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Long intergenic non-coding RNAs (lincRNAs) are appearing as an important class of regulatory RNAs with a variety of biological functions. The aim of this study was to identify the lincRNA profile in the dengue vector Aedes aegypti and evaluate their potential role in host-pathogen interaction. The majority of previous RNA-Seq transcriptome studies in Ae. aegypti have focused on the expression pattern of annotated protein coding genes under different biological conditions. Here, we used 35 publically available RNA-Seq datasets with relatively high depth to screen the Ae. aegypti genome for lincRNA discovery. This led to the identification of 3,482 putative lincRNAs. These lincRNA genes displayed a slightly lower GC content and shorter transcript lengths compared to protein-encoding genes. Ae. aegypti lincRNAs also demonstrate low evolutionary sequence conservation even among closely related species such as Culex quinquefasciatus and Anopheles gambiae. We examined their expression in dengue virus serotype 2 (DENV-2) and Wolbachia infected and non-infected adult mosquitoes and Aa20 cells. The results revealed that DENV-2 infection increased the abundance of a number of host lincRNAs, from which some suppress viral replication in mosquito cells. RNAi-mediated silencing of lincRNA_1317 led to enhancement in viral replication, which possibly indicates its potential involvement in the host anti-viral defense. A number of lincRNAs were also differentially expressed in Wolbachia-infected mosquitoes. The results will facilitate future studies to unravel the function of lncRNAs in insects and may prove to be beneficial in developing new ways to control vectors or inhibit replication of viruses in them.

Wolbachia pipientis is an intracellular endosymbiotic bacterium that blocks the replication of several arboviruses in transinfected Aedes aegypti mosquitoes, yet its antiviral mechanism remains unknown. For the first time, we employed Nanopore direct RNA sequencing technology to investigate the impact of w AlbB strain of Wolbachia on the host’s N 6 -methyladenosine (m 6 A) machinery and post-transcriptional modification landscape. Our study revealed that Wolbachia infection elevates the expression of genes involved in the mosquito’s m 6 A methyltransferase complex. However, knocking down these m 6 A-related genes did not affect Wolbachia density. Nanopore sequencing identified 1,392 differentially modified m 6 A DRACH motifs on mosquito transcripts, with 776 showing increased and 616 showing decreased m 6 A levels due to Wolbachia . These m 6 A sites were predominantly enriched in coding sequences and 3′-untranslated regions. Gene Ontology analysis revealed that genes with reduced m 6 A levels were over-represented in functional GO terms associated with purine nucleotide binding functions critical in the post-transcriptional modification process of m 6 A. Differential gene expression analysis of the Nanopore data uncovered that a total of 643 protein-coding genes were significantly differentially expressed, 427 were downregulated, and 216 were upregulated. Several classical and non-classical immune-related genes were amongst the downregulated DEGs. Notably, it revealed a critical host factor, transmembrane protein 41B (TMEM41B), which is required for flavivirus infection, was upregulated and methylated in the presence of Wolbachia . Indeed, there is a strong correlation between gene expression being upregulated in genes with both increased and decreased levels of m 6 A modification, respectively. Our findings underscore Wolbachia ’s ability to modulate many intracellular aspects of its mosquito host by influencing post-transcriptional m 6 A modifications and gene expression, and it unveils a potential link behind its antiviral properties.

The corpora allata - corpora cardiaca (CA-CC) is an endocrine gland complex that regulates mosquito development and reproduction through the synthesis of juvenile hormone (JH). Epoxidase (Epox) is a key enzyme in the production of JH. We recently utilized CRISPR/Cas9 to establish an epoxidase- deficient ( epox −/− ) Aedes aegypti line. The CA from epox −/− mutants do not synthesize epoxidated JH III but methyl farneosate (MF), a weak agonist of the JH receptor, and therefore have reduced JH signalling. Illumina sequencing was used to examine the differences in gene expression between the CA-CC from wild type ( WT ) and epox −/− adult female mosquitoes. From 18,034 identified genes, 317 were significantly differentially expressed. These genes are involved in many biological processes, including the regulation of cell proliferation and apoptosis, energy metabolism, and nutritional uptake. In addition, the same CA-CC samples were also used to examine the microRNA (miRNA) profiles of epox −/− and WT mosquitoes. A total of 197 miRNAs were detected, 24 of which were differentially regulated in epox −/− mutants. miRNA binding sites for these particular miRNAs were identified using an in silico approach; they target a total of 101 differentially expressed genes. Our results suggest that a lack of epoxidase, besides affecting JH synthesis, results in the diminishing of JH signalling that have significant effects on Ae. aegypti CA-CC transcriptome profiles, as well as its miRNA repertoire.

