Genomic analysis of two phlebotomine sand fly vectors of Leishmania from the New and Old World.

Phlebotomine sand flies are of global significance as important vectors of human disease, transmitting bacterial, viral, and protozoan pathogens, including the kinetoplastid parasites of the genus Leishmania, the causative agents of devastating diseases collectively termed leishmaniasis. More than 40 pathogenic Leishmania species are transmitted to humans by approximately 35 sand fly species in 98 countries with hundreds of millions of people at risk around the world. No approved efficacious vaccine exists for leishmaniasis and available therapeutic drugs are either toxic and/or expensive, or the parasites are becoming resistant to the more recently developed drugs. Therefore, sand fly and/or reservoir control are currently the most effective strategies to break transmission. To better understand the biology of sand flies, including the mechanisms involved in their vectorial capacity, insecticide resistance, and population structures we sequenced the genomes of two geographically widespread and important sand fly vector species: Phlebotomus papatasi, a vector of Leishmania parasites that cause cutaneous leishmaniasis, (distributed in Europe, the Middle East and North Africa) and Lutzomyia longipalpis, a vector of Leishmania parasites that cause visceral leishmaniasis (distributed across Central and South America). We categorized and curated genes involved in processes important to their roles as disease vectors, including chemosensation, blood feeding, circadian rhythm, immunity, and detoxification, as well as mobile genetic elements. We also defined gene orthology and observed micro-synteny among the genomes. Finally, we present the genetic diversity and population structure of these species in their respective geographical areas. These genomes will be a foundation on which to base future efforts to prevent vector-borne transmission of Leishmania parasites.

Host Cytoskeleton Gene Expression Is Correlated with the Formation of Ascovirus Reproductive Viral Vesicles.

Ascoviruses are large DNA viruses that primarily infect lepidopteran larvae. They differ markedly from other plant or animal viruses by initiating replication in the nucleus, then inducing nuclear lysis followed by extensive cellular hypertrophy and subsequent cleavage of the entire enlarged cell into numerous viral vesicles. Most progeny virions are assembled in these vesicles as they circulate in the hemolymph. Here, we report transcriptome studies of host cytoskeletal genes in larvae infected with ascoviruses from 6 h to 21 days post-infection (dpi). We focused on the cabbage looper, Trichoplusia ni, infected with the Trichoplusia ni ascovirus (TnAV), along with supporting studies on the fall armyworm, Spodoptera frugiperda, infected with the Spodoptera frugiperda ascovirus (SfAV). In T. ni, many cytoskeleton genes were upregulated at 48 hours post-infection (hpi), including 29 tubulins, 21 actins, 21 dyneins, and 13 kinesins. Mitochondrial genes were upregulated as much as two-fold at 48 hpi and were expressed at levels comparable to controls in both T. ni and S. frugiperda, even after 21 dpi, when several cytoskeleton genes remained upregulated. Our studies suggest a temporal correlation between increases in the expression of certain host cytoskeletal genes and viral vesicle formation. However, these results need confirmation through functional genetic studies of proteins encoded by these genes.

Early in vivo transcriptome of Trichoplusia ni ascovirus core genes.

Ascoviruses are large double-stranded DNA insect viruses that destroy the nucleus and transform each cell into 20 or more viral vesicles for replication. In the present study we used RNA-sequencing to compare the expression of Trichoplusia ni ascovirus 6a1 (TnAV-6a1) core genes during the first week of infection, with emphasis on the first 48 h, comparing transcript levels in major somatic tissues (epidermis, tracheal matrix and fat body), the sites infected initially, with those of the haemolymph, where viral vesicles circulate and most replication occurs. By 48 h post-infection (p.i.), only 26 genes were expressed in somatic tissues at ≥5 log2 reads per kilobase per million, whereas in the haemolymph 48 genes were expressed at a similar level by the same time. Early and high expression of TnAV caspase-2-like gene occurred in all tissues, implying it is required for replication, but that it is probably not associated with apoptosis induction, which occurs in infections of Spodoptera frugiperda ascovirus 1 a (SfAV-1a), the ascovirus type species. Other highly expressed viral genes at 48 h p.i. in viral vesicles included a dynein-like beta chain and lipid-modifying enzymes, suggesting their importance to vesicle formation and growth as well as virion synthesis. Finally, as occurs in SfAV expression, we found bicistronic and tricistronic mRNA messages produced by TnAV.

Extended in vivo transcriptomes of two ascoviruses with different tissue tropisms reveal alternative mechanisms for enhancing virus reproduction in hemolymph.

Ascoviruses are large dsDNA viruses characterized by the extraordinary changes they induce in cellular pathogenesis and architecture whereby after nuclear lysis and extensive hypertrophy, each cell is cleaved into numerous vesicles for virion reproduction. However, the level of viral replication and transcription in vesicles compared to other host tissues remains uncertain. Therefore, we applied RNA-Sequencing to compare the temporal transcriptome of Spodoptera frugiperda ascovirus (SfAV) and Trichoplusia ni ascovirus (TnAV) at 7, 14, and 21 days post-infection (dpi). We found most transcription occurred in viral vesicles, not in initial tissues infected, a remarkably novel reproduction mechanism compared to all other viruses and most other intracellular pathogens. Specifically, the highest level of viral gene expression occurred in hemolymph, for TnAV at 7 dpi, and SfAV at 14 dpi. Moreover, we found that host immune genes were partially down-regulated in hemolymph, where most viral replication occurred in highly dense accumulations of vesicles.

Gene expression profiling reveals candidate genes for defining spider silk gland types.

Molecular studies of the secretory glands involved in spider silk production have revealed candidate genes for silk synthesis and a complicated history of spider silk gene evolution. However, differential gene expression profiles of the multiple silk gland types within an individual orb-web weaving spider are lacking. Each of these gland types produces a functionally distinct silk type. Comparison of gene expression among spider silk gland types would provide insight into the genes that define silk glands generally from non-silk gland tissues, and the genes that define silk glands from each other. Here, we perform 3' tag digital gene expression profiling of the seven silk gland types of the silver garden orb weaver Argiope argentata. Five of these gland types produce silks that are non-adhesive fibers, one silk includes both fibers and glue-like adhesives, and one silk is exclusively glue-like. We identify 1275 highly expressed, significantly upregulated, and tissue specific silk gland specific transcripts (SSTs). These SSTs include seven types of spider silk protein encoding genes known as spidroin genes. We find that the fiber-producing major ampullate and minor ampullate silk glands have more similar expression profiles than any other pair of glands. We also find that a subset of the SSTs is enriched for transmembrane transport and oxidoreductases, and that these transcripts highlight differences and similarities among the major ampullate, minor ampullate, and aggregate silk glands. Furthermore, we show that the wet glue-producing aggregate glands have the most unique SSTs, but still share some SSTs with fiber producing glands. Aciniform glands were the only gland type to share a majority of SSTs with other silk gland types, supporting previous hypotheses that duplication of aciniform glands and subsequent divergence of the duplicates gave rise to the multiple silk gland types within an individual spider.

Two repeated motifs enriched within some enhancers and origins of replication are bound by SETMAR isoforms in human colon cells.

Setmar is a gene specific to simian genomes. The function(s) of its isoforms are poorly understood and their existence in healthy tissues remains to be validated. Here we profiled SETMAR expression and its genome-wide binding landscape in colon tissue. We found isoforms V3 and V6 in healthy and tumour colon tissues as well as incell lines. In two colorectal cell lines SETMAR binds to several thousand Hsmar1 and MADE1 terminal ends, transposons mostly located in non-genic regions of active chromatin including in enhancers. It also binds to a 12-bp motifs similar to an inner motif in Hsmar1 and MADE1 terminal ends. This motif is interspersed throughout the genome and is enriched in GC-rich regions as well as in CpG islands that contain constitutive replication origins. It is also found in enhancers other than those associated with Hsmar1 and MADE1. The role of SETMAR in the expression of genes, DNA replication and in DNA repair are discussed.

The C-terminal Domain of piggyBac Transposase Is Not Required for DNA Transposition.

PiggyBac(PB)-like elements (pble) are members of a eukaryotic DNA transposon family. This family is of interest to evolutionary genomics because pble transposases have been domesticated at least 9 times in vertebrates. The amino acid sequence of pble transposases can be split into three regions: an acidic N-terminal domain (~100 aa), a central domain (~400 aa) containing a DD[D/E] catalytic triad, and a cysteine-rich domain (CRD; ~90 aa). Two recent reports suggested that a functional CRD is required for pble transposase activity. Here we found that two CRD-deficient pble transposases, a PB variant and an isoform encoded by the domesticated PB-derived vertebrate transposase gene 5 (pgbd5) trigger transposition of the Ifp2 pble. When overexpressed in HeLa cells, these CRD-deficient transposases can insert Ifp2 elements with proper and improper transposon ends, associated with deleterious effects on cells. Finally, we found that mouse CRD-deficient transposase Pgbd5, as well as PB, do not insert pbles at random into chromosomes. Transposition events occurred more often in genic regions, in the neighbourhood of the transcription start sites and were often found in genes predominantly expressed in the human central nervous system.

Mitochondrial and Innate Immunity Transcriptomes from Spodoptera frugiperda Larvae Infected with the Spodoptera frugiperda Ascovirus.

Ascoviruses are large, enveloped DNA viruses that induce remarkable changes in cellular architecture during which the cell is partitioned into numerous vesicles for viral replication. Previous studies have shown that these vesicles arise from a process resembling apoptosis yet which differs after nuclear lysis in that mitochondria are not degraded but are modified by the virus, changing in size, shape, and motility. Moreover, infection does not provoke an obvious innate immune response. Thus, we used in vivo RNA sequencing to determine whether infection by the Spodoptera frugiperda ascovirus 1a (SfAV-1a) modified expression of host mitochondrial, cytoskeletal, and innate immunity genes. We show that transcripts from many mitochondrial genes were similar to those from uninfected controls, whereas others increased slightly during vesicle formation, including those for ATP6, ATP8 synthase, and NADH dehydrogenase subunits, supporting electron microscopy (EM) data that these organelles were conserved for virus replication. Transcripts from 58 of 106 cytoskeletal genes studied increased or decreased more than 2-fold postinfection. More than half coded for mitochondrial motor proteins. Similar increases occurred for innate immunity transcripts and their negative regulators, including those for Toll, melanization, and phagocytosis pathways. However, those for many antimicrobial peptides, such as moricin, increased more than 20-fold. In addition, transcripts for gloverin-3, spod_x_tox, Hdd23, and lebocin, also antimicrobial, increased more than 20-fold. Interestingly, a phenoloxidase inhibitor transcript increased 12-fold, apparently to interfere with melanization. SfAV-1a destroys most fat body cells by 7 days postinfection, so innate immunity gene transcripts apparently occur in remaining cells in this tissue and possibly other major tissues, namely, epidermis and tracheal matrix.IMPORTANCE Ascoviruses are large DNA viruses that infect insects, inducing a cellular pathology that resembles apoptosis but which differs by causing enormous cellular hypertrophy followed by cleavage of the cell into numerous viral vesicles for replication. Previous EM studies suggest that mitochondria are important for vesicle formation. Transcriptome analyses of Spodoptera frugiperda larvae infected with SfAV-1a showed that mitochondrial transcripts were similar to those from uninfected controls or increased slightly during vesicle formation, especially for ATP6, ATP8 synthase, and NADH dehydrogenase subunits. This pattern resembles that for chronic disease-inducing viruses, which conserve mitochondria, differing markedly from viruses causing short-term viral diseases, which degrade mitochondrial DNA. Though mitochondrial transcript increases were low, our results demonstrate that SfAV-1a alters host mitochondrial expression more than any other virus. Regarding innate immunity, although SfAV-1a destroys most fat body cells, certain immunity genes were highly upregulated (greater than 20-fold), suggesting that these transcripts may originate from other tissues.

