Dynamic expression of miRNAs across immature and adult stages of the malaria mosquito Anopheles stephensi (original) (raw)
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Background: Over the past several years, thousands of microRNAs (miRNAs) have been identified in the genomes of various insects through cloning and sequencing or even by computational prediction. However, the number of miRNAs identified in anopheline species is low and little is known about their role. The mosquito Anopheles funestus is one of the dominant malaria vectors in Africa, which infects and kills millions of people every year. Therefore, small RNA molecules isolated from the four life stages (eggs, larvae, pupae and unfed adult females) of An. funestus were sequenced using next generation sequencing technology. Results: High throughput sequencing of four replicates in combination with computational analysis identified 107 mature miRNA sequences expressed in the An. funestus mosquito. These include 20 novel miRNAs without sequence identity in any organism and eight miRNAs not previously reported in the Anopheles genus but are known in non-anopheles mosquitoes. Finally, the changes in the expression of miRNAs during the mosquito development were determined and the analysis showed that many miRNAs have stage-specific expression, and are co-transcribed and co-regulated during development. Conclusions: This study presents the first direct experimental evidence of miRNAs in An. funestus and the first profiling study of miRNA associated with the maturation in this mosquito. Overall, the results indicate that miRNAs play important roles during the growth and development. Silencing such molecules in a specific life stage could decrease the vector population and therefore interrupt malaria transmission.
Scientific Reports, 2017
Drastic changes in gene expression occur after adult female mosquitoes take a blood meal and use the nutrients for egg maturation. A growing body of evidence indicates that microRNAs (miRNAs) contribute to this tightly controlled tissue-and stage-specific gene expression. To investigate the role of miRNAs, we monitored miRNA expression in the mosquito Anopheles gambiae during the 72-h period immediately after blood feeding. We also measured the association of miRNAs with Argonaute 1 (Ago1) and Argonaute 2 (Ago2) to assess the functional status of individual miRNA species. Overall, 173 mature miRNAs were precipitated with Ago1 and Ago2, including 12 new miRNAs, the orthologs of which are found thus far only in other Anopheles species. Ago1 is the predominant carrier of miRNAs in Anopheles gambiae. The abundance and Ago loading of most of the mature miRNAs were relatively stable after blood ingestion. However, miRNAs of the miR-309/286/2944 cluster were considerably upregulated after blood feeding. Injection of the specific antagomir for miR-309 resulted in smaller developing oocytes and ultimately fewer eggs. In addition, the Ago association of some miRNAs was not proportional to their cellular abundance, suggesting that integration of miRNAs into the Ago complexes is regulated by additional mechanisms. The Anopheles gambiae mosquito is the major vector of human malaria in Africa. Malaria is caused by protozoan parasites belonging to the genus Plasmodium and is spread between hosts by the bites of parasite-infected female mosquitoes. Female adults of An. gambiae must obtain blood from vertebrates to acquire nutrients for egg production. The female mosquitoes digest a protein-rich blood meal, greater than their original body weight, within 48 h after ingestion and use the nutrients to synthesize egg yolk proteins. Substantial physiological changes occur after blood ingestion in mosquitoes, and over 50% of all genes show significant variation in transcript accumulation 1. Recent studies suggest that blood digestion and egg production in An. gambiae and other mosquito species are also regulated by mosquito microRNAs (miRNAs) 2. Expression of some miRNAs is considerably altered after blood ingestion in An. gambiae 3 , An. stephensi 4 , Aedes aegypti 5 , and Aedes albopictus 6. Functional studies in Ae. aegypti have shown that perturbation of individual miRNAs in female mosquitoes leads to defects in blood digestion and egg production 7-10. miRNAs are small endogenous non-coding RNAs that play important roles in post-transcriptional gene regulation in plants and animals 11. Most miRNAs are transcribed by RNA polymerase II as primary-miRNA (pri-miRNA) transcripts. The canonical miRNA pathway converts the pri-miRNA hairpins into ∼22-nucleotide (nt) mature duplex miRNAs via consecutive cleavages by two RNase III enzymes, Drosha and Dicer, with the help of some double-stranded RNA-binding proteins 12. The two strands of the mature miRNA duplex are denoted by the suffix-5p or-3p, depending on whether they originate from the 5′ or 3′ end of the pri-miRNA. Only one strand of the miRNA duplex is usually loaded onto an Argonaute (Ago) protein as part of the miRNA-induced silencing complex (miRISC). The strand in miRISC is known as the guide strand; the other strand (the passenger strand) is excluded and subsequently degraded 12. The guide strand directs the miRISC to messenger RNA
Proceedings of the National Academy of Sciences of the United States of America, 2015
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...
For many arthropods, including insects, diapause is the primary mechanism for survival during unfavorable seasons. Although the exogenous signals and endogenous hormones that induce and regulate diapause are well-characterized, we still lack a mechanistic understanding of how environmental information is translated into molecular regulators of the diapause pathway. However, short, non-coding microRNAs (miRNAs) are likely involved in generating both the arrested egg follicle development and fat hypertrophy in diapausing females of the Northern house mosquito, Culex pipiens. To determine whether miRNAs might respond to changes in day length and/or regulate diapause pathways, we measured the abundance of candidate miRNAs in diapausing and nondiapausing females of Cx. pipiens across the adult lifespan. Of the selected miRNAs nearly all were more abundant in nondiapausing females relative to diapausing females, but at different times. Specifically, miR-13b-3p, miR-14-3p, miR-277-3p, and ...
Blood feeding and Plasmodium infection alters the miRNome of Anopheles stephensi
PloS one, 2014
Blood feeding is an integral process required for physiological functions and propagation of the malaria vector Anopheles. During blood feeding, presence of the malaria parasite, Plasmodium in the blood induces several host effector molecules including microRNAs which play important roles in the development and maturation of the parasite within the mosquito. The present study was undertaken to elucidate the dynamic expression of miRNAs during gonotrophic cycle and parasite development in Anopheles stephensi. Using next generation sequencing technology, we identified 126 miRNAs of which 17 were novel miRNAs. The miRNAs were further validated by northern hybridization and cloning. Blood feeding and parasitized blood feeding in the mosquitoes revealed regulation of 13 and 16 miRNAs respectively. Expression profiling of these miRNAs revealed that significant miRNAs were down-regulated upon parasitized blood feeding with a repertoire of miRNAs showing stage specific up-regulation. Expression profiles of significantly modulated miRNAs were further validated by real time PCR. Target prediction of regulated miRNAs revealed overlapping targeting by different miRNAs. These targets included several metabolic pathways including metabolic, redox homeostasis and protein processing machinery components. Our analysis revealed tight regulation of specific miRNAs post blood feeding and parasite infection in An. stephensi. Such regulated expression suggests possible role of these miRNAs during gonotrophic cycle in mosquito. Another set of miRNAs were also significantly regulated at 42 h and 5 days post infection indicating parasite stage-specific role of host miRNAs. This study will result in better understanding of the role of miRNAs during gonotrophic cycle and parasite development in mosquito and can probably facilitate in devising novel malaria control strategies at vector level. Citation: Jain S, Rana V, Shrinet J, Sharma A, Tridibes A, et al. (2014) Blood Feeding and Plasmodium Infection Alters the miRNome of Anopheles stephensi. PLoS ONE 9(5): e98402.
Proceedings of the National Academy of Sciences of the United States of America, 2016
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 se...
RNA biology, 2015
Females of the hematophagous mosquito species require a vertebrate blood meal to supply amino acids and other nutrients necessary for egg development, serving as the driving force for the spread of many vector-borne diseases in humans. Blood digestion utilizes both early and late phase serine proteases (SPs) that are differentially regulated at the transcriptional and post-transcriptional level. To uncover the regulatory complexity of SPs in the female mosquito midgut, we investigated involvement of miRNAs in regulating the juvenile hormone (JH)-controlled chymotrypsin-like SP, JHA15. We identified regulatory regions complementary to the mosquito-specific miRNA, miR-1890, within the 3' UTR of JHA15 mRNA. The level of the JHA15 transcript is highest post eclosion and drastically declines post blood meal (PBM), exhibiting an opposite trend to miR-1890 that peaks at 24 h PBM. Depletion of miR-1890 results in defects in blood digestion, ovary development and egg deposition. JHA15 mR...
2022
Background: microRNAs (miRNAs) are small endogenous non-coding RNAs that play important roles in many developments, differentiation, apoptosis, and innate immunity in insects. Pyrethroid resistance has become the unique largest obstacle for the mosquito control, and An. sinensis is the main malaria vector mosquito in Eastern and other Asian countries. Methods: We investigated miRNA expression profiles associated with pyrethroid-resistant in An. sinensis by Illumina Hiseq sequencing. Meanwhile, we applied bioinformatics methods to identify miRNAs and their target genes associated with pyrethroid resistance in. miRNAs selected in the bioinformatics approach were confirmed by RT-qPCR. In addition, dual luciferase reporter was also performed to verify the interaction of miRNA and its target genes.Results: We obtained 7,120,612- 7,157,914 clean RNA sequencing reads from the different sample sequencing. A total of 329 miRNAs (included 246 novel ones) were identified by Illumina Hiseq sequ...