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

Conceptual examination of emotion-related terms in Japanese: In the psychological studies of expressive behavior

DNA-seq library statistics. Total number of reads, percentage of mapped reads and percentage of properly paired reads from DNA-seq of 16 individual Ae. albopictus mosquitoes (Foshan strain). (DOCX 20 kb

Multidisciplinary investigation on "An improved genome for the Asian tiger mosquito Aedes albopictus and its applications in studying endogenous viral sequences"

Mosquito-borne diseases, including arboviral diseases such as Dengue, Chikungunya and Zika, have increased their world-wide incidence in the past 50 years and currently account for about 17% of all infectious diseases globally. Urbanization, globalization, increased international mobility and the widespread distribution of the main arboviral vectors, the mosquitoes Aedes aegypti and Aedes albopictus are all factors that have contributed to the (re)-emergence of arboviral diseases. Effective therapies and vaccines are limited for most arboviruses. Accordingly, vector control is the primary way to prevent transmission of arboviruses to humans. A deep understanding of the interaction and co-evolution between viruses and mosquito vectors is expected to aid in the development of novel transmission control strategies. The capacity of mosquitoes to support viral replication and transmission is called vector competence and is a dynamic and variable trait affected by many factors, suggesting an “arms race” between mosquitoes and viruses. The main arboviral vector in Europe is the invasive species Ae. albopictus, which has increasingly received attention from the scientific community due to its quick worldwide spread from south East Asia in the past 50 years. Nonretroviral RNA endogenous viral elements (nrEVEs) with similarities to the non-retroviral RNA viruses of the Flaviviridae and Rhabdoviridae family have been found with high frequency in Ae. albopictus mosquitoes. These viral integrations often interact with the most-recently characterized of the three RNA interference (RNAi) pathways: the PIWI-interacting RNA (piRNA) pathway. Most, but not all, nrEVEs in the genome of Ae. albopictus map next to transposable elements (TEs) fragments in piRNA clusters and produce piRNAs, small molecules that associate with Argonaute proteins of the PIWI clade to silence TEs based on sequence complementarity to piRNAs. In addition to its canonical role in preserving genome integrity, the piRNA pathway has antiviral activity in Aedes spp. mosquitoes. Despite the abundance of nrEVEs the biology, functional role, and patterns of integration in wild mosquito populations are still relatively unexplored. nrEVEs could be expressed and influence the phenotype of mosquitoes acting as a form of host antiviral immunity. A difficulty in studying the Ae. albopictus genome was the absence of a high-quality reference genome. During my PhD, I focused my attention on Ae. albopictus to primarily improve knowledge of its genome. I contributed to the sequencing, assembly, and annotation of a new reference genome for Ae. albopictus based on long-reads sequencing technologies; I coordinated an international consortium to annotate and characterize genomic features and their expression and produce a physical map of the genome. The availability of this new assembly allowed me to ask more specific questions on the landscape of viral integrations. A newly developed bioinformatic pipeline was combined with molecular biology techniques to identify viral sequences integrated into the genomes of wild-collected mosquitoes from different geographical locations. I also used the new genome assembly to reconstruct and identify RNA viruses in mosquito small-RNA sequencing data. I correlated the viruses identified in the mosquitoes with their population-specific nrEVE landscape, under the hypothesis that the pattern of nrEVEs is shaped by exposure to viruses. Lastly, I applied the CRISPR-Cas9 genome editing technology on Ae. albopictus embryos to modify a viral integration and a piRNA cluster and test the hypothesis that viral integrations have a role as immunity effectors against cognate viral infections. Overall, results gained through my PhD activities will enhance our understanding on the genome structure of Ae. albopictus and the importance of repetitive elements like viral integrations in the context of its biology.Mosquito-borne diseases, including arboviral diseases such as Dengue, Chikungunya and Zika, have increased their world-wide incidence in the past 50 years and currently account for about 17% of all infectious diseases globally. Urbanization, globalization, increased international mobility and the widespread distribution of the main arboviral vectors, the mosquitoes Aedes aegypti and Aedes albopictus are all factors that have contributed to the (re)-emergence of arboviral diseases. Effective therapies and vaccines are limited for most arboviruses. Accordingly, vector control is the primary way to prevent transmission of arboviruses to humans. A deep understanding of the interaction and co-evolution between viruses and mosquito vectors is expected to aid in the development of novel transmission control strategies. The capacity of mosquitoes to support viral replication and transmission is called vector competence and is a dynamic and variable trait affected by many factors, suggesting an “arms race” between mosquitoes and viruses. The main arboviral vector in Europe is the invasive species Ae. albopictus, which has increasingly received attention from the scientific community due to its quick worldwide spread from south East Asia in the past 50 years. Nonretroviral RNA endogenous viral elements (nrEVEs) with similarities to the non-retroviral RNA viruses of the Flaviviridae and Rhabdoviridae family have been found with high frequency in Ae. albopictus mosquitoes. These viral integrations often interact with the most-recently characterized of the three RNA interference (RNAi) pathways: the PIWI-interacting RNA (piRNA) pathway. Most, but not all, nrEVEs in the genome of Ae. albopictus map next to transposable elements (TEs) fragments in piRNA clusters and produce piRNAs, small molecules that associate with Argonaute proteins of the PIWI clade to silence TEs based on sequence complementarity to piRNAs. In addition to its canonical role in preserving genome integrity, the piRNA pathway has antiviral activity in Aedes spp. mosquitoes. Despite the abundance of nrEVEs the biology, functional role, and patterns of integration in wild mosquito populations are still relatively unexplored. nrEVEs could be expressed and influence the phenotype of mosquitoes acting as a form of host antiviral immunity. A difficulty in studying the Ae. albopictus genome was the absence of a high-quality reference genome. During my PhD, I focused my attention on Ae. albopictus to primarily improve knowledge of its genome. I contributed to the sequencing, assembly, and annotation of a new reference genome for Ae. albopictus based on long-reads sequencing technologies; I coordinated an international consortium to annotate and characterize genomic features and their expression and produce a physical map of the genome. The availability of this new assembly allowed me to ask more specific questions on the landscape of viral integrations. A newly developed bioinformatic pipeline was combined with molecular biology techniques to identify viral sequences integrated into the genomes of wild-collected mosquitoes from different geographical locations. I also used the new genome assembly to reconstruct and identify RNA viruses in mosquito small-RNA sequencing data. I correlated the viruses identified in the mosquitoes with their population-specific nrEVE landscape, under the hypothesis that the pattern of nrEVEs is shaped by exposure to viruses. Lastly, I applied the CRISPR-Cas9 genome editing technology on Ae. albopictus embryos to modify a viral integration and a piRNA cluster and test the hypothesis that viral integrations have a role as immunity effectors against cognate viral infections. Overall, results gained through my PhD activities will enhance our understanding on the genome structure of Ae. albopictus and the importance of repetitive elements like viral integrations in the context of its biology

Endogenous viral elements in mosquito genomes: current knowledge and outstanding questions

Integrations from non-retroviral RNA viruses (nrEVEs) have been identified across several taxa, including mosquitoes. Amongst all Culicinae species, the viral vectors Aedes aegypti and Aedes albopictus stand out for their high number of nrEVEs. In addition, Aedes nrEVEs are enriched in piRNA clusters and generate piRNAs that can silence incoming viral genomes. As such, nrEVEs represent a new form of inherited antiviral immunity. To propel this discovery into novel transmission-blocking vector control strategies, a deeper understanding of nrEVE biology and evolution is essential because differences in the landscape of nrEVEs have been identified in wild-caught mosquitoes, the piRNA profile of nrEVEs is not homogeneous and nrEVEs outside piRNA clusters exist and are expressed at the mRNA level. Here we summarise current knowledge on nrEVEs in mosquitoes and we point out the many unanswered questions and potentials of these genomic elements

Leaning Into the Bite: The piRNA Pathway as an Exemplar for the Genetic Engineering Need in Mosquitoes

The piRNA pathway is a specialized small RNA interference that in mosquitoes is mechanistically distant from analogous biology in the Drosophila model. Current genetic engineering methods, such as targeted genome manipulation, have a high potential to tease out the functional complexity of this intricate molecular pathway. However, progress in utilizing these methods in arthropod vectors has been geared mostly toward the development of new vector control strategies rather than to study cellular functions. Herein we propose that genetic engineering methods will be essential to uncover the full functionality of PIWI/piRNA biology in mosquitoes and that extending the applications of genetic engineering on other aspects of mosquito biology will grant access to a much larger pool of knowledge in disease vectors that is just out of reach. We discuss motivations for and impediments to expanding the utility of genetic engineering to study the underlying biology and disease transmission and describe specific areas where efforts can be placed to achieve the full potential for genetic engineering in basic biology in mosquito vectors. Such efforts will generate a refreshed intellectual source of novel approaches to disease control and strong support for the effective use of approaches currently in development

Virome and nrEVEome diversity of Aedes albopictus mosquitoes from La Reunion Island and China

Background Aedes albopictus is a public health threat for its worldwide spread and ability to transmit arboviruses. Understanding mechanisms of mosquito immunity can provide new tools to control arbovirus spread. The genomes of Aedes mosquitoes contain hundreds of nonretroviral endogenous viral elements (nrEVEs), which are enriched in piRNA clusters and produce piRNAs, with the potential to target cognate viruses. Recently, one nrEVE was shown to limit cognate viral infection through nrEVE-derived piRNAs. These findings suggest that nrEVEs constitute an archive of past viral infection and that the landscape of viral integrations may be variable across populations depending on their viral exposure. Methods We used bioinformatics and molecular approaches to identify known and novel (i.e. absent in the reference genome) viral integrations in the genome of wild collected Aedes albopictus mosquitoes and characterize their virome. Results We showed that the landscape of viral integrations is dynamic with seven novel viral integrations being characterized, but does not correlate with the virome, which includes both viral species known and unknown to infect mosquitoes. However, the small RNA coverage profile of nrEVEs and the viral genomic contigs we identified confirmed an interaction among these elements and the piRNA and siRNA pathways in mosquitoes. Conclusions Mosquitoes nrEVEs have been recently described as a new form of heritable, sequence-specific mechanism of antiviral immunity. Our results contribute to understanding the dynamic distribution of nrEVEs in the genomes of wild Ae. albopictus and their interaction with mosquito viruses

Profile of Small RNAs, vDNA Forms and Viral Integrations in Late Chikungunya Virus Infection of Aedes albopictus Mosquitoes

International audienceThe Asian tiger mosquito Aedes albopictus is contributing to the (re)-emergence of Chikungunya virus (CHIKV). To gain insights into the molecular underpinning of viral persistence, which renders a mosquito a life-long vector, we coupled small RNA and whole genome sequencing approaches on carcasses and ovaries of mosquitoes sampled 14 days post CHIKV infection and investigated the profile of small RNAs and the presence of vDNA fragments. Since Aedes genomes harbor nonretroviral Endogenous Viral Elements (nrEVEs) which confers tolerance to cognate viral infections in ovaries, we also tested whether nrEVEs are formed after CHIKV infection. We show that while small interfering (si)RNAs are evenly distributed along the full viral genome, PIWI-interacting (pi)RNAs mostly arise from a ~1000 bp window, from which a unique vDNA fragment is identified. CHIKV infection does not result in the formation of new nrEVEs, but piRNAs derived from existing nrEVEs correlate with differential expression of an endogenous transcript. These results demonstrate that all three RNAi pathways contribute to the homeostasis during the late stage of CHIKV infection, but in different ways, ranging from directly targeting the viral sequence to regulating the expression of mosquito transcripts and expand the role of nrEVEs beyond immunity against cognate viruses

Additional file 2: Table S2. of Comparative genomics shows that viral integrations are abundant and express piRNAs in the arboviral vectors Aedes aegypti and Aedes albopictus

List of viral genomes used for in silico screening of Dipteran genomes. The only DNA arbovirus is in yellow. (XLSX 38 kb