Variations in genome size between wild and domesticated lineages of fowls belonging to the Gallus gallus species.

Efforts to elucidate the causes of biological differences between wild fowls and their domesticated relatives, the chicken, have to date mainly focused on the identification of single nucleotide mutations. Other types of genomic variations have however been demonstrated to be important in avian evolution and associated to variations in phenotype. They include several types of sequences duplicated in tandem that can vary in their repetition number. Here we report on genome size differences between the red jungle fowl and several domestic chicken breeds and selected lines. Sequences duplicated in tandem such as rDNA, telomere repeats, satellite DNA and segmental duplications were found to have been significantly re-shaped during domestication and subsequently by human-mediated selection. We discuss the extent to which changes in genome organization that occurred during domestication agree with the hypothesis that domesticated animal genomes have been shaped by evolutionary forces aiming to adapt them to anthropized environments.

Sequence properties of certain GC rich avian genes, their origins and absence from genome assemblies: case studies.

BACKGROUND: More and more eukaryotic genomes are sequenced and assembled, most of them presented as a complete model in which missing chromosomal regions are filled by Ns and where a few chromosomes may be lacking. Avian genomes often contain sequences with high GC content, which has been hypothesized to be at the origin of many missing sequences in these genomes. We investigated features of these missing sequences to discover why some may not have been integrated into genomic libraries and/or sequenced. RESULTS: The sequences of five red jungle fowl cDNA models with high GC content were used as queries to search publicly available datasets of Illumina and Pacbio sequencing reads. These were used to reconstruct the leptin, TNFα, MRPL52, PCP2 and PET100 genes, all of which are absent from the red jungle fowl genome model. These gene sequences displayed elevated GC contents, had intron sizes that were sometimes larger than non-avian orthologues, and had non-coding regions that contained numerous tandem and inverted repeat sequences with motifs able to assemble into stable G-quadruplexes and intrastrand dyadic structures. Our results suggest that Illumina technology was unable to sequence the non-coding regions of these genes. On the other hand, PacBio technology was able to sequence these regions, but with dramatically lower efficiency than would typically be expected. CONCLUSIONS: High GC content was not the principal reason why numerous GC-rich regions of avian genomes are missing from genome assembly models. Instead, it is the presence of tandem repeats containing motifs capable of assembling into very stable secondary structures that is likely responsible.

Ecdysis triggering hormone receptors regulate male courtship behavior via antennal lobe interneurons in Drosophila.

Ecdysis triggering hormone receptors (ETHR) regulate the behavioral sequence necessary for cuticle shedding. Recent reports have documented functions for ETHR signaling in adult Drosophila melanogaster. In this study, we report that ETHR silencing in local interneurons of the antennal lobes and fruitless neurons leads to sharply increased rates of male-male courtship. RNAseq analysis of ETHR knockdown flies reveals differential expression of genes involved in axon guidance, courtship behavior and chemosensory functions. Our findings indicate an important role for ETHR in regulation of Drosophila courtship behavior through chemosensory processing in the antennal lobe.

But where did the centromeres go in the chicken genome models?

The chicken genome was the third vertebrate to be sequenced. To date, its sequence and feature annotations are used as the reference for avian models in genome sequencing projects developed on birds and other Sauropsida species, and in genetic studies of domesticated birds of economic and evolutionary biology interest. Therefore, an accurate description of this genome model is important to a wide number of scientists. Here, we review the location and features of a very basic element, the centromeres of chromosomes in the galGal5 genome model. Centromeres are elements that are not determined by their DNA sequence but by their epigenetic status, in particular by the accumulation of the histone-like protein CENP-A. Comparison of data from several public sources (primarily marker probes flanking centromeres using fluorescent in situ hybridization done on giant lampbrush chromosomes and CENP-A ChIP-seq datasets) with galGal5 annotations revealed that centromeres are likely inappropriately mapped in 9 of the 16 galGal5 chromosome models in which they are described. Analysis of karyology data confirmed that the location of the main CENP-A peaks in chromosomes is the best means of locating the centromeres in 25 galGal5 chromosome models, the majority of which (16) are fully sequenced and assembled. This data re-analysis reaffirms that several sources of information should be examined to produce accurate genome annotations, particularly for basic structures such as centromeres that are epigenetically determined.

