Transcriptome Analysis of Aedes aegypti Transgenic Mosquitoes with Altered Immunity

The mosquito immune system is involved in pathogen-elicited defense responses. The NF-κB factors REL1 and REL2 are downstream transcription activators of Toll and IMD immune pathways, respectively. We have used genome-wide microarray analyses to characterize fat-body-specific gene transcript repertoires activated by either REL1 or REL2 in two transgenic strains of the mosquito Aedes aegypti. Vitellogenin gene promoter was used in each transgenic strain to ectopically express either REL1 (REL1+) or REL2 (REL2+) in a sex, tissue, and stage specific manner. There was a significant change in the transcript abundance of 297 (79 up- and 218 down-regulated) and 299 (123 up- and 176 down-regulated) genes in fat bodies of REL1+ and REL2+, respectively. Over half of the induced genes had predicted functions in immunity, and a large group of these was co-regulated by REL1 and REL2. By generating a hybrid transgenic strain, which ectopically expresses both REL1 and REL2, we have shown a synergistic action of these NF-κB factors in activating immune genes. The REL1+ immune transcriptome showed a significant overlap with that of cactus (RNAi)-depleted mosquitoes (50%). In contrast, the REL2+ -regulated transcriptome differed from the relatively small group of gene transcripts regulated by RNAi depletion of a putative inhibitor of the IMD pathway, caspar (35 up- and 140 down-regulated), suggesting that caspar contributes to regulation of a subset of IMD-pathway controlled genes. Infections of the wild type Ae. aegypti with Plasmodium gallinaceum elicited the transcription of a distinct subset of immune genes (76 up- and 25 down-regulated) relative to that observed in REL1+ and REL2+ mosquitoes. Considerable overlap was observed between the fat body transcriptome of Plasmodium-infected mosquitoes and that of mosquitoes with transiently depleted PIAS, an inhibitor of the JAK-STAT pathway. PIAS gene silencing reduced Plasmodium proliferation in Ae. aegypti, indicating the involvement of the JAK-STAT pathway in anti-Plasmodium defense in this infection model

Identical Functional Organization of Nonpolytene and Polytene Chromosomes in Drosophila melanogaster

Salivary gland polytene chromosomes demonstrate banding pattern, genetic meaning of which is an enigma for decades. Till now it is not known how to mark the band/interband borders on physical map of DNA and structures of polytene chromosomes are not characterized in molecular and genetic terms. It is not known either similar banding pattern exists in chromosomes of regular diploid mitotically dividing nonpolytene cells. Using the newly developed approach permitting to identify the interband material and localization data of interband-specific proteins from modENCODE and other genome-wide projects, we identify physical limits of bands and interbands in small cytological region 9F13-10B3 of the X chromosome in D. melanogaster, as well as characterize their general molecular features. Our results suggests that the polytene and interphase cell line chromosomes have practically the same patterns of bands and interbands reflecting, probably, the basic principle of interphase chromosome organization. Two types of bands have been described in chromosomes, early and late-replicating, which differ in many aspects of their protein and genetic content. As appeared, origin recognition complexes are located almost totally in the interbands of chromosomes

MicroRNA-277 targets insulin-like peptides 7 and 8 to control lipid metabolism and reproduction in Aedes aegypti mosquitoes

Hematophagous female mosquitoes transmit numerous devastating human diseases, including malaria, dengue fever, Zika virus, and others. Because of their obligatory requirement of a vertebrate blood meal for reproduction, these mosquitoes need a lot of energy; therefore, understanding the molecular mechanisms linking metabolism and reproduction is of particular importance. Lipids are the major energy store providing the fuel required for host seeking and reproduction. They are essential components of the fat body, a metabolic tissue that is the insect analog of vertebrate liver and adipose tissue. In this study, we found that microRNA-277 (miR-277) plays an important role in regulating mosquito lipid metabolism. The genetic disruption of miR-277 using the CRISPR-Cas9 system led to failures in both lipid storage and ovary development. miR-277 mimic injection partially rescued these phenotypic manifestations. Examination of subcellular localization of FOXO protein via CRISPR-assisted, single-stranded oligodeoxynucleotide-mediated homology-directed repair revealed that insulin signaling is up-regulated in response to miR-277 depletion. In silico target prediction identified that insulin-like peptides 7 and 8 (ilp7 and ilp8) are putative targets of miR-277; RNA immunoprecipitation and a luciferase reporter assay confirmed that ilp7 and ilp8 are direct targets of this miRNA. CRISPR-Cas9 depletion of ilp7 and ilp8 led to metabolic and reproductive defects. These depletions identified differential actions of ILP7 and ILP8 in lipid homeostasis and ovarian development. Thus, miR-277 plays a critical role in mosquito lipid metabolism and reproduction by targeting ilp7 and ilp8, and serves as a monitor to control ILP7 and ILP8 mRNA levels

Regulation of the gut-specific carboxypeptidase: A study using the binary Gal4/UAS system in the mosquito Aedes aegypti

