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

Abstract

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 antagomirsilenced 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.Homeobox protein gene | microRNA | CRISPR/Cas9 | ovary | fast evolution T ransmission of mosquito-borne diseases brings enormous human suffering, with more than 1 million deaths worldwide annually. The yellow fever mosquito Aedes aegypti has reemerged as one of the most dangerous vectors of human diseases, transmitting Dengue fever, Yellow fever, Chikungunya, and Zika virus (1-4). Because of the lack of effective vaccines and increasing drug resistance in pathogens, biological control is considered one of the most promising strategies for preventing disease transmission. In particular, female hematophagous mosquitoes rely on acquisition of blood to initiate a series of physiological events promoting egg development (5, 6). Therefore, understanding the molecular mechanisms underlying ovarian activation is of great significance for the development of effective approaches to control mosquito-borne diseases (7,8).MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression at the posttranscriptional level through translational repression or mRNA decay (9). Hence, miRNAs play significant roles in governing multiple functions in animals and plants via integrating sophisticated miRNA-mRNA regulatory networks (10, 11...

Construction of an efficient genomic editing system with CRISPR/Cas9 in the vector mosquito Aedes albopictus

“…Previous studies have demonstrated the effectiveness of genome editing with the CRISPR/Cas9 system in other vector mosquitoes such as Anopheles stephensi (Gantz et al ., ), Anopheles gambiae (Hammond et al ., ), Ae. agypti (Basu et al ., ; Hall et al ., ; Zhang et al ., ; Li et al ., ) and Culex quinquefasciatus (Itokawa et al ., ). These studies demonstrated that the rate of mutagenesis remarkably varies across mosquito species, target DNA sites, and even sgRNAs targeting the same genomic targets.…”

Germline Cas9 Expression Yields Highly Efficient Genome Engineering in a Major Worldwide Disease Vector,Aedes aegypti

“…Previous studies have demonstrated effective CRISPR/Cas9 genome editing in the mosquito Ae. aegypti (30)(31)(32)(33)(34), however these studies utilized a non-transgenic source of Cas9 limiting both survivorship and editing efficiencies. To overcome these previous limitations, and to reduce the complexity of injecting multiple components (i.e.…”

“…To overcome the significant limitations posed by previous genome editing tools, the clustered regularly interspaced short palindromic repeats/CRISPR-associated sequence 9 (CRISPR/Cas9) system, originally discovered in bacteria and archaea (14)(15)(16)(17)(18)(19)(20), has been adapted as a programmable (20,21) precision genome editing tool in a diversity of organisms (22)(23)(24)(25)(26)(27)(28)(29), including mosquitoes (30)(31)(32)(33)(34). Briefly, the CRISPR/Cas9 system is guided by a chimeric programmable synthetic short guide RNA (sgRNA) (20) that binds Cas9 directing it to a user specified genomic DNA target sequence, via Watson-Crick base pairing between the sgRNA and the target DNA sequence, thereby generating site-specific double-strand (ds) dsDNA breaks.…”

“…Importantly, CRISPR-mediated genome engineering of organisms has been achieved in a variety of different ways. For example, direct injection of in vitro purified sgRNAs combined with either purified Cas9 RNA, recombinant Cas9 protein, or even with Cas9 expression plasmids, have all been successful for a variety of organisms (29,32,(35)(36)(37)(38)(39)(40), including mosquitoes (30)(31)(32)(33)(34). However, the rate of mutagenesis and lethality have varied widely both within and among these different studies, and such discrepancies are likely due to either the methods used to introduce the editing components, the variability in sgRNA functionality against target sites, or even unavoidable variability from manual injection of the components (41).…”

Disruption of sex‐specific doublesex exons results in male‐ and female‐specific defects in the black cutworm, Agrotis ipsilon

“…24,26,27 However, it is not easy to use these methods in other lepidopterans. A recently developed genome-editing method, the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system, [28][29][30] has been successfully used as a new method for investigating gene function in various Diptera, [31][32][33][34][35] Amphipoda, 36 Diplostraca, 37 Decapoda, 38 Coleoptera, 39 Lepidoptera 27,[40][41][42][43][44][45][46] and Orthoptera. 47,48 Successful applications of CRISPR/Cas9 in insect pest species could provide a promising alternative strategy for genetic manipulation of agricultural insect pests.…”