Transcriptome Analysis of the Spodoptera frugiperda Ascovirus In Vivo Provides Insights into How Its Apoptosis Inhibitors and Caspase Promote Increased Synthesis of Viral Vesicles and Virion Progeny.

Ascoviruses are double-stranded DNA (dsDNA) viruses that attack caterpillars and differ from all other viruses by inducing nuclear lysis followed by cleavage of host cells into numerous anucleate vesicles in which virus replication continues as these grow in the blood. Ascoviruses are also unusual in that most encode a caspase or caspase-like proteins. A robust cell line to study the novel molecular biology of ascovirus replication in vitro is lacking. Therefore, we used strand-specific transcriptome sequencing (RNA-Seq) to study transcription in vivo in third instars of Spodoptera frugiperda infected with the type species, Spodoptera frugiperda ascovirus1a (SfAV-1a), sampling transcripts at different time points after infection. We targeted transcription of two types of SfAV-1a genes; first, 44 core genes that occur in several ascovirus species, and second, 26 genes predicted in silico to have metabolic functions likely involved in synthesizing viral vesicle membranes. Gene cluster analysis showed differences in temporal expression of SfAV-1a genes, enabling their assignment to three temporal classes: early, late, and very late. Inhibitors of apoptosis (IAP-like proteins; ORF016, ORF025, and ORF074) were expressed early, whereas its caspase (ORF073) was expressed very late, which correlated with apoptotic events leading to viral vesicle formation. Expression analysis revealed that a Diedel gene homolog (ORF121), the only known "virokine," was highly expressed, implying that this ascovirus protein helps evade innate host immunity. Lastly, single-nucleotide resolution of RNA-Seq data revealed 15 bicistronic and tricistronic messages along the genome, an unusual occurrence for large dsDNA viruses.IMPORTANCE Unlike all other DNA viruses, ascoviruses code for an executioner caspase, apparently involved in a novel cytopathology in which viral replication induces nuclear lysis followed by cell cleavage, yielding numerous large anucleate viral vesicles that continue to produce virions. Our transcriptome analysis of genome expression in vivo by the Spodoptera frugiperda ascovirus shows that inhibitors of apoptosis are expressed first, enabling viral replication to proceed, after which the SfAV-1a caspase is synthesized, leading to viral vesicle synthesis and subsequent extensive production of progeny virions. Moreover, we detected numerous bicistronic and tricistronic mRNA messages in the ascovirus transcriptome, implying that ascoviruses use other noncanonical translational mechanisms, such as internal ribosome entry sites (IRESs). These results provide the first insights into the molecular biology of a unique coordinated gene expression pattern in which cell architecture is markedly modified, more than in any other known eukaryotic virus, to promote viral reproduction and transmission.

The whole genome sequence of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), reveals insights into the biology and adaptive evolution of a highly invasive pest species.

BACKGROUND: The Mediterranean fruit fly (medfly), Ceratitis capitata, is a major destructive insect pest due to its broad host range, which includes hundreds of fruits and vegetables. It exhibits a unique ability to invade and adapt to ecological niches throughout tropical and subtropical regions of the world, though medfly infestations have been prevented and controlled by the sterile insect technique (SIT) as part of integrated pest management programs (IPMs). The genetic analysis and manipulation of medfly has been subject to intensive study in an effort to improve SIT efficacy and other aspects of IPM control. RESULTS: The 479 Mb medfly genome is sequenced from adult flies from lines inbred for 20 generations. A high-quality assembly is achieved having a contig N50 of 45.7 kb and scaffold N50 of 4.06 Mb. In-depth curation of more than 1800 messenger RNAs shows specific gene expansions that can be related to invasiveness and host adaptation, including gene families for chemoreception, toxin and insecticide metabolism, cuticle proteins, opsins, and aquaporins. We identify genes relevant to IPM control, including those required to improve SIT. CONCLUSIONS: The medfly genome sequence provides critical insights into the biology of one of the most serious and widespread agricultural pests. This knowledge should significantly advance the means of controlling the size and invasive potential of medfly populations. Its close relationship to Drosophila, and other insect species important to agriculture and human health, will further comparative functional and structural studies of insect genomes that should broaden our understanding of gene family evolution.