Pathogen transmission by mosquitoes is tightly linked to blood feeding which, in turn, is required for egg development. Studies of these processes would greatly benefit from genetic methods, such as the binary Gal4/UAS system. The latter has been well established for model organisms, but its availability is limited for mosquitoes. The objective of this study was to develop the blood-meal-activated, gut-specific Gal4/UAS system for the yellow-fever mosquito Aedes aegypti and utilize it to investigate the regulation of gut-specific gene expression. A 1.1-kb, 5(') upstream region of the carboxypeptidase A (CP) gene was used to genetically engineer the CP-Gal4 driver mosquito line. The CP-Gal4 specifically activated the Enhanced Green Fluorescent Protein (EGFP) reporter only after blood feeding in the gut of the CP-Gal4 > UAS-EGFP female Ae. aegypti. We used this system to study the regulation of CP gene expression. In vitro treatments with either amino acids (AAs) or insulin stimulated expression of the CP-Gal4 > UAS-EGFP transgene; no effect was observed with 20-hydroxyecdysone (20E) treatments. The transgene activation by AAs and insulin was blocked by rapamycin, the inhibitor of the Target-of-Rapamycin (TOR) kinase. RNA interference (RNAi) silence of the insulin receptor (IR) reduced the expression of the CP-Gal4 > UAS-EGFP transgene. Thus, in vitro and in vivo experiments have revealed that insulin and TOR pathways control expression of the digestive enzyme CP. In contrast, 20E, the major regulator of post-blood-meal vitellogenic events in female mosquitoes, has no role in regulating the expression of this gene. This novel CP-Gal4/UAS system permits functional testing of midgut-specific genes that are involved in blood digestion and interaction with pathogens in Ae. aegypti mosquitoes

MicroRNA-8 targets the Wingless signaling pathway in the female mosquito fat body to regulate reproductive processes

Female mosquitoes require a blood meal for reproduction, and this blood meal provides the underlying mechanism for the spread of many important vector-borne diseases in humans. A deeper understanding of the molecular mechanisms linked to mosquito blood meal processes and reproductive events is of particular importance for devising innovative vector control strategies. We found that the conserved microRNA miR-8 is an essential regulator of mosquito reproductive events. Two strategies to inhibit miR-8 function in vivo were used for functional characterization: systemic antagomir depletion and spatiotemporal inhibition using the miRNA sponge transgenic method in combination with the yeast transcriptional activator gal4 protein/upstream activating sequence system. Depletion of miR-8 in the female mosquito results in defects related to egg development and deposition. We used a multialgorithm approach for miRNA target prediction in mosquito 3' UTRs and experimentally verified secreted wingless-interacting molecule (swim) as an authentic target of miR-8. Our findings demonstrate that miR-8 controls the activity of the long-range Wingless (Wg) signaling by regulating Swim expression in the female fat body. We discovered that the miR-8/Wg axis is critical for the proper secretion of lipophorin and vitellogenin by the fat body and subsequent accumulation of these yolk protein precursors by developing oocytes

microRNA-309 targets the Homeobox gene SIX4 and controls ovarian development in the mosquito Aedes aegypti

Obligatory blood-triggered reproductive strategy is an evolutionary adaptation of mosquitoes for rapid egg development. It contributes to the vectorial capacity of these insects. Therefore, understanding the molecular mechanisms underlying reproductive processes is of particular importance. Here, we report that microRNA-309 (miR-309) plays a critical role in mosquito reproduction. A spatiotemporal expression profile of miR-309 displayed its blood feeding-dependent onset and ovary-specific manifestation in female Aedes aegypti mosquitoes. Antagomir silencing of miR-309 impaired ovarian development and resulted in nonsynchronized follicle growth. Furthermore, the genetic disruption of miR-309 by CRISPR/Cas9 system led to the developmental failure of primary follicle formation. Examination of genomic responses to miR-309 depletion revealed that several pathways associated with ovarian development are down-regulated. Comparative analysis of genes obtained from the high-throughput RNA sequencing of ovarian tissue from the miR-309 antagomir-silenced mosquitoes with those from the in silico computation target prediction identified that the gene-encoding SIX homeobox 4 protein (SIX4) is a putative target of miR-309. Reporter assay and RNA immunoprecipitation confirmed that SIX4 is a direct target of miR-309. RNA interference of SIX4 was able to rescue phenotypic manifestations caused by miR-309 depletion. Thus, miR-309 plays a critical role in mosquito reproduction by targeting SIX4 in the ovary and serves as a regulatory switch permitting a stage-specific degradation of the ovarian SIX4 mRNA. In turn, this microRNA (miRNA)-targeted degradation is required for appropriate initiation of a blood feeding-triggered phase of ovarian development, highlighting involvement of this miRNA in mosquito reproduction

MicroRNA-8 targets the Wingless signaling pathway in the female mosquito fat body to regulate reproductive processes

Female mosquitoes require a blood meal for reproduction, and this blood meal provides the underlying mechanism for the spread of many important vector-borne diseases in humans. A deeper understanding of the molecular mechanisms linked to mosquito blood meal processes and reproductive events is of particular importance for devising innovative vector control strategies. We found that the conserved microRNA miR-8 is an essential regulator of mosquito reproductive events. Two strategies to inhibit miR-8 function in vivo were used for functional characterization: systemic antagomir depletion and spatiotemporal inhibition using the miRNA sponge transgenic method in combination with the yeast transcriptional activator gal4 protein/upstream activating sequence system. Depletion of miR-8 in the female mosquito results in defects related to egg development and deposition. We used a multialgorithm approach for miRNA target prediction in mosquito 3′ UTRs and experimentally verified secreted wingless-interacting molecule (swim) as an authentic target of miR-8. Our findings demonstrate that miR-8 controls the activity of the long-range Wingless (Wg) signaling by regulating Swim expression in the female fat body. We discovered that the miR-8/Wg axis is critical for the proper secretion of lipophorin and vitellogenin by the fat body and subsequent accumulation of these yolk protein precursors by developing oocytes