Sequencing and Analysis of the Mediterranean Amphioxus (Branchiostoma lanceolatum) Transcriptome

BACKGROUND: The basally divergent phylogenetic position of amphioxus (Cephalochordata), as well as its conserved morphology, development and genetics, make it the best proxy for the chordate ancestor. Particularly, studies using the amphioxus model help our understanding of vertebrate evolution and development. Thus, interest for the amphioxus model led to the characterization of both the transcriptome and complete genome sequence of the American species, Branchiostoma floridae. However, recent technical improvements allowing induction of spawning in the laboratory during the breeding season on a daily basis with the Mediterranean species Branchiostoma lanceolatum have encouraged European Evo-Devo researchers to adopt this species as a model even though no genomic or transcriptomic data have been available. To fill this need we used the pyrosequencing method to characterize the B. lanceolatum transcriptome and then compared our results with the published transcriptome of B. floridae. RESULTS: Starting with total RNA from nine different developmental stages of B. lanceolatum, a normalized cDNA library was constructed and sequenced on Roche GS FLX (Titanium mode). Around 1.4 million of reads were produced and assembled into 70,530 contigs (average length of 490 bp). Overall 37% of the assembled sequences were annotated by BlastX and their Gene Ontology terms were determined. These results were then compared to genomic and transcriptomic data of B. floridae to assess similarities and specificities of each species. CONCLUSION: We obtained a high-quality amphioxus (B. lanceolatum) reference transcriptome using a high throughput sequencing approach. We found that 83% of the predicted genes in the B. floridae complete genome sequence are also found in the B. lanceolatum transcriptome, while only 41% were found in the B. floridae transcriptome obtained with traditional Sanger based sequencing. Therefore, given the high degree of sequence conservation between different amphioxus species, this set of ESTs may now be used as the reference transcriptome for the Branchiostoma genus

Target-site resistance mutations (kdr and RDL), but not metabolic resistance, negatively impact male mating competiveness in the malaria vector Anopheles gambiae

The implementation of successful insecticide resistance management strategies for malaria control is currently hampered by poor understanding of the fitness cost of resistance on mosquito populations, including their mating competiveness. To fill this knowledge gap, coupled and uncoupled Anopheles gambiae s.l. males (all M form (Anopheles coluzzii)) were collected from mating swarms in Burkina Faso. This multiple insecticide resistant population exhibited high 1014F kdrR allele frequencies (460%) and RDLR (480%) in contrast to the Ace-1R allele (o6%). Kdr heterozygote males were more likely to mate than homozygote resistant (OR=2.36; Po0.001), suggesting a negative impact of kdr on An. coluzzii mating ability. Interestingly, heterozygote males were also more competitive than homozygote susceptible (OR=3.26; P=0.006), suggesting a heterozygote advantage effect. Similarly, heterozygote RDLR/RDLS were also more likely to mate than homozygote-resistant males (OR=2.58; P=0.007). Furthermore, an additive mating disadvantage was detected in male homozygotes for both kdr/RDL-resistant alleles. In contrast, no fitness difference was observed for the Ace-1 mutation. Comparative microarray-based genome-wide transcription analysis revealed that metabolic resistance did not significantly alter the mating competitiveness of male An. coluzzii mosquitoes. Indeed, no significant difference of expression levels was observed for the main metabolic resistance genes, suggesting that metabolic resistance has a limited impact on male mating competiveness. In addition, specific gene classes/GO terms associated with mating process were detected including sensory perception and peroxidase activity. The detrimental impact of insecticide resistance on mating competiveness observed here suggests that resistance management strategies such as insecticide rotation could help reverse the resistance, if implemented early

Fortunella margarita Transcriptional Reprogramming Triggered by Xanthomonas citri subsp. citri

<p>Abstract</p> <p>Background</p> <p>Citrus canker disease caused by the bacterial pathogen <it>Xanthomonas citri </it>subsp. <it>citri (</it>Xcc) <it>has </it>become endemic in areas where high temperature, rain, humidity, and windy conditions provide a favourable environment for the dissemination of the bacterium. Xcc is pathogenic on many commercial citrus varieties but appears to elicit an incompatible reaction on the citrus relative <it>Fortunella margarita </it>Swing (kumquat), in the form of a very distinct delayed necrotic response. We have developed subtractive libraries enriched in sequences expressed in kumquat leaves during both early and late stages of the disease. The isolated differentially expressed transcripts were subsequently sequenced. Our results demonstrate how the use of microarray expression profiling can help assign roles to previously uncharacterized genes and elucidate plant pathogenesis-response related mechanisms. This can be considered to be a case study in a citrus relative where high throughput technologies were utilized to understand defence mechanisms in <it>Fortunella </it>and citrus at the molecular level.</p> <p>Results</p> <p><b>cDNAs from sequenced kumquat libraries (ESTs) made from subtracted RNA populations, healthy vs. infected, were used to make this microarray</b>. Of 2054 selected genes on a customized array, 317 were differentially expressed (P < 0.05) in Xcc challenged kumquat plants compared to mock-inoculated ones. This study identified components of the incompatible interaction such as reactive oxygen species (ROS) and programmed cell death (PCD). Common defence mechanisms and a number of resistance genes were also identified. In addition, there were a considerable number of differentially regulated genes that had no homologues in the databases. This could be an indication of either a specialized set of genes employed by kumquat in response to canker disease or new defence mechanisms in citrus.</p> <p>Conclusion</p> <p>Functional categorization of kumquat Xcc-responsive genes revealed an enhanced defence-related metabolism as well as a number of resistant response-specific genes in the kumquat transcriptome in response to Xcc inoculation. Gene expression profile(s) were analyzed to assemble a comprehensive and inclusive image of the molecular interaction in the kumquat/Xcc system. This was done in order to elucidate molecular mechanisms associated with the development of the hypersensitive response phenotype in kumquat leaves. These data will be used to perform comparisons among citrus species to evaluate means to enhance the host immune responses against bacterial diseases.</p

Development of a cDNA microarray for the measurement of gene expression in the sheep scab mite Psoroptes ovis

Background: Sheep scab is caused by the ectoparasitic mite Psoroptes ovis which initiates a profound cutaneous inflammatory response, leading to the development of the skin lesions which are characteristic of the disease. Existing control strategies rely upon injectable endectocides and acaricidal dips but concerns over residues, eco-toxicity and the development of acaricide resistance limit the sustainability of this approach. In order to identify alternative means of disease control, a deeper understanding of both the parasite and its interaction with the host are required. Methods: Herein we describe the development and utilisation of an annotated P. ovis cDNA microarray containing 3,456 elements for the measurement of gene expression in this economically important ectoparasite. The array consists of 981 P. ovis EST sequences printed in triplicate along with 513 control elements. Array performance was validated through the analysis of gene expression differences between fed and starved P. ovis mites. Results: Sequences represented on the array include homologues of major house dust mite allergens and tick salivary proteins, along with factors potentially involved in mite reproduction and xenobiotic metabolism. In order to validate the performance of this unique resource under biological conditions we used the array to analyse gene expression differences between fed and starved P. ovis mites. These analyses identified a number of house dust mite allergen homologues up-regulated in fed mites and P. ovis transcripts involved in stress responses, autophagy and chemosensory perception up-regulated in starved mites. Conclusion: The P. ovis cDNA microarray described here has been shown to be both robust and reproducible and will enable future studies to analyse gene expression in this important ectoparasite

A proteomics approach to decipher the molecular nature of planarian stem cells

Background In recent years, planaria have emerged as an important model system for research into stem cells and regeneration. Attention is focused on their unique stem cells, the neoblasts, which can differentiate into any cell type present in the adult organism. Sequencing of the Schmidtea mediterranea genome and some expressed sequence tag projects have generated extensive data on the genetic profile of these cells. However, little information is available on their protein dynamics. Results We developed a proteomic strategy to identify neoblast-specific proteins. Here we describe the method and discuss the results in comparison to the genomic high-throughput analyses carried out in planaria and to proteomic studies using other stem cell systems. We also show functional data for some of the candidate genes selected in our proteomic approach. Conclusions We have developed an accurate and reliable mass-spectra-based proteomics approach to complement previous genomic studies and to further achieve a more accurate understanding and description of the molecular and cellular processes related to the neoblasts

The genomes of two key bumblebee species with primitive eusocial organization

Background: The shift from solitary to social behavior is one of the major evolutionary transitions. Primitively eusocial bumblebees are uniquely placed to illuminate the evolution of highly eusocial insect societies. Bumblebees are also invaluable natural and agricultural pollinators, and there is widespread concern over recent population declines in some species. High-quality genomic data will inform key aspects of bumblebee biology, including susceptibility to implicated population viability threats. Results: We report the high quality draft genome sequences of Bombus terrestris and Bombus impatiens, two ecologically dominant bumblebees and widely utilized study species. Comparing these new genomes to those of the highly eusocial honeybee Apis mellifera and other Hymenoptera, we identify deeply conserved similarities, as well as novelties key to the biology of these organisms. Some honeybee genome features thought to underpin advanced eusociality are also present in bumblebees, indicating an earlier evolution in the bee lineage. Xenobiotic detoxification and immune genes are similarly depauperate in bumblebees and honeybees, and multiple categories of genes linked to social organization, including development and behavior, show high conservation. Key differences identified include a bias in bumblebee chemoreception towards gustation from olfaction, and striking differences in microRNAs, potentially responsible for gene regulation underlying social and other traits. Conclusions: These two bumblebee genomes provide a foundation for post-genomic research on these key pollinators and insect societies. Overall, gene repertoires suggest that the route to advanced eusociality in bees was mediated by many small changes in many genes and processes, and not by notable expansion or depauperation

A first genome assembly of the barley fungal pathogen Pyrenophora teres f. teres

Background: Pyrenophora teres f. teres is a necrotrophic fungal pathogen and the cause of one of barley’s most important diseases, net form of net blotch. Here we report the first genome assembly for this species based solely on short Solexa sequencing reads of isolate 0-1. The assembly was validated by comparison to BAC sequences, ESTs, orthologous genes and by PCR, and complemented by cytogenetic karyotyping and the first genome-wide genetic map for P. teres f. teres. Results: The total assembly was 41.95 Mbp and contains 11,799 gene models of 50 amino acids or more. Comparison against two sequenced BACs showed that complex regions with a high GC content assembled effectively. Electrophoretic karyotyping showed distinct chromosomal polymorphisms between isolates 0-1 and 15A, and cytological karyotyping confirmed the presence of at least nine chromosomes. The genetic map spans 2477.7 cM and is composed of 243 markers in 25 linkage groups, and incorporates SSR markers developed from the assembly. Among predicted genes, non-ribosomal peptide synthetases and efflux pumps in particular appear to have undergone a P. teres f. teres-specific expansion of non-orthologous gene families. Conclusions: This study demonstrates that paired-end Solexa sequencing can successfully capture coding regions of a filamentous fungal genome. The assembly contains a plethora of predicted genes that have been implicated in a necrotrophic lifestyle and pathogenicity and presents a significant resource for examining the bases for P. teres f. teres pathogenicity

Transcriptome Profiling of Citrus Fruit Response to Huanglongbing Disease

Huanglongbing (HLB) or “citrus greening” is the most destructive citrus disease worldwide. In this work, we studied host responses of citrus to infection with Candidatus Liberibacter asiaticus (CaLas) using next-generation sequencing technologies. A deep mRNA profile was obtained from peel of healthy and HLB-affected fruit. It was followed by pathway and protein-protein network analysis and quantitative real time PCR analysis of highly regulated genes. We identified differentially regulated pathways and constructed networks that provide a deep insight into the metabolism of affected fruit. Data mining revealed that HLB enhanced transcription of genes involved in the light reactions of photosynthesis and in ATP synthesis. Activation of protein degradation and misfolding processes were observed at the transcriptomic level. Transcripts for heat shock proteins were down-regulated at all disease stages, resulting in further protein misfolding. HLB strongly affected pathways involved in source-sink communication, including sucrose and starch metabolism and hormone synthesis and signaling. Transcription of several genes involved in the synthesis and signal transduction of cytokinins and gibberellins was repressed while that of genes involved in ethylene pathways was induced. CaLas infection triggered a response via both the salicylic acid and jasmonic acid pathways and increased the transcript abundance of several members of the WRKY family of transcription factors. Findings focused on the fruit provide valuable insight to understanding the mechanisms of the HLB-induced fruit disorder and eventually developing methods based on small molecule applications to mitigate its devastating effects on fruit production

Transcriptomic Signatures of Ash (Fraxinus spp.) Phloem

Ash (Fraxinus spp.) is a dominant tree species throughout urban and forested landscapes of North America (NA). The rapid invasion of NA by emerald ash borer (Agrilus planipennis), a wood-boring beetle endemic to Eastern Asia, has resulted in the death of millions of ash trees and threatens billions more. Larvae feed primarily on phloem tissue, which girdles and kills the tree. While NA ash species including black (F. nigra), green (F. pennsylvannica) and white (F. americana) are highly susceptible, the Asian species Manchurian ash (F. mandshurica) is resistant to A. planipennis perhaps due to their co-evolutionary history. Little is known about the molecular genetics of ash. Hence, we undertook a functional genomics approach to identify the repertoire of genes expressed in ash phloem.Using 454 pyrosequencing we obtained 58,673 high quality ash sequences from pooled phloem samples of green, white, black, blue and Manchurian ash. Intriguingly, 45% of the deduced proteins were not significantly similar to any sequences in the GenBank non-redundant database. KEGG analysis of the ash sequences revealed a high occurrence of defense related genes. Expression analysis of early regulators potentially involved in plant defense (i.e. transcription factors, calcium dependent protein kinases and a lipoxygenase 3) revealed higher mRNA levels in resistant ash compared to susceptible ash species. Lastly, we predicted a total of 1,272 single nucleotide polymorphisms and 980 microsatellite loci, among which seven microsatellite loci showed polymorphism between different ash species.The current transcriptomic data provide an invaluable resource for understanding the genetic make-up of ash phloem, the target tissue of A. planipennis. These data along with future functional studies could lead to the identification/characterization of defense genes involved in resistance of ash to A. planipennis, and in future ash breeding programs for marker development

Tissue-Specific Transcriptomics of the Exotic Invasive Insect Pest Emerald Ash Borer (Agrilus planipennis)

BACKGROUND: The insect midgut and fat body represent major tissue interfaces that deal with several important physiological functions including digestion, detoxification and immune response. The emerald ash borer (Agrilus planipennis), is an exotic invasive insect pest that has killed millions of ash trees (Fraxinus spp.) primarily in the Midwestern United States and Ontario, Canada. However, despite its high impact status little knowledge exists for A. planipennis at the molecular level. METHODOLOGY AND PRINCIPAL FINDINGS: Newer-generation Roche-454 pyrosequencing was used to obtain 126,185 reads for the midgut and 240,848 reads for the fat body, which were assembled into 25,173 and 37,661 high quality expressed sequence tags (ESTs) for the midgut and the fat body of A. planipennis larvae, respectively. Among these ESTs, 36% of the midgut and 38% of the fat body sequences showed similarity to proteins in the GenBank nr database. A high number of the midgut sequences contained chitin-binding peritrophin (248)and trypsin (98) domains; while the fat body sequences showed high occurrence of cytochrome P450s (85) and protein kinase (123) domains. Further, the midgut transcriptome of A. planipennis revealed putative microbial transcripts encoding for cell-wall degrading enzymes such as polygalacturonases and endoglucanases. A significant number of SNPs (137 in midgut and 347 in fat body) and microsatellite loci (317 in midgut and 571 in fat body) were predicted in the A. planipennis transcripts. An initial assessment of cytochrome P450s belonging to various CYP clades revealed distinct expression patterns at the tissue level. CONCLUSIONS AND SIGNIFICANCE: To our knowledge this study is one of the first to illuminate tissue-specific gene expression in an invasive insect of high ecological and economic consequence. These findings will lay the foundation for future gene expression and functional studies in A. planipennis