Zika virus (ZIKV), a flavivirus transmitted primarily by Aedes aegypti, has recently spread globally in an unprecedented fashion, yet we have a poor understanding of host-microbe interactions in this system. To gain insights into the interplay between ZIKV and the mosquito, we sequenced the small RNA profiles in ZIKV-infected and non-infected Ae. aegypti mosquitoes at 2, 7 and 14 days post-infection. ZIKA induced an RNAi response in the mosquito with virus-derived short interfering RNAs and PIWI-interacting RNAs dramatically increased in abundance post-infection. Further, we found 17 host microRNAs (miRNAs) that were modulated by ZIKV infection at all time points. Strikingly, many of these regulated miRNAs have been reported to have their expression altered by dengue and West Nile viruses, while the response was divergent from that induced by the alphavirus Chikungunya virus in mosquitoes. This suggests that conserved miRNA responses occur within mosquitoes in response to flavivirus infection. This study expands our understanding of ZIKV-vector interactions and provides potential avenues to be further investigated to target ZIKV in the mosquito host.

Mapping small reads to genome reference is an essential and more common approach to identify microRNAs (miRNAs) in an organism. Using closely related species genomes as proxy references can facilitate miRNA expression studies in non-model species that their genomes are not available. However, the level of error this introduces is mostly unknown, as this is the result of evolutionary distance between the proxy reference and the species of interest. To evaluate the accuracy of miRNA discovery pipelines in non-model organisms, small RNA library data from a mosquito, Aedes aegypti, were mapped to three well annotated insect genomes as proxy references using miRanalyzer with two strict and loose mapping criteria. In addition, another web-based miRNA discovery pipeline (DSAP) was used as a control for program performance. Using miRanalyzer, more than 80% reduction was observed in the number of mapped reads using strict criterion when proxy genome references were used; however, only 20% reduction was recorded for mapped reads to other species known mature miRNA datasets. Except a few changes in ranking, mapping criteria did not make any significant differences in the profile of the most abundant miRNAs in A. aegypti when its original or a proxy genome was used as reference. However, more variation was observed in miRNA ranking profile when DSAP was used as analysing tool. Overall, the results also suggested that using a proxy reference did not change the most abundant miRNAs' differential expression profiles when infected or non-infected libraries were compared. However, usage of a proxy reference could provide about 67% of the original outcome from more extremely up- or down-regulated miRNA profiles. Although using closely related species genome incurred some losses in the number of miRNAs, the most abundant miRNAs along with their differential expression profile would be acceptable based on the sensitivity level of each project.

Native Australian soldier flies, Inopus spp. (Diptera: Stratiomyidae), are agricultural pests of economic importance to the sugarcane industry. A screen of the salivary gland transcriptome of Inopus flavus (James) revealed the presence of viral RNA belonging to a potentially novel member of the family Dicistroviridae. The complete genome sequence consists of 9793 nucleotides with two open reading frames. The genome includes two potential internal ribosomal entry sites (IRESs): one within the 5’ UTR and the other in the intergenic region (IGR). Virus particles purified from infected larvae and visualised by electron microscopy were found to be icosahedral, non-enveloped, and 30 nm in diameter.

Vector-borne viruses pose great risks to human health. Zika virus has recently emerged as a global threat, rapidly expanding its distribution. Understanding the interactions of the virus with mosquito vectors at the molecular level is vital for devising new approaches in inhibiting virus transmission. In this study, we embarked on analyzing the transcriptional response of Aedes aegypti mosquitoes to Zika virus infection. Results showed large changes in both coding and long noncoding RNAs. Analysis of these genes showed similarities with other flaviviruses, including dengue virus, which is transmitted by the same mosquito vector. The outcomes provide a global picture of changes in the mosquito vector in response to Zika virus infection. Zika virus (ZIKV) of the Flaviviridae family is a recently emerged mosquito-borne virus that has been implicated in the surge of the number of microcephaly instances in South America. The recent rapid spread of the virus led to its declaration as a global health emergency by the World Health Organization. The virus is transmitted mainly by the mosquito Aedes aegypti , which is also the vector of dengue virus; however, little is known about the interactions of the virus with the mosquito vector. In this study, we investigated the transcriptome profiles of whole A. aegypti mosquitoes in response to ZIKV infection at 2, 7, and 14 days postinfection using transcriptome sequencing. Results showed changes in the abundance of a large number of transcripts at each time point following infection, with 18 transcripts commonly changed among the three time points. Gene ontology analysis revealed that most of the altered genes are involved in metabolic processes, cellular processes, and proteolysis. In addition, 486 long intergenic noncoding RNAs that were altered upon ZIKV infection were identified. Further, we found changes of a number of potential mRNA target genes correlating with those of altered host microRNAs. The outcomes provide a basic understanding of A. aegypti responses to ZIKV and help to determine host factors involved in replication or mosquito host antiviral response against the virus. IMPORTANCE Vector-borne viruses pose great risks to human health. Zika virus has recently emerged as a global threat, rapidly expanding its distribution. Understanding the interactions of the virus with mosquito vectors at the molecular level is vital for devising new approaches in inhibiting virus transmission. In this study, we embarked on analyzing the transcriptional response of Aedes aegypti mosquitoes to Zika virus infection. Results showed large changes in both coding and long noncoding RNAs. Analysis of these genes showed similarities with other flaviviruses, including dengue virus, which is transmitted by the same mosquito vector. The outcomes provide a global picture of changes in the mosquito vector in response to Zika virus infection.

