Regulatory role of cellular and viral microRNAs in insect-virus interactions

The discovery of microRNAs (miRNAs) and their functions has led to a paradigm shift in our understanding of the regulation of gene expression, adding an extra layer of complexity for the mechanisms of gene expression. Both cellular and virus encoded miRNAs play important roles in virus-host interactions that may affect virus replication and the outcome of infection. Recent developments in RNA-seq platforms and bioinformatics tools have accelerated the discovery of miRNAs, their targets, and a myriad of associated research in various species. Here, recent findings and developments in miRNA research pertinent to insect host-virus interactions are reviewed and analyzed

Role of MicroRNAs in Insect Host–Microorganism Interactions

MicroRNAs (miRNAs) have appeared as important regulators of various biological processes including development, cancer, immunity, and host–microorganism interactions. Accumulating evidence demonstrates the differential expression of host miRNAs upon infection by various microorganisms and the involvement of microorganism-encoded miRNAs in host manipulation. Some of these alterations could be part of a host response to an infection to limit replication and dissemination of the microorganism or, conversely, due to manipulation of the host miRNA pathway by the microorganism to facilitate its replication. Insights into the role of miRNAs in host defense responses and host manipulation by microorganisms will enable a better understanding of host–microorganism interactions

Improving group functioning in solving realistic problems

Group work in educational settings has several advantages, but it is also associated with some difficulties, most commonly concerns about group functioning. The aim of this study was to improve group functioning in solving realistic problems, using a combination of: 1) team skill training prior to group work; 2) peer and self-assessment to evaluate contribution to group work; and 3) lecturer designed versus studentdesigned problems. The results showed positive effects on group functioning. After initial team skill training, peer assessments and moderation of marks based on those assessments stimulated the so-called free loaders to improve their contributions to group work, which is a result that has been demonstrated in only a few studies. In addition, students showed some preference for designing their own problems and they thought the learning outcomes were higher than from lecturer-designed problems

Improving Group Functioning in Solving Realistic Problems

Abstract Group work in educational settings has several advantages, but it is also associated with some difficulties, most commonly concerns about group functioning. The aim of this study was to improve group functioning in solving realistic problems, using a combination of: 1) team skill training prior to group work; 2) peer and selfassessment to evaluate contribution to group work; and 3) lecturer-designed versus student-designed problems. The results showed positive effects on group functioning. After initial team skill training, peer assessments and moderation of marks based on those assessments stimulated the so-called free loaders to improve their contributions to group work, which is a result that has been demonstrated in only a few studies. In addition, students showed some preference for designing their own problems and they thought the learning outcomes were higher than from lecturer-designed problems. Abstract Group work in educational settings has several advantages, but it is also associated with some difficulties, most commonly concerns about group functioning. The aim of this study was to improve group functioning in solving realistic problems, using a combination of: 1) team skill training prior to group work; 2) peer and self-assessment to evaluate contribution to group work; and 3) lecturer-designed versus studentdesigned problems. The results showed positive effects on group functioning. After initial team skill training, peer assessments and moderation of marks based on those assessments stimulated the so-called free loaders to improve their contributions to group work, which is a result that has been demonstrated in only a few studies. In addition, students showed some preference for designing their own problems and they thought the learning outcomes were higher than from lecturer-designed problems. Keyword

Regulation of arginine methyltransferase 3 by a Wolbachia-induced microRNA in Aedes aegypti and its effect on Wolbachia and dengue virus replication

The gram-negative endosymbiotic bacteria, Wolbachia, have been found to colonize a wide range of invertebrates, including over 40% of insect species. Best known for host reproductive manipulations, some strains of Wolbachia have been shown to reduce the host life span by about 50% and inhibit replication and transmission of dengue virus (DENV) in the mosquito vector, Aedes aegypti. The molecular mechanisms underlying these effects still are not well understood. Our previous studies showed that Wolbachia uses host microRNAs (miRNAs) to manipulate host gene expression for its efficient maintenance and limiting replication of DENV in Ae. aegypti. Protein arginine methyltransferases are structurally and functionally conserved proteins from yeast to human. In mammals, it has been reported that protein arginine methyltransferases such as PRMT1, 5 and 6 could regulate replication of different viruses. Ae. aegypti contains eight members of protein arginine methyltransferases (AaArgM1-8). Here, we show that the wMelPop strain of Wolbachia introduced into Ae. aegypti significantly induces the expression of AaArgM3. Interestingly, we found that Wolbachia uses aae-miR-2940, which is highly upregulated in Wolbachia-infected mosquitoes, to upregulate the expression of AaArgM3. Silencing of AaArgM3 in a mosquito cell line led to the inhibition of Wolbachia replication, but had no effect on the replication of DENV. These results provide further evidence that Wolbachia uses the host miRNAs to manipulate host gene expression and facilitate colonization in Ae. aegypti mosquito

