Search

The mosquito Aedes aegypti is the primary vector of human arboviral diseases caused by dengue, chikungunya and Zika viruses. Many studies have shown the potential roles of small RNA molecules such as microRNA, small interfering RNA and PIWI-interacting RNA in vector mosquitoes. The function of tRNA fragments (tRF), the newly discovered class of small RNAs, in mosquitoes is not known. In this study, we show that specific tRFs are expressed in significantly differential manner between males and females of Ae. aegypti strains. Specific tRFs also show differential response during developmental transition from larvae to adults, as well as after blood feeding of adult females. The expression pattern of tRFs upon blood feeding varied depending upon if the blood contained dengue virus, and also if the females were treated with antibiotic prior to feeding to cleanse of the gut bacteria. Our findings show that a single tRF derived from the precursor sequences of a tRNA-Gly was differentially expressed between males and females, developmental transitions and also upon blood feeding by females of two laboratory strains that vary in midgut susceptibility to dengue virus infection. The multifaceted functional implications of this specific tRF suggest that biogenesis of small regulatory molecules from a tRNA can have wide ranging effects on key aspects of Ae. aegypti vector biology., Author summary The mosquito Aedes aegypti is a major vector of arboviral diseases in subtropics and tropics. The confounding effects of immature development and adult microbiome on the ability of Ae. aegypti to transmit diseases (vector competence) have gained renewed attention in the recent years. However, the molecular nature of these links/ effects remains unknown. This is major gap in knowledge regarding how vector competence is regulated at molecular level, and how that regulation may be variable among different strains of this mosquito. In this study, we investigated expression of newly discovered class of small RNAs, called tRNA fragments (tRF) in Ae. aegypti strains. Based on small RNA sequencing and bioinformatics analyses, we show that tRFs are expressed in Ae. aegypti, and they are associated with significant changes in expression between males and females, during development stages, and post blood feeding responses. A single tRF showed association with sex-biased expression, developmental regulation and in response to blood meals between Moyo-S and Moyo-R strains that differ in midgut susceptibility to dengue virus. The findings of this study are expected to guide future research efforts directed toward examining detailed regulatory mechanisms of tRFs in vector competence of Ae. aegypti to disease transmission.

Although many similarities in arthropod CNS development exist, differences in axonogenesis and the formation of midline cells, which regulate axon growth, have been observed. For example, axon growth patterns in the ventral nerve cord of Artemia franciscana differ from that of Drosophila melanogaster. Despite such differences, conserved molecular marker expression at the midline of several arthropod species indicates that midline cells may be homologous in distantly related arthropods. However, data from additional species are needed to test this hypothesis. In this investigation, nerve cord formation and the putative homology of midline cells were examined in distantly related arthropods, including: long- and short-germ insects (D. melanogaster, Aedes aeygypti, and Tribolium castaneum), branchiopod crustaceans (A. franciscana and Triops longicauditus), and malacostracan crustaceans (Porcellio laevis and Parhyale hawaiensis). These comparative analyses were aided by a cross-reactive antibody generated against the Netrin (Net) protein, a midline cell marker and regulator of axonogenesis. The mechanism of nerve cord formation observed in Artemia is found in Triops, another branchiopod, but is not found in the other arthropods examined. Despite divergent mechanisms of midline cell formation and nerve cord development, Net accumulation is detected in a well-conserved subset of midline cells in branchiopod crustaceans, malacostracan crustaceans, and insects. Notably, the Net accumulation pattern is also conserved at the midline of the amphipod P. hawaiensis, which undergoes split germ-band development. Conserved Net accumulation patterns indicate that arthropod midline cells are homologous, and that Nets function to regulate commissure formation during CNS development of Tetraconata.