Background An optimal starting point for relating genome function to organismal biology is a high-quality nuclear genome assembly, and long-read sequencing is revolutionizing the production of this genomic resource in insects. Despite this, nuclear genome assemblies have been under-represented for agricultural insect pests, particularly from the order Coleoptera. Here we present a de novo genome assembly and structural annotation for the coconut rhinoceros beetle, Oryctes rhinoceros (Coleoptera: Scarabaeidae), based on Oxford Nanopore Technologies (ONT) long-read data generated from a wild-caught female, as well as the assembly process that also led to the recovery of the complete circular genome assemblies of the beetle’s mitochondrial genome and that of the biocontrol agent, Oryctes rhinoceros nudivirus (OrNV). As an invasive pest of palm trees, O. rhinoceros is undergoing an expansion in its range across the Pacific Islands, requiring new approaches to management that may include strategies facilitated by genome assembly and annotation. Results High-quality DNA isolated from an adult female was used to create four ONT libraries that were sequenced using four MinION flow cells, producing a total of 27.2 Gb of high-quality long-read sequences. We employed an iterative assembly process and polishing with one lane of high-accuracy Illumina reads, obtaining a final size of the assembly of 377.36 Mb that had high contiguity (fragment N50 length = 12 Mb) and accuracy, as evidenced by the exceptionally high completeness of the benchmarked set of conserved single-copy orthologous genes (BUSCO completeness = 99.1%). These quality metrics place our assembly ahead of the published Coleopteran genomes, including that of an insect model, the red flour beetle ( Tribolium castaneum ). The structural annotation of the nuclear genome assembly contained a highly-accurate set of 16,371 protein-coding genes, with only 2.8% missing BUSCOs, and the expected number of non-coding RNAs. The number and structure of paralogous genes in a gene family like Sigma GST is lower than in another scarab beetle ( Onthophagus taurus ), but higher than in the red flour beetle ( Tribolium castaneum ), which suggests expansion of this GST class in Scarabaeidae. The quality of our gene models was also confirmed with the correct placement of O. rhinoceros among other members of the rhinoceros beetles (subfamily Dynastinae) in a phylogeny based on the sequences of 95 protein-coding genes in 373 beetle species from all major lineages of Coleoptera. Finally, we provide a list of 30 candidate dsRNA targets whose orthologs have been experimentally validated as highly effective targets for RNAi-based control of several beetles. Conclusions The genomic resources produced in this study form a foundation for further functional genetic research and management programs that may inform the control and surveillance of O. rhinoceros populations, and we demonstrate the efficacy of de novo genome assembly using long-read ONT data from a single field-caught insect.

In Australia, Soldier flies ( spp.) are economically significant pests of sugarcane that currently lack a viable management strategy. Despite various research efforts, the mechanisms underlying the damage caused by soldier fly larvae remain poorly understood. Our study aims to explore whether this damage is associated with the transmission of plant viruses during larval feeding. We also explore the larval transcriptome to identify any entomopathogenic viruses with the potential to be used as biocontrol agents in future pest management programs. Seven novel virus sequences are identified and characterised using de novo assembly of RNA-Seq data obtained from salivary glands of larvae. The novel virus sequences belong to different virus families and are tentatively named SF-associated anphevirus (SFaAV), SF-associated orthomyxo-like virus (SFaOV), SF-associated narna-like virus (SFaNV), SF-associated partiti-like virus (SFaPV), SF-associated toti-like virus (SFaTV-1 and SFaTV-2) and SF-associated densovirus (SFaDV). These newly identified viruses are more likely insect-associated viruses, as phylogenetic analyses show that they cluster with other insect-specific viruses. Small RNA analysis indicates prominent peaks at both 21 nt and 26-29 nt, suggesting the activation of host siRNA and piwiRNA pathways. Our study helps to improve understanding of the virome of soldier flies and could identify insect viruses for deployment in novel pest management strategies.

MicroRNAs (miRNAs) are small non-coding RNAs that play important roles in many biological processes such as development, cell signaling and immune response. Small RNA deep sequencing technology provided an opportunity for a thorough survey of the miRNA profile of a mosquito cell line from Aedes aegypti. We characterized the miRNA composition of the nucleus and the cytoplasm of uninfected cells and compared it with the one of cells infected with the endosymbiotic bacterium Wolbachia strain wMelPop-CLA. We found an overall increase of small RNAs between 18 and 28 nucleotides in both cellular compartments in Wolbachia-infected cells and identified specific miRNAs induced and/or suppressed by the Wolbachia infection. We discuss the mechanisms that the cell may use to shuttle miRNAs between the cytoplasm and the nucleus. In addition, we identified piRNAs that changed their abundance in response to Wolbachia infection. The miRNAs and piRNAs identified in this study provide promising leads for investigations into the host-endosymbiont interactions and for better understanding of how Wolbachia manipulates the host miRNA machinery in order to facilitate its persistent replication in infected cells.