberculosis by Mycobacterium or typhoid fever by Salmonella. To deal with such successful pathogens therapeutically, the knowledge of a host-pathogen interaction system becomes primarily essential, which further depends on the use of a model system. A well characterized pathogen, namely Salmonella, suits the role of a model for this purpose, which can infect a wide array of hosts causing a variety of diseases. This review focuses on various such aspects of research on Salmonella which are useful for studying the pathogenesis of other intracellular pathogens.

Colored polydimethylsiloxane micropillar arrays for high throughput measurements of forces applied by genetic model organisms

Measuring forces applied by multi-cellular organisms is valuable in investigating biomechanics of their locomotion. Several technologies have been developed to measure such forces, for example, strain gauges, micro-machined sensors, and calibrated cantilevers. We introduce an innovative combination of techniques as a high throughput screening tool to assess forces applied by multiple genetic model organisms. First, we fabricated colored Polydimethylsiloxane (PDMS) micropillars where the color enhances contrast making it easier to detect and track pillar displacement driven by the organism. Second, we developed a semiautomated graphical user interface to analyze the images for pillar displacement, thus reducing the analysis time for each animal to minutes. The addition of color reduced the Young's modulus of PDMS. Therefore, the dye-PDMS composite was characterized using Yeoh's hyperelastic model and the pillars were calibrated using a silicon based force sensor. We used our device to measure forces exerted by wild type and mutant Caenorhabditis elegans moving on an agarose surface. Wild type C. elegans exert an average force of similar to 1 mu N on an individual pillar and a total average force of similar to 7.68 mu N. We show that the middle of C. elegans exerts more force than its extremities. We find that C. elegans mutants with defective body wall muscles apply significantly lower force on individual pillars, while mutants defective in sensing externally applied mechanical forces still apply the same average force per pillar compared to wild type animals. Average forces applied per pillar are independent of the length, diameter, or cuticle stiffness of the animal. We also used the device to measure, for the first time, forces applied by Drosophila melanogaster larvae. Peristaltic waves occurred at 0.4Hz applying an average force of similar to 1.58 mu N on a single pillar. Our colored microfluidic device along with its displacement tracking software allows us to measure forces applied by multiple model organisms that crawl or slither to travel through their environment. (C) 2015 AIP Publishing LLC.

Limitations of Non Model-Based Recognition Schemes

Different approaches to visual object recognition can be divided into two general classes: model-based vs. non model-based schemes. In this paper we establish some limitation on the class of non model-based recognition schemes. We show that every function that is invariant to viewing position of all objects is the trivial (constant) function. It follows that every consistent recognition scheme for recognizing all 3-D objects must in general be model based. The result is extended to recognition schemes that are imperfect (allowed to make mistakes) or restricted to certain classes of objects.

Speciation in Red Algae: Members of the Ceramiales as Model Organisms

Red algae (Rhodophyta) are an ancient group with unusual morphological, biochemical, and life-history features including a complete absence of flagella. Although the red algae present many opportunities for studying speciation, this has rarely been explicitly addressed. Here, we examine an aspect of paternal gene flow by determining fertilization success of female Neosiphonia harveyi (Ceramiales), which retains a morphological record of all successful and unsuccessful female gametes. High fertilization rates were observed except when there were no males at all within the tidepool, or in a submerged marina environment. Small numbers of reproductive males were able to saturate fertilization rates, suggesting that limited availability of sperm may be less significant in red algae than previously thought. In another member of the Ceramiales, Antithamnion, relatively large chromosomes permit karyological identification of polyploids. The Western Pacific species Antithamnion sparsum is closely related to the diploid species Antithamnion defectum, known only from the Eastern Pacific, and appears to have evolved from it. Molecular evidence suggests that A. sparsum is an autopolyploid, and that the European species known as Antithamnion densum is divergent from the A. sparsum/defectum complex.