Background and purpose: Allergic inflammation and autoimmune diseases, such as atopic dermatitis, psoriasis and multiple sclerosis (MS), involve both mast cell and T-cell activation. However, possible interactions between the two and the mechanism of such activations are largely unknown. Experimental approach: Human umbilical cord blood-derived cultured mast cells (hCBMCs) and Jurkat T cells were incubated separately or together, following activation with myelin basic protein (MBP), as well as with or without pretreatment with the flavonoid luteolin for 15 min. The supernatant fluid was assayed for inflammatory mediators released from mast cells and interleukin (IL)-2 release from Jurkat cells. Key results: MBP (10 μM) stimulates hCBMCs to release IL-6, IL-8, transforming growth factor (TGF)-β1, tumour necrosis factor-α (TNF-α), vascular endothelial growth factor (VEGF), histamine and tryptase (n=6, P

Although mosquito genome projects uncovered orthologues of many known developmental regulatory genes, extremely little is known about the development of vector mosquitoes. Here, we investigate the role of the Netrin receptor frazzled (fra) during embryonic nerve cord development of two vector mosquito species. Fra expression is detected in neurons just prior to and during axonogenesis in the embryonic ventral nerve cord of Aedes aegypti (dengue vector) and Anopheles gambiae (malaria vector). Analysis of fra function was investigated through siRNA-mediated knockdown in Ae. aegypti embryos. Confirmation of fra knockdown, which was maintained throughout embryogenesis, indicated that microinjection of siRNA is an effective method for studying gene function in Ae. aegypti embryos. Loss of fra during Ae. aegypti development results in thin and missing commissural axons. These defects are qualitatively similar to those observed in Dr. melanogaster fra null mutants. However, the Aa. aegypti knockdown phenotype is stronger and bears resemblance to the Drosophila commissureless mutant phenotype. The results of this investigation, the first targeted knockdown of a gene during vector mosquito embryogenesis, suggest that although Fra plays a critical role during development of the Ae. aegypti ventral nerve cord, mechanisms regulating embryonic commissural axon guidance have evolved in distantly related insects.

INTRODUCTIONBlood-feeding mosquitoes, including the dengue and yellow fever vector Aedes aegypti, transmit many of the world’s deadliest diseases. Such diseases have resurged in developing countries and pose clear threats for epidemic outbreaks in developed countries. Recent mosquito genome projects have stimulated interest in the potential for arthropod-borne disease control by genetic manipulation of vector insects, and genes that regulate development are of particular interest. This protocol describes methods for culturing Ae. aegypti and includes a procedure for egg collection that can be used in conjunction with fixation, immunohistochemistry, and in situ protocols.

INTRODUCTIONBlood-feeding mosquitoes, including the dengue and yellow fever vector Aedes aegypti, transmit many of the world’s deadliest diseases. Such diseases have resurged in developing countries and pose clear threats for epidemic outbreaks in developed countries. Recent mosquito genome projects have stimulated interest in the potential for arthropod-borne disease control by genetic manipulation of vector insects, and genes that regulate development are of particular interest. In recent years, RNA interference (RNAi) has proven to be an effective strategy for inhibiting gene function in many organisms. This protocol describes a method for knockdown of embryonic genes in Ae. aegypti embryos by microinjection of small interfering RNA (siRNA) designed to target a specific gene of interest. The procedure includes a strategy for siRNA design, microinjection, and measurement of knockdown effectiveness.

Blood-feeding mosquitoes, including the dengue and yellow fever vector Aedes aegypti, transmit many of the world's deadliest diseases. Such diseases have resurged in developing countries and pose clear threats for epidemic outbreaks in developed countries. Recent mosquito genome projects have stimulated interest in the potential for arthropod-borne disease control by genetic manipulation of vector insects, and gene products that regulate development are of particular interest. This protocol for immunohistochemical analysis of protein expression can be used to analyze expression of developmental proteins of interest in Ae. aegypti embryos, larvae, and pupae, which will be critical for the development of markers for particular developing tissues.

Blood-feeding mosquitoes, including the dengue and yellow fever vector Aedes aegypti, transmit many of the world's deadliest diseases. Such diseases have resurged in developing countries and pose clear threats for epidemic outbreaks in developed countries. Recent mosquito genome projects have stimulated interest in the potential for arthropod-borne disease control by genetic manipulation of vector insects. Targets of particular interest include genes that regulate development. However, although the Ae. aegypti genome project uncovered homologs of many known developmental regulatory genes, little is known of the genetic regulation of development in Ae. aegypti or other vector mosquitoes. This article provides an overview of the background, husbandry, and potential uses of Ae. aegypti as a model species. Methods for culturing, collecting and fixing developing tissues, analyzing gene and protein expression, and knocking down genes are permitting detailed analyses of the functions of developmental regulatory genes and the selective inhibition of such genes during Ae. aegypti development. This methodology, much of which is applicable to other mosquito species, is useful to both the comparative development and vector research communities.

Blood-feeding mosquitoes, including the dengue and yellow fever vector Aedes aegypti, transmit many of the world's deadliest diseases. Such diseases have resurged in developing countries and pose clear threats for epidemic outbreaks in developed countries. Recent mosquito genome projects have stimulated interest in the potential for arthropod-borne disease control by genetic manipulation of vector insects, and genes that regulate development are of particular interest. This protocol describes a method for fixation and dissection of Ae. aegypti embryos, larvae, and pupae. Tissue processed in this manner can be used subsequently for in situ hybridization detection of mRNA or immunohistochemical analysis of protein expression.

BackgroundDespite the devastating global impact of mosquito-borne illnesses on human health, very little is known about mosquito developmental biology. In this investigation, functional genetic analysis of embryonic salivary gland development was performed inAedes aegypti, the dengue and yellow fever vector and an emerging model for vector mosquito development. Although embryonic salivary gland development has been well studied inDrosophila melanogaster, little is known about this process in mosquitoes or other arthropods.ResultsMosquitoes possess orthologs of many genes that regulateDrosophila melanogasterembryonic salivary gland development. The expression patterns of a large subset of these genes were assessed duringAe. aegyptidevelopment. These studies identified a set of molecular genetic markers for the developing mosquito salivary gland. Analysis of marker expression allowed for tracking of the progression ofAe. aegyptisalivary gland development in embryos. InDrosophila,the salivary glands develop from placodes located in the ventral neuroectoderm. However, inAe. aegypti,salivary marker genes are not expressed in placode-like patterns in the ventral neuroectoderm. Instead, marker gene expression is detected in salivary gland rudiments adjacent to the proventriculus. These observations highlighted the need for functional genetic characterization of mosquito salivary gland development. An siRNA- mediated knockdown strategy was therefore employed to investigate the role of one of the marker genes,cyclic-AMP response element binding protein A(Aae crebA),duringAe. aegyptisalivary gland development.These experiments revealed thatAae crebAencodes a key transcriptional regulator of the secretory pathway in the developingAe. aegyptisalivary gland.ConclusionsThe results of this investigation indicated that the initiation of salivary gland development inAe. aegyptisignificantly differs from that ofD. melanogaster. Despite these differences, some elements of salivary gland development, including the ability of CrebA to regulate secretory gene expression, are conserved between the two species. These studies underscore the need for further analysis of mosquito developmental genetics and may foster comparative studies of salivary gland development in additional insect species.

INTRODUCTIONBlood-feeding mosquitoes, including the dengue and yellow fever vector Aedes aegypti, transmit many of the world’s deadliest diseases. Such diseases have resurged in developing countries and pose clear threats for epidemic outbreaks in developed countries. Recent mosquito genome projects have stimulated interest in the potential for arthropod-borne disease control by genetic manipulation of vector insects, and genes that regulate development are of particular interest. This protocol describes a method for fixation and dissection of Ae. aegypti embryos, larvae, and pupae. Tissue processed in this manner can be used subsequently for in situ hybridization detection of mRNA or immunohistochemical analysis of protein expression.

INTRODUCTIONBlood-feeding mosquitoes, including the dengue and yellow fever vector Aedes aegypti, transmit many of the world’s deadliest diseases. Such diseases have resurged in developing countries and pose clear threats for epidemic outbreaks in developed countries. Recent mosquito genome projects have stimulated interest in the potential for arthropod-borne disease control by genetic manipulation of vector insects. Targets of particular interest include genes that regulate development. However, although the Ae. aegypti genome project uncovered homologs of many known developmental regulatory genes, little is known of the genetic regulation of development in Ae. aegypti or other vector mosquitoes. This article provides an overview of the background, husbandry, and potential uses of Ae. aegypti as a model species. Methods for culturing, collecting and fixing developing tissues, analyzing gene and protein expression, and knocking down genes are permitting detailed analyses of the functions of developmental regulatory genes and the selective inhibition of such genes during Ae. aegypti development. This methodology, much of which is applicable to other mosquito species, is useful to both the comparative development and vector research communities.

INTRODUCTIONBlood-feeding mosquitoes, including the dengue and yellow fever vector Aedes aegypti, transmit many of the world’s deadliest diseases. Such diseases have resurged in developing countries and pose clear threats for epidemic outbreaks in developed countries. Recent mosquito genome projects have stimulated interest in the potential for arthropod-borne disease control by genetic manipulation of vector insects, and genes that regulate development are of particular interest. This protocol for whole-mount in situ hybridization can be used to analyze gene expression in Ae. aegypti embryos and larvae, a critical aspect of understanding developmental gene function in this vector mosquito.

INTRODUCTIONBlood-feeding mosquitoes, including the dengue and yellow fever vector Aedes aegypti, transmit many of the world’s deadliest diseases. Such diseases have resurged in developing countries and pose clear threats for epidemic outbreaks in developed countries. Recent mosquito genome projects have stimulated interest in the potential for arthropod-borne disease control by genetic manipulation of vector insects, and gene products s that regulate development are of particular interest. This protocol for immunohistochemical analysis of protein expression can be used to analyze expression of developmental proteins of interest in Ae. aegypti embryos, larvae, and pupae, which will be critical for the development of markers for particular developing tissues.

Mast cells are important effector cells in IgE- and Th2-mediated immune responses. Mast cells are located close to substance P (SP) containing nerves. SP can activate connective tissue-type mast cells, but not mucosal mast cells. Here we examined the effect of SP on (a) mediator release from human umbilical cord blood-derived cultured mast cells (hCBMCs) grown in the presence of stem cell factor (SCF), IL-6 plus IL-4; (b) IL-2 from activated T cells alone or together with mast cells. SP dose-dependently released histamine, tryptase and IL-8 (at 10 μM, release was 72.8 ± 7.42%, 5390 ± 954 ng/106 cells and 1140 ± 103 pg/106cells, respectively) compared to controls (6.3 ± 2.4 %, 397 ± 235 ng/106 cells and 583 ± 40 pg/106 cells, respectively). Addition of mast cells to T cells, activated by anti-CD3/anti-CD28, for 24 h stimulated IL-2 production from 38.4 ± 9.2 pg/ml to 1450 ± 63 pg/ml (p90%) both SP-induced hCBMCs activation and T cell IL-2 release. Mast cells can superactivate T cells and could constitute a new therapeutic target for diseases such as multiple sclerosis. Luteolin could be used to inhibit this interaction. This work was supported by Theta Biomedical Consulting and Development Co., Inc. (Brookline, MA).

Although vector mosquitoes transmit deadly diseases, little is known about their development. Analysis of mosquito developmental genetics will advance the study of insect evolutionary development and may reveal novel vector control strategies. We recently described methods for functional analysis of embryonic genes in Aedes aegypti, the dengue fever vector and an emerging arthropod model. Although it was anticipated that Drosophila developmental genes and their functions would be generally well conserved in mosquitoes, our analyses are revealing striking differences. For example, knockdown of axon guidance genes in A. aegypti embryos indicates that nerve cord development has diverged in insects. We are now examining development of the A. aegypti olfactory system and salivary gland, two tissues of vector importance. Large-scale comparative analysis of developmental genes in D. melanogaster and three vector mosquito genomes suggests that genetic regulation of the development of these and other tissues has diverged. Lineage specific duplication, expansion, and loss of genes critical for Drosophila development were observed. Sequence analyses suggest that some processes, including salivary gland development, may be under more stringent selection forces than others. Comparative analysis of predicted miRNA binding sites suggests that the repertoire of developmental genes targeted by miRNAs is species-specific. Our comparative analyses are providing insight into the evolution of insect developmental genes and processes and will promote the design of additional functional genetic studies in mosquitoes.