The Juan non-LTR retrotransposon in mosquitoes: genomic impact, vertical transmission and indications of recent and widespread activity

<p>Abstract</p> <p>Background</p> <p>In contrast to DNA-mediated transposable elements (TEs), retrotransposons, particularly non-long terminal repeat retrotransposons (non-LTRs), are generally considered to have a much lower propensity towards horizontal transfer. Detailed studies on site-specific non-LTR families have demonstrated strict vertical transmission. More studies are needed with non-site-specific non-LTR families to determine whether strict vertical transmission is a phenomenon related to site specificity or a more general characteristic of all non-LTRs. <it>Juan </it>is a Jockey clade non-LTR retrotransposon first discovered in mosquitoes that is widely distributed in the mosquito family <it>Culicidae</it>. Being a non-site specific non-LTR, <it>Juan </it>offers an opportunity to further investigate the hypothesis that non-LTRs are genomic elements that are primarily vertically transmitted.</p> <p>Results</p> <p>Systematic analysis of the ~1.3 Gbp <it>Aedes aegypti </it>(<it>Ae. aegypti</it>) genome sequence suggests that <it>Juan-A </it>is the only <it>Juan</it>-type non-LTR in <it>Aedes aegypti</it>. <it>Juan-A </it>is highly reiterated and comprises approximately 3% of the genome. Using minimum cutoffs of 90% length and 70% nucleotide (nt) identity, 663 copies were found by BLAST using the published <it>Juan-A </it>sequence as the query. All 663 copies are at least 95% identical to <it>Juan-A</it>, while 378 of these copies are 99% identical to <it>Juan-A</it>, indicating that the <it>Juan-A </it>family has been transposing recently in evolutionary history. Using the 0.34 Kb 5' UTR as the query, over 2000 copies were identified that may contain internal promoters, leading to questions on the genomic impact of <it>Juan-A</it>. <it>Juan </it>sequences were obtained by PCR, library screening, and database searches for 18 mosquito species of six genera including <it>Aedes</it>, <it>Ochlerotatus</it>, <it>Psorophora</it>, <it>Culex</it>, <it>Deinocerites</it>, and <it>Wyeomyia</it>. Comparison of host and <it>Juan </it>phylogenies shows overall congruence with few exceptions.</p> <p>Conclusion</p> <p><it>Juan-A </it>is a major genomic component in <it>Ae. aegypti </it>and it has been retrotransposing recently in evolutionary history. There are also indications that <it>Juan </it>has been recently active in a wide range of mosquito species. Furthermore, our research demonstrates that a Jockey clade non-LTR without target site-specificity has been sustained by vertical transmission in the mosquito family. These results strengthen the argument that non-LTRs tend to be genomic elements capable of persistence by vertical descent over a long evolutionary time.</p

Odorant Receptor C-Terminal Motifs in Divergent Insect Species

Insect odorant receptors are a large family of seven transmembrane proteins believed to be G-protein coupled receptors. The peptide sequences of two odorant receptors within a given species may share as little as 17% identity, and there is limited similarity between receptors of divergent species. One exception is DmOr83b, which is found in Drosophila melanogaster and is highly conserved in at least ten other insect species. DmOr83b is broadly expressed in most of the olfactory sensory neurons of D. melanogaster at most developmental stages, while other odorant receptors tend to have more restricted and specific expression patterns. DmOr83b is critical for D. melanogaster olfaction, and it is involved in properly localizing other odorant receptors possibly by forming heterodimers with these receptors. The C-terminal region has been implicated as sites for such heterodimer formation. Multiple em for motif elicitation (MEME), a hidden markov model based program, was used to uncover three conserved motifs in the C-termini of a vast majority of the odorant receptor peptides from Anopheles gambiae, D. melanogaster, and Apis mellifera. These motifs are also found in DmOr83b and its orthologs and the order of these motifs is conserved as well. The conservation of these motifs among divergent odorant receptors in divergent species suggests functional importance. We propose that these motifs are involved in receptor- receptor protein interactions, contributing to the heterodimer formation between DmOr83b (or its orthologs) and other odorant receptors

Evolutionary analysis of the kinesin light chain genes in the yellow fever mosquito Aedes aegypti: gene duplication as a source for novel early zygotic genes

<p>Abstract</p> <p>Background</p> <p>The maternal zygotic transition marks the time at which transcription from the zygotic genome is initiated and a subset of maternal RNAs are progressively degraded in the developing embryo. A number of early zygotic genes have been identified in <it>Drosophila melanogaster </it>and comparisons to sequenced mosquito genomes suggest that some of these early zygotic genes such as <it>bottleneck </it>are fast-evolving or subject to turnover in dipteran insects. One objective of this study is to identify early zygotic genes from the yellow fever mosquito <it>Aedes aegypti </it>to study their evolution. We are also interested in obtaining early zygotic promoters that will direct transgene expression in the early embryo as part of a <it>Medea </it>gene drive system.</p> <p>Results</p> <p>Two novel early zygotic kinesin light chain genes we call <it>AaKLC2.1 </it>and <it>AaKLC2.2 </it>were identified by transcriptome sequencing of <it>Aedes aegypti </it>embryos at various time points. These two genes have 98% nucleotide and amino acid identity in their coding regions and show transcription confined to the early zygotic stage according to gene-specific RT-PCR analysis. These <it>AaKLC2 </it>genes have a paralogous gene (<it>AaKLC1</it>) in <it>Ae. aegypti</it>. Phylogenetic inference shows that an ortholog to the <it>AaKLC2 </it>genes is only found in the sequenced genome of <it>Culex quinquefasciatus</it>. In contrast, <it>AaKLC1 </it>gene orthologs are found in all three sequenced mosquito species including <it>Anopheles gambiae</it>. There is only one KLC gene in <it>D. melanogaster </it>and other sequenced holometabolous insects that appears to be similar to <it>AaKLC1</it>. Unlike <it>AaKLC2</it>, <it>AaKLC1 </it>is expressed in all life stages and tissues tested, which is consistent with the expression pattern of the <it>An. gambiae </it>and <it>D. melanogaster </it>KLC genes. Phylogenetic inference also suggests that <it>AaKLC2 </it>genes and their likely <it>C. quinquefasciatus </it>ortholog are fast-evolving genes relative to the highly conserved <it>AaKLC1-like </it>paralogs. Embryonic injection of a luciferase reporter under the control of a 1 kb fragment upstream of the <it>AaKLC2.1 </it>start codon shows promoter activity at least as early as 3 hours in the developing <it>Ae. aegypti </it>embryo. The <it>AaKLC2.1 </it>promoter activity reached ~1600 fold over the negative control at 5 hr after egg deposition.</p> <p>Conclusions</p> <p>Transcriptome profiling by use of high throughput sequencing technologies has proven to be a valuable method for the identification and discovery of early and transient zygotic genes. The evolutionary investigation of the KLC gene family reveals that duplication is a source for the evolution of new genes that play a role in the dynamic process of early embryonic development. <it>AaKLC2.1 </it>may provide a promoter for early zygotic-specific transgene expression, which is a key component of the <it>Medea </it>gene drive system.</p

Expression Analysis and Knockdown of Two Antennal Odorant-Binding Protein Genes in Aedes aegypti