Study of pesticide effects on non-target organisms

A utilização insustentável de pesticidas, especialmente em zonas com elevado valor ecológico constitui uma ameaça à integridade dos ecossistemas. Sendo um problema à escala mundial, e também no contexto nacional, o presente trabalho pretende ser um contributo para a avaliação dos efeitos de pesticidas em organismos não alvo terrestres e, principalmente, aquáticos, em contextos de progressiva relevância ecológica. Neste sentido, o estudo foi direccionado para áreas (A1 e A2) integradas numa zona agrícola extensa em Portugal, utilizada para a produção de milho e, principalmente, de arroz (Baixo Mondego), a qual sustenta uma elevada biodiversidade. O estudo teve início na área A1, onde a monitorização físico-química e os ensaios com amostras naturais (ensaios WET - whole effluent tests) provenientes desta área evidenciaram que, apesar da ausência de pesticidas, as amostras de água colhidas no canal que atravessava os arrozais foram as mais nocivas para o crescimento de Pseudokirchneriella subcapitata e Chlorella vulgaris. Uma vez que outras fontes de contaminação (produção de gado) actuavam em A1, o estudo prosseguiu apenas na área A2. Assim, em A2, começou-se por determinar a toxicidade individual e da mistura de dois herbicidas formulados aplicados nos campos de arroz (Viper®) e milho (Mikado®) em condições laboratoriais. Viper® foi o herbicida mais tóxico, tanto para o crescimento de P. subcapitata e C. vulgaris, como para a sobrevivência, reprodução e crescimento de Daphnia longispina e Daphnia magna. Adicionalmente, estimou-se que a mistura Viper®/Mikado® induz efeitos antagonistas no crescimento de P. subcapitata e efeitos sinérgicos no crescimento de C. vulgaris e na sobrevivência dos dafnídeos. A avaliação da toxicidade destes herbicidas formulados e seus ingredientes activos no comportamento de minhocas terrestres (Eisenia andrei), usando solos naturais, demonstrou que Viper® e penoxsulam causaram uma % de evitamento superior nos organismos expostos. Contudo, o risco para E. andrei será à partida reduzido se as taxas de aplicação dos herbicidas forem respeitadas. Ensaios WET foram novamente usados para testar amostras naturais da área A2. Verificou-se que a qualidade do sistema aquático e do arrozal diminuiu durante a estação agrícola, em paralelo com a presença de nutrientes e pesticidas. O crescimento algal foi inibido, apesar dos parâmetros de história de vida dos dafnídeos terem sido estimulados. O resultado desta avaliação subestimou, em certos casos, os impactos reais causados pela aplicação de pesticidas. A avaliação in situ simultânea à aplicação de herbicidas nos arrozais demonstrou que os efeitos registados foram de facto restritos aos pulsos de herbicidas. A inibição das taxas de alimentação de D. longispina e D. magna forneceram um sinal precoce de alterações no sistema, seguido pela diminuição da sua sobrevivência e do crescimento de P. subcapitata. Em suma, as diferentes fases da avaliação efectuada confirmaram a existência de condições desfavoráveis devido às práticas agrícolas, reforçando a necessidade de se conjugar ensaios laboratoriais com avaliações in situ de maior relevância ecológica, para reduzir o grau de incerteza aliado à determinação dos riscos.

Quantitative analysis of indexed publications on seventeen model organisms in nine countries, from 1974 to 2006

Developed countries have an even distribution of published papers on the seventeen model organisms. Developing countries have biased preferences for a few model organisms which are associated with endemic human diseases. A variant of the Hirsch-index, that we call the mean (mo)h-index (""model organism h-index""), shows an exponential relationship with the amount of papers published in each country on the selected model organisms. Developing countries cluster together with low mean (mo)h-indexes, even those with high number of publications. The growth curves of publications on the recent model Caenorhabditis elegans in developed countries shows different formats. We also analyzed the growth curves of indexed publications originating from developing countries. Brazil and South Korea were selected for this comparison. The most prevalent model organisms in those countries show different growth curves when compared to a global analysis, reflecting the size and composition of their research communities.

Toxicity of the therapeutic potassium permanganate to tilapia Oreochromis niloticus and to non-target organisms Ceriodaphnia dubia (microcrustacean cladocera) and Pseudokirchneriella subcapitata (green microalgae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)