Identification of microRNAs expressed in two mosquito vectors, Aedes albopictus and Culex quinquefasciatus

Abstract

Background: MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression in a variety of organisms, including insects, vertebrates, and plants. miRNAs play important roles in cell development and differentiation as well as in the cellular response to stress and infection. To date, there are limited reports of miRNA identification in mosquitoes, insects that act as essential vectors for the transmission of many human pathogens, including flaviviruses. West Nile virus (WNV) and dengue virus, members of the Flaviviridae family, are primarily transmitted by Aedes and Culex mosquitoes. Using high-throughput deep sequencing, we examined the miRNA repertoire in Ae. albopictus cells and Cx. quinquefasciatus mosquitoes.

Results: We identified a total of 65 miRNAs in the Ae. albopictus C7/10 cell line and 77 miRNAs in Cx. quinquefasciatus mosquitoes, the majority of which are conserved in other insects such as Drosophila melanogaster and Anopheles gambiae. The most highly expressed miRNA in both mosquito species was miR-184, a miRNA conserved from insects to vertebrates. Several previously reported Anopheles miRNAs, including miR-1890 and miR-1891, were also found in Culex and Aedes, and appear to be restricted to mosquitoes. We identified seven novel miRNAs, arising from nine different precursors, in C7/10 cells and Cx. quinquefasciatus mosquitoes, two of which have predicted orthologs in An. gambiae. Several of these novel miRNAs reside within a ~350 nt long cluster present in both Aedes and Culex. miRNA expression was confirmed by primer extension analysis. To determine whether flavivirus infection affects miRNA expression, we infected female Culex mosquitoes with WNV. Two miRNAs, miR-92 and miR-989, showed significant changes in expression levels following WNV infection.

Conclusions: Aedes and Culex mosquitoes are important flavivirus vectors. Recent advances in both mosquito genomics and high-throughput sequencing technologies enabled us to interrogate the miRNA profile in these two species. Here, we provide evidence for over 60 conserved and seven novel mosquito miRNAs, expanding upon our current understanding of insect miRNAs. Undoubtedly, some of the miRNAs identified will have roles not only in mosquito development, but also in mediating viral infection in the mosquito host.

Figure 1

Deep sequencing of small RNA populations in Ae. albopictus C7/10 cells and Cx. quinquefasciatus mosquitoes. Size distributions of small RNA libraries (18-28 nt) from A) C7/10 cells (Aedes) and B) Cx. quinquefasciatus (Culex). C) Frequencies of read counts for individual, conserved miRNAs present in C7/10 cells and Culex mosquitoes. Expression levels, based on read counts, of individual miRNAs are separated into several ranges and span over 5 orders of magnitude. D) Breakdown of the total number of reads obtained for each library. The number of reads mapping to miRNA and miRNA* strands is reported.

Figure 2

Culex miRNA sequence variations. A) The total number of canonical miRNA sequence reads, annotated in Table 2, is set to "0" on the x-axis. Differences in the total numbers of canonical 3' versus 5' miRNA ends are due to greater diversity at the 3' end of a given miRNA. Negative numbers on the x-axis indicate 3' or 5' nucleotide truncations in a miRNA sequence, while positive numbers indicate 3' or 5' nucleotide additions. 58 Culex miRNAs (sequenced at least 10 times) are collectively represented. The numbers of reads with 3' or 5' nucleotide truncations or additions for individual miRNAs, B) miR-1, C) miR-210, and D) miR-252 are shown.

Figure 3

Primer extension analysis confirms miRNA expression. Total RNA was isolated from A) C7/10 cells or C7/10-WNV312 cells persistently infected with WNV replicons and B, C) Cx. quinquefasciatus mosquitoes fed a non-infectious blood meal (Culex) or a blood meal containing WNV-NY99 (Culex-WNV). 10 μg (A, C) or 4 μg (B) of RNA was used for primer extension to detect miRNAs. For each miRNA, free probe with no RNA is shown as a negative control. Ethidum bromide stained rRNA is shown as loading control.

Figure 4

Identification of novel mosquito miRNAs. Primer extension analysis was used to confirm the expression of A) three novel Ae. albopictus miRNAs and C) one novel Cx. quinquefasciatus miRNAs. Total RNA was isolated from C7/10 cells or Cx. quinquefasciatus mosquitoes as described in Figure 3 and Methods. B) and D) Predicted pre-miRNA stem-loop structures for each novel miRNA. Ae. albopictus miRNAs were mapped to the Ae. aegypti genome, and therefore may not reflect the actual pre-miRNA structures. Mature miRNA sequences are shown in red, while corresponding miRNA* sequences identified in each library are shown in blue. For miR-2951, asterisks indicate additional 5' nucleotides present in a lower percentage of the reads mapping to each miRNA compared to the canonical sequence annotated in Tables 1 and 2. Ae. albopictus miR-2951 differs by one nucleotide from the Ae. aegypti genome. E) Pre-miRNA structures for two novel orthologous miRNAs mapping to the A. gambiae genome. The predicted mature miRNAs, based on sequence conservation, are shown in red.

Figure 5

Novel mosquito miRNAs are clustered. A) Predicted pre-miRNA stem-loop structures for cqu-miR-2941-1, cqu-miR-2941-2, and cqu-miR-2952. B) Primer extension analysis confirms miR-2941 expression in total RNA isolated from Cx. quinquefasciatus adult female blood-fed mosquitoes (see Methods). C) Predicted pre-miRNA stem-loop structures for aae-miR-2941-1, and aae-miR-2941-2. As in Figure 4, mature miRNAs are shown in red, and miRNA* strands, when identified in a library, are shown in blue. D) Genomic location of the miR-2941 clusters in the Cx. quinquefasciatus and Ae. aegypti genomes. Numbers on the bottom indicate the nucleotide distance between pri-miRNA stem-loops for each miRNA.