The presence and expression of odorant-binding proteins (OBPs) in the olfactory organs suggest that they play an important role in mosquito olfaction. However, no direct evidence has been found for their involvement in the host-seeking behavior of mosquitoes. It is important to establish a method in which a loss-of-function test can be performed to determine the possible role of these genes in olfaction. In this study, a double subgenomic Sindbis virus expression system was used to reduce the expression of two Obp genes in Aedes aegypti L (Diptera: Culicidae), AaegObp1 and AaegObp2. Quantitative real-time PCR analysis showed predominant expression of both genes in the female antennae, the primary olfactory tissue of mosquitoes. Moreover, at 11 days post virus-inoculation, the mRNA levels of AaegObp1 and AaegObp2 were significantly reduced in olfactory tissues of recombinant virus-inoculated female mosquitoes compared to that of controls by approximately 8 and 100-fold, respectively. These data suggest that the double subgenomic Sindbis virus expression system can be efficiently used to knockdown Obp gene expression in olfactory tissues of mosquitoes. We discuss the potential for a systematic analysis of the molecular players involved in mosquito olfaction using this newly developed technique. Such analysis will provide an important step to interfere with the host-seeking behavior of mosquitoes to prevent the transmission of diseases

Protein kinase C-dependent signaling controls the midgut epithelial barrier to malaria parasite infection in anopheline mosquitoes.

Anopheline mosquitoes are the primary vectors of parasites in the genus Plasmodium, the causative agents of malaria. Malaria parasites undergo a series of complex transformations upon ingestion by the mosquito host. During this process, the physical barrier of the midgut epithelium, along with innate immune defenses, functionally restrict parasite development. Although these defenses have been studied for some time, the regulatory factors that control them are poorly understood. The protein kinase C (PKC) gene family consists of serine/threonine kinases that serve as central signaling molecules and regulators of a broad spectrum of cellular processes including epithelial barrier function and immunity. Indeed, PKCs are highly conserved, ranging from 7 isoforms in Drosophila to 16 isoforms in mammals, yet none have been identified in mosquitoes. Despite conservation of the PKC gene family and their potential as targets for transmission-blocking strategies for malaria, no direct connections between PKCs, the mosquito immune response or epithelial barrier integrity are known. Here, we identify and characterize six PKC gene family members--PKCδ, PKCε, PKCζ, PKD, PKN, and an indeterminate conventional PKC--in Anopheles gambiae and Anopheles stephensi. Sequence and phylogenetic analyses of the anopheline PKCs support most subfamily assignments. All six PKCs are expressed in the midgut epithelia of A. gambiae and A. stephensi post-blood feeding, indicating availability for signaling in a tissue that is critical for malaria parasite development. Although inhibition of PKC enzymatic activity decreased NF-κB-regulated anti-microbial peptide expression in mosquito cells in vitro, PKC inhibition had no effect on expression of a panel of immune genes in the midgut epithelium in vivo. PKC inhibition did, however, significantly increase midgut barrier integrity and decrease development of P. falciparum oocysts in A. stephensi, suggesting that PKC-dependent signaling is a negative regulator of epithelial barrier function and a potential new target for transmission-blocking strategies

Direct sequencing and expression analysis of a large number of miRNAs in Aedes aegypti and a multi-species survey of novel mosquito miRNAs

<p>Abstract</p> <p>Background</p> <p>MicroRNAs (miRNAs) are a novel class of gene regulators whose biogenesis involves hairpin structures called precursor miRNAs, or pre-miRNAs. A pre-miRNA is processed to make a miRNA:miRNA* duplex, which is then separated to generate a mature miRNA and a miRNA*. The mature miRNAs play key regulatory roles during embryonic development as well as other cellular processes. They are also implicated in control of viral infection as well as innate immunity. Direct experimental evidence for mosquito miRNAs has been recently reported in anopheline mosquitoes based on small-scale cloning efforts.</p> <p>Results</p> <p>We obtained approximately 130, 000 small RNA sequences from the yellow fever mosquito, <it>Aedes aegypti</it>, by 454 sequencing of samples that were isolated from mixed-age embryos and midguts from sugar-fed and blood-fed females, respectively. We also performed bioinformatics analysis on the <it>Ae. aegypti </it>genome assembly to identify evidence for additional miRNAs. The combination of these approaches uncovered 98 different pre-miRNAs in <it>Ae. aegypti </it>which could produce 86 distinct miRNAs. Thirteen miRNAs, including eight novel miRNAs identified in this study, are currently only found in mosquitoes. We also identified five potential revisions to previously annotated miRNAs at the miRNA termini, two cases of highly abundant miRNA* sequences, 14 miRNA clusters, and 17 cases where more than one pre-miRNA hairpin produces the same or highly similar mature miRNAs. A number of miRNAs showed higher levels in midgut from blood-fed female than that from sugar-fed female, which was confirmed by northern blots on two of these miRNAs. Northern blots also revealed several miRNAs that showed stage-specific expression. Detailed expression analysis of eight of the 13 mosquito-specific miRNAs in four divergent mosquito genera identified cases of clearly conserved expression patterns and obvious differences. Four of the 13 miRNAs are specific to certain lineage(s) within mosquitoes.</p> <p>Conclusion</p> <p>This study provides the first systematic analysis of miRNAs in <it>Ae. aegypti </it>and offers a substantially expanded list of miRNAs for all mosquitoes. New insights were gained on the evolution of conserved and lineage-specific miRNAs in mosquitoes. The expression profiles of a few miRNAs suggest stage-specific functions and functions related to embryonic development or blood feeding. A better understanding of the functions of these miRNAs will offer new insights in mosquito biology and may lead to novel approaches to combat mosquito-borne infectious diseases.</p

Insertion polymorphisms of SINE200 retrotransposons within speciation islands of Anopheles gambiae molecular forms

<p>Abstract</p> <p>Background</p> <p>SINEs (Short INterspersed Elements) are homoplasy-free and co-dominant genetic markers which are considered to represent useful tools for population genetic studies, and could help clarifying the speciation processes ongoing within the major malaria vector in Africa, <it>Anopheles gambiae </it>s.s. Here, we report the results of the analysis of the insertion polymorphism of a nearly 200 bp-long SINE (<it>SINE200</it>) within genome areas of high differentiation (i.e. "speciation islands") of M and S <it>A. gambiae </it>molecular forms.</p> <p>Methods</p> <p>A <it>SINE</it>-PCR approach was carried out on thirteen <it>SINE200 </it>insertions in M and S females collected along the whole range of distribution of <it>A. gambiae </it>s.s. in sub-Saharan Africa. Ten specimens each for <it>Anopheles arabiensis</it>, <it>Anopheles melas, Anopheles quadriannulatus </it>A and 15 M/S hybrids from laboratory crosses were also analysed.</p> <p>Results</p> <p>Eight loci were successfully amplified and were found to be specific for <it>A. gambiae </it>s.s.: 5 on 2L chromosome and one on X chromosome resulted monomorphic, while two loci positioned respectively on 2R (i.e. <it>S200 </it>2R12D) and X (i.e. <it>S200 </it>X6.1) chromosomes were found to be polymorphic. <it>S200 </it>2R12D was homozygote for the insertion in most S-form samples, while intermediate levels of polymorphism were shown in M-form, resulting in an overall high degree of genetic differentiation between molecular forms (Fst = 0.46 p < 0.001) and within M-form (Fst = 0.46 p < 0.001). The insertion of <it>S200 </it>X6.1 was found to be fixed in all M- and absent in all S-specimens. This led to develop a novel easy-to-use PCR approach to straightforwardly identify <it>A. gambiae </it>molecular forms. This novel approach allows to overcome the constraints associated with markers on the rDNA region commonly used for M and S identification. In fact, it is based on a single copy and irreversible <it>SINE200 </it>insertion and, thus, is not subjected to peculiar evolutionary patterns affecting rDNA markers, e.g. incomplete homogenization of the arrays through concerted evolution and/or mixtures of M and S IGS-sequences among the arrays of single chromatids.</p> <p>Conclusion</p> <p>The approach utilized allowed to develop new easy-to-use co-dominant markers for the analysis of genetic differentiation between M and S-forms and opens new perspectives in the study of the speciation process ongoing within <it>A. gambiae</it>.</p

Co-targeting of DNA, RNA, and protein molecules provides optimal outcomes for treating osteosarcoma and pulmonary metastasis in spontaneous and experimental metastasis mouse models.

Metastasis is a major cause of mortality for cancer patients and remains as the greatest challenge in cancer therapy. Driven by multiple factors, metastasis may not be controlled by the inhibition of single target. This study was aimed at assessing the hypothesis that drugs could be rationally combined to co-target critical DNA, RNA and protein molecules to achieve "saturation attack" against metastasis. Independent actions of the model drugs DNA-intercalating doxorubicin, RNA-interfering miR-34a and protein-inhibiting sorafenib on DNA replication, RNA translation and protein kinase signaling in highly metastatic, human osteosarcoma 143B cells were demonstrated by the increase of γH2A.X foci formation, reduction of c-MET expression and inhibition of Erk1/2 phosphorylation, respectively, and optimal effects were found for triple-drug combination. Consequently, triple-drug treatment showed a strong synergism in suppressing 143B cell proliferation and the greatest effects in reducing cell invasion. Compared to single- and dual-drug treatment, triple-drug therapy suppressed pulmonary metastases and orthotopic osteosarcoma progression to significantly greater degrees in orthotopic osteosarcoma xenograft/spontaneous metastases mouse models, while none showed significant toxicity. In addition, triple-drug therapy improved the overall survival to the greatest extent in experimental metastases mouse models. These findings demonstrate co-targeting of DNA, RNA and protein molecules as a novel therapeutic strategy for the treatment of metastasis

A Multi-Robot Cooperation Framework for Sewing Personalized Stent Grafts

This paper presents a multi-robot system for manufacturing personalized medical stent grafts. The proposed system adopts a modular design, which includes: a (personalized) mandrel module, a bimanual sewing module, and a vision module. The mandrel module incorporates the personalized geometry of patients, while the bimanual sewing module adopts a learning-by-demonstration approach to transfer human hand-sewing skills to the robots. The human demonstrations were firstly observed by the vision module and then encoded using a statistical model to generate the reference motion trajectories. During autonomous robot sewing, the vision module plays the role of coordinating multi-robot collaboration. Experiment results show that the robots can adapt to generalized stent designs. The proposed system can also be used for other manipulation tasks, especially for flexible production of customized products and where bimanual or multi-robot cooperation is required.Comment: 10 pages, 12 figures, accepted by IEEE Transactions on Industrial Informatics, Key words: modularity, medical device customization, multi-robot system, robot learning, visual servoing, robot sewin

Improved reference genome of the arboviral vector Aedes albopictus

Background: The Asian tiger mosquito Aedes albopictus is globally expanding and has become the main vector for human arboviruses in Europe. With limited antiviral drugs and vaccines available, vector control is the primary approach to prevent mosquito-borne diseases. A reliable and accurate DNA sequence of the Ae. albopictus genome is essential to develop new approaches that involve genetic manipulation of mosquitoes. Results: We use long-read sequencing methods and modern scaffolding techniques (PacBio, 10X, and Hi-C) to produce AalbF2, a dramatically improved assembly of the Ae. albopictus genome. AalbF2 reveals widespread viral insertions, novel microRNAs and piRNA clusters, the sex-determining locus, and new immunity genes, and enables genome-wide studies of geographically diverse Ae. albopictus populations and analyses of the developmental and stage-dependent network of expression data. Additionally, we build the first physical map for this species with 75% of the assembled genome anchored to the chromosomes. Conclusion: The AalbF2 genome assembly represents the most up-to-date collective knowledge of the Ae. albopictus genome. These resources represent a foundation to improve understanding of the adaptation potential and the epidemiological relevance of this species and foster the development of innovative control measures