Deep landscape update of dispersed and tandem repeats in the genome model of the red jungle fowl, Gallus gallus, using a series of de novo investigating tools.

BACKGROUND: The program RepeatMasker and the database Repbase-ISB are part of the most widely used strategy for annotating repeats in animal genomes. They have been used to show that avian genomes have a lower repeat content (8-12 %) than the sequenced genomes of many vertebrate species (30-55 %). However, the efficiency of such a library-based strategies is dependent on the quality and completeness of the sequences in the database that is used. An alternative to these library based methods are methods that identify repeats de novo. These alternative methods have existed for a least a decade and may be more powerful than the library based methods. We have used an annotation strategy involving several complementary de novo tools to determine the repeat content of the model genome galGal4 (1.04 Gbp), including identifying simple sequence repeats (SSRs), tandem repeats and transposable elements (TEs). RESULTS: We annotated over one Gbp. of the galGal4 genome and showed that it is composed of approximately 19 % SSRs and TEs repeats. Furthermore, we estimate that the actual genome of the red jungle fowl contains about 31-35 % repeats. We find that library-based methods tend to overestimate TE diversity. These results have a major impact on the current understanding of repeats distributions throughout chromosomes in the red jungle fowl. CONCLUSIONS: Our results are a proof of concept of the reliability of using de novo tools to annotate repeats in large animal genomes. They have also revealed issues that will need to be resolved in order to develop gold-standard methodologies for annotating repeats in eukaryote genomes.

The future of transposable element annotation and their classification in the light of functional genomics - what we can learn from the fables of Jean de la Fontaine?

Transposable element (TE) science has been significantly influenced by the pioneering ideas of David Finnegan near the end of the last century, as well as by the classification systems that were subsequently developed. Today, whole genome TE annotation is mostly done using tools that were developed to aid gene annotation rather than to specifically study TEs. We argue that further progress in the TE field is impeded both by current TE classification schemes and by a failure to recognize that TE biology is fundamentally different from that of multicellular organisms. Novel genome wide TE annotation methods are helping to redefine our understanding of TE sequence origins and evolution. We briefly discuss some of these new methods as well as ideas for possible alternative classification schemes. Our hope is to encourage the formation of a society to organize a larger debate on these questions and to promote the adoption of standards for annotation and an improved TE classification.

Spider Transcriptomes Identify Ancient Large-Scale Gene Duplication Event Potentially Important in Silk Gland Evolution.

The evolution of specialized tissues with novel functions, such as the silk synthesizing glands in spiders, is likely an influential driver of adaptive success. Large-scale gene duplication events and subsequent paralog divergence are thought to be required for generating evolutionary novelty. Such an event has been proposed for spiders, but not tested. We de novo assembled transcriptomes from three cobweb weaving spider species. Based on phylogenetic analyses of gene families with representatives from each of the three species, we found numerous duplication events indicative of a whole genome or segmental duplication. We estimated the age of the gene duplications relative to several speciation events within spiders and arachnids and found that the duplications likely occurred after the divergence of scorpions (order Scorpionida) and spiders (order Araneae), but before the divergence of the spider suborders Mygalomorphae and Araneomorphae, near the evolutionary origin of spider silk glands. Transcripts that are expressed exclusively or primarily within black widow silk glands are more likely to have a paralog descended from the ancient duplication event and have elevated amino acid replacement rates compared with other transcripts. Thus, an ancient large-scale gene duplication event within the spider lineage was likely an important source of molecular novelty during the evolution of silk gland-specific expression. This duplication event may have provided genetic material for subsequent silk gland diversification in the true spiders (Araneomorphae).