Downregulation of Aedes aegypti chromodomain helicase DNA binding protein 7/Kismet by Wolbachia and its effect on dengue virus replication

Dengue virus (DENV) is a mosquito-transmitted virus imposing a significant burden on human health around the world. Since current control strategies are not sufficient, there is an urgent need to find alternative methods to control DENV transmission. It has been demonstrated that introduction of Wolbachia pipientis in Aedes aegypti mosquitoes can impede DENV transmission with the mechanism(s) not fully understood. Recently, a number of studies have found the involvement of chromodomain DNA binding helicases in case of Human Immunodeficiency virus (HIV) and Influenza A virus infection. In this study, we have identified three chromodomain helicase DNA binding protein (CHD) genes in Ae. aegypti and looked at their response in the case of Wolbachia and DENV infections. Foremost amongst them we have found that AeCHD7/Kismet is significantly downregulated in the presence of Wolbachia infection only in female mosquitoes. Furthermore, AeCHD7 levels showed significant increase during DENV infection, and AeCHD7 depletion led to severe reduction in the replication of DENV. Our data have identified AeCHD7 as a novel Ae. aegypti host factor that is important for DENV replication, and Wolbachia downregulates it, which may contribute towards the mechanism(s) of limiting DENV replication

Aedes Anphevirus: an insect-specific virus distributed worldwide in Aedes aegypti mosquitoes that has complex interplays with Wolbachia and dengue virus infection in cells

Insect specific viruses (ISVs) of the yellow fever mosquito Aedes aegypti have been demonstrated to modulate transmission of arboviruses such as dengue virus (DENV) and West Nile virus by the mosquito. The diversity and composition of the virome of Ae. aegypti, however, remains poorly understood. In this study, we characterised Aedes anphevirus (AeAV), a negative-sense RNA virus from the order Mononegavirales. AeAV identified from Aedes cell lines were infectious to both Ae. aegypti and Aedes albopictus cells, but not to three mammalian cell lines. To understand the incidence and genetic diversity of AeAV, we assembled 17 coding-complete and two partial genomes of AeAV from available RNA-Seq data. AeAV appears to transmit vertically and be present in laboratory colonies, wild-caught mosquitoes and cell lines worldwide. Phylogenetic analysis of AeAV strains indicates that as the Ae. aegypti mosquito has expanded into the Americas and Asia-Pacific, AeAV has evolved into monophyletic African, American and Asia-Pacific lineages. The endosymbiotic bacterium Wolbachia pipientis restricts positive-sense RNA viruses in Ae. aegypti. Re-analysis of a small RNA library of Ae. aegypti cells co-infected with AeAV and Wolbachia produces an abundant RNAi response consistent with persistent virus replication. We found Wolbachia enhances replication of AeAV when compared to a tetracycline cleared cell line, and AeAV modestly reduces DENV replication in vitro. The results from our study improve understanding of the diversity and evolution of the virome of Ae. aegypti and adds to previous evidence that shows Wolbachia does not restrict a range of negative strand RNA viruses. IMPORTANCE: The mosquito Aedes aegypti transmits a number of arthropod-borne viruses (arboviruses) such as dengue virus and Zika virus. Mosquitoes also harbour insect-specific viruses that may affect replication of pathogenic arboviruses in their body. Currently, however, there are only a handful of insect-specific viruses described from Ae. aegypti in the literature. Here, we characterise a novel negative strand virus, Aedes anphevirus (AeAV). Meta-analysis of Ae. aegypti samples showed that it is present in Ae. aegypti mosquitoes worldwide and is vertically transmitted. Wolbachia transinfected mosquitoes are currently being used in biocontrol as they effectively block transmission of several positive sense RNA viruses in mosquitoes. Our results demonstrate that Wolbachia enhances the replication of AeAV and modestly reduces dengue virus replication in a cell line model. This study expands our understanding of the virome in Ae. aegypti as well as providing insight into the complexity of the Wolbachia virus restriction phenotype

Wolbachia suppresses cell fusing agent virus in mosquito cells

The genus Flavivirus contains a large number of positive-sense ssRNA viruses. While some are transmitted by mosquitoes or other arthropods and are pathogenic to humans and animals (e. g. dengue and Zika viruses), some are insect-specific and do not replicate in vertebrate cells. These are known as insect-specific flaviviruses (ISFs). Cell fusing agent virus (CFAV) was the first described ISF, which was detected in an Aedes aegypti cell line, Aag2. Here, we investigated the effect of Wolbachia, a widespread endosymbiont of many insect species, that is known to block replication of several pathogenic flaviviruses, on CFAV. Our results demonstrated that, in mosquito cells, Wolbachia vastly suppresses replication of CFAV, with significantly less CFAV viral interfering small RNAs produced in the cells. However, removal of Wolbachia with tetracycline led to increased CFAV replication. These results suggest that Wolbachia is also able to suppress an ISF

Wolbachia endosymbiont of the horn fly Haematobia irritans irritans: a supergroup A strain with multiple horizontally acquired cytoplasmic incompatibility genes

The horn fly, Haematobia irritans irritans, is a hematophagous parasite of livestock distributed throughout Europe, Africa, Asia, and the Americas. Welfare losses on livestock due to horn fly infestation are estimated to cost between USD 1-2.5 billion annually in North America and Brazil. The endosymbiotic bacterium Wolbachia pipientis is a maternally inherited manipulator of reproductive biology in arthropods and naturally infects laboratory colonies of horn flies from Kerrville, USA and Alberta, Canada, but has also been identified in wild-caught samples from Canada, USA, Mexico and Hungary. Re-assembly of PacBio long-read and Illumina genomic DNA libraries from the Kerrville H. i. irritans genome project allowed for a complete and circularised 1.3 Mb Wolbachia genome (wIrr). Annotation of wIrr yielded 1249 coding genes, 34 tRNAs, three rRNAs, and five prophage regions. Comparative genomics and whole genome Bayesian evolutionary analysis of wIrr compared to published Wolbachia genomes suggests that wIrr is most closely related to and diverged from Wolbachia supergroup A strains known to infect Drosophila spp. Whole-genome synteny analyses between wIrr and closely related genomes indicates that wIrr has undergone significant genome rearrangements while maintaining high nucleotide identity. Comparative analysis of the cytoplasmic incompatibility (CI) genes of wIrr suggests two phylogenetically distinct CI loci and acquisition of another CifB homolog from phylogenetically distant supergroup A Wolbachia strains suggesting horizontal acquisition of these loci. The wIrr genome provides a resource for future examination of the impact Wolbachia may have in both biocontrol and potential insecticide resistance of horn flies

A comparative analysis of corpora allata-corpora cardiaca microRNA repertories revealed significant changes during mosquito metamorphosis

The corpora allata (CA) are a pair of endocrine glands with neural connections to the brain and close association with another neuroendocrine organ, the corpora cardiaca (CC). The CA from adult female Aedes aegypti mosquitoes synthesize fluctuating levels of juvenile hormone (JH), which have been linked to the ovarian development and are influenced by nutritional signals. In this study, we investigated the potential involvement of microRNAs (miRNAs), a type of small non-coding RNAs, in the regulation of gene expression in CA-CC complexes during mosquito reproductive development, at stages with distinct JH biosynthesis patterns. We analyzed the miRNA repertoires expressed in the CA-CC of pupae, sugar-fed and blood-fed female Ae. aegypti. In total, 156 mature miRNAs were detected in the CA-CC, with 84 displaying significant differences in expression among the three CA-CC developmental stages. There were more miRNAs that were expressed in pupae, and decreased or were absent after adult emergence, when compared with changes between CA-CC of sugar and blood-fed females. Analysis of the genes identified as potential targets for the CA-CC miRNA repertoires classified them into the broad categories of metabolism, information storage and processing, and cellular processes and signaling; with genes involved in cellular processes and signaling representing the largest portion. Among them, the signal-transduction mechanisms and intracellular trafficking, secretion and vesicular transport contained almost 55% of the genes' targets. A substantial number of miRNAs were differentially abundant in the libraries of the three developmental stages, and those changes were much more notable when pupae and adult stages were compared. We detected putative binding sites for some of the most abundant miRNAs on genes encoding JH biosynthetic enzymes and CC neuropeptides. These studies should help us to gain a better understanding of the regulation of CA-CC activity mediated by miRNAs during major developmental stages in mosquitoes