Discovery of 98 distinct pre-miRNA sequences in Ae. aegypti
As shown in Table , we have uncovered 98 different pre-miRNAs in Ae. aegypti
which could produce 86 distinct miRNAs. Some of the 98 pre-miRNA sequences produce identical miRNAs and miRNA*s. Also included in Table are 20 distinct miRNA* sequences that were uncovered by small RNA sequencing. Eighty-nine of the Ae. aegypti
miRNA and miRNA* sequences showed a perfect match to small RNA sequences from at least one of the three samples (Table ). There are clear variations of sequence counts among different miRNA species in these samples. However, to gain quantitative insights in the relative abundance of these miRNAs, further investigations are needed using methods such as northern blot, primer extension, and direct sequencing of millions of small RNA reads [11
]. Twenty-nine of the 98 pre-miRNAs do not have small RNA sequences in the embryo and midgut samples. However, these 29 pre-miRNAs all form hairpins and are conserved among Ae. aegypti
, Cx. quinquefasciatus
, and An. gambiae
, the three mosquito species with sequenced genomes.
Sequence, location, and expression of miRNAs in Aedes aegypti.
Possible revisions at the ends of known miRNAs and cases of abundant miRNA* sequences
As shown in Table , there are a few cases where the Ae. aegypti miRNA sequences, as indicated by direct sequencing, start or end with one or a few extra nucleotides compared to the known miRNAs reported from D. melanogaster or anopheline mosquitoes (miRBase). To minimize the contribution of sequencing error, we only consider cases where there are at least six such sequences in the 454 database and these differences are the majority. These miRNAs include aae-miR-2a, aae-miR-210, aae-miR-263b, aae-miR-281, and aae-miR-283. Because internal sequence variations between miRNAs from different species could simply result from species differences, we did not include in the above list the aae-miRNAs that only had internal sequence variations compared to known miRNAs. On the other hand, shifts at the 5' or 3' ends could either suggest a difference between species or imprecise annotation at the miRNA termini. Thus the above 5 aae-miRNAs provide leads to further studies to investigate whether these previously reported miRNA sequences need to be revised.
In vast majority of the cases, mature miRNAs are much more abundant than miRNA*. However, miR-281* and miR-1175* are at least a few dozen fold more abundant than their miRNA sequences. In both cases, the miRNA and the miRNA* sequences are 100% identical among Ae. aegypti, Cx. quinquiefasciatus, and An. gambiae. It is therefore possible that miR-281* and miR-1175* are functional. There are a few other cases in which the miRNA* is more abundant than the miRNA sequences (Table ). However, the numbers of hits are low in these cases and it is difficult to assess how significant the differences may be.
miRNA gene clusters and duplications: evolutionary implications
There are 14 clusters of miRNAs that are defined as more than one miRNA hairpin within 10 kb [29
]. Twelve of these clusters have members that are separated by less than one kb. All these clusters can be identified in Table and Additional file 1
by tracking and sorting the contigs and start and end positions of the pre-miRNAs. Two previously identified clusters are worth noting. The first is the cluster that includes miR-9b, miR-79, and miR-306. We previously identified this cluster in both An. gambiae
and D. melanogaster
and we thought miR-306 was missing in the Ae. aegypti
]. However, small RNA sequencing and closer analysis of the Ae. aegypti
assembly suggest that miR-306 is indeed present in Ae. aegypti
and the relative positions of the three miRNAs in the cluster are conserved among all three species. The aae-miR-306 shows 2 mismatches in the 22 bp overlap compared to aga-miR-306 and dme-miR-306.
The second cluster includes miR-12 and miR-283, which flank either miR-304 in D. melanogaster
or miR-1889 in An. gambiae
]. MiR-1889 has similarity to the reverse-complementary sequence of miR-304 but the difference is significant enough for miRbase to assign a unique name for it. Given that the D. melanogaster
miR-304 and the An. gambiae
miR-1889 are flanked by the same miRNAs and that this miRNA cluster is found in an intron of orthologous genes in the two species, miR-304 and miR-1889 may have a common origin. Through small RNA sequencing and a closer analysis of the genome assembly, we identified miR-1889 in Ae. aegypti
(Table and Additional file 1
), which was previously thought to be missing in the Ae. aegypti
]. The newly identified aae-miR-1889 shows 3 mismatches in the 21 bp overlap with aga-miR-1889. The identification of aae-miR-1889 further supports the strand orientation of the mosquito miR-1889. It is tempting to suggest that one of the two miRNA hairpins, miR-304 or miR-1889, was inverted during evolution. It is also possible that fruit flies and mosquitoes utilize different strands of the hairpin as mature miRNA. Wecurrently do not have evidence to support either of these hypotheses.
There are 17 cases where one of the pre-miRNAs is duplicated and thus more than one pre-miRNA hairpin produces the same or highly similar mature miRNAs. These pre-miRNAs are shown either with a suffix of "-1" and "-2" for hairpins that produce identical miRNAs or with a suffix of "a" or "b" for hairpins that produce highly similar miRNAs (Table ). These miRNAs are a rich source for future comparative analysis to uncover the evolutionary patterns of miRNA duplication and the process of creating novel miRNAs in mosquitoes. It remains to be determined whether the rather common miRNA duplication observed in Ae. aegypti
reflect the importance of duplication for the generation of new miRNAs in mosquitoes. In this regard, it is interesting to note that while duplication is a common mechanism to generate new miRNAs in plants (e.g., [30
]), duplication was thought to be not important in Drosophila
Novel miRNAs that are potentially specific to mosquitoes
Nine of the 98 pre-miRNA hairpins are novel and currently have only been found in mosquitoes. These nine pre-miRNAs produce seven distinct mature miRNAs (miR-M1, -M2, -M3, -M4a and -M4b; miR-N1 and miR-N2). All seven mature miRNAs have multiple hits from small RNA sequencing, confirming their status as miRNAs. A few of these also have hits in the miRNA* strand. Shown in Figures and are the sequence alignments of the novel pre-miRNA sequences discovered in this study and the hairpins they form. Two physically linked pre-miRNA hairpins (miR-M1-1 and miR-M1-2) produce the same miR-M1 in Ae. aegypti. Two physically linked pre-miRNA hairpins produce similar but not identical miR-M4a and miR-M4b. MiR-M1, -M2, -M3, -M4a and -M4b are found in all three available mosquito genome assemblies.
Figure 1 Alignments and stem-loop structures of five novel mosquito pre-miRNAs. See Table 1 for naming and sequence locations of these miRNAs. Left panels are the hairpin structures. Right panels are the sequence alignments between Ae. aegypti miRNAs (aae-miRNAs) (more ...)
Figure 2 Alignments and stem-loop structures of a novel miRNA cluster within the intron of a gene encoding a transcription factor. See Table 1 for naming and sequence locations of these miRNAs. There are two hairpins for the same miR-N1 (-1 and -2) in both Ae. (more ...)
Two physically linked pre-miRNA hairpins (miR-N1-1 and miR-N1-2) produce the same miR-N1 and they are also in close proximity to the miR-N2 hairpin in Ae. aegypti. The three hairpins are in the first intron of a gene in Ae. aegypti (Vectorbase Gene ID AAEL009263) encoding a putative transcription factor with a basic leucine zipper domain. Sequence analysis suggests that miR-N1 is found in the orthologous gene in Cx. quinquefasciatus but not found in An. gambiae. MiR-N2 is only found in Ae. aegypti. MiR-N1 also exists in two hairpins in the intron of the homologous gene in Cx. quinquefasciatusand there is a third hairpin that has a predicted miRNA with a similar 5' sequence as miR-N1. We name this miRNA miR-N3 and it is only found in Cx. quinquefasciatus (Vectorbase Gene ID CPIJ000468). Cqu-miR-N3 is not listed in Table , which only shows miRNAs from Ae. aegypti.
Thus, we have identified eight novel mosquito-specific miRNAs in this study. We define "mosquito-specific" miRNAs here as those that are currently only found in mosquitoes. BLAST searches using low stringent parameters (word size at seven, e-value cut-off at 10) failed to identify any reliable homologues from miRBase or non-redundant GenBank sequences. We also performed oligomap comparisons [31
] of the "mosquito-specific" miRNAs to all miRBase sequences using default parameters and did not identify any match in any other organism. Oligomap [31
] is designed for comparisons of short sequences such as miRNAs, allowing gaps and mismatches. Taken together, the evidence indicates that what we are reporting in this study are novel miRNAs. This study increased the number of novel miRNAs that are only found in mosquitoes from five [21
] to 13. It is important to emphasize that some of these so-called "mosquito-specific" miRNAs may be discovered outside mosquitoes as future efforts of genome and small RNA sequencing expand to more and more organisms.
Expression patterns of conserved miRNAs
We chose nine conserved miRNAs and eight mosquito-specific miRNAs for further analysis using northern blot to confirm the small RNA sequencing results and to determine the expression patterns of these miRNAs in different developmental stages. Expression analysis of the eight mosquito-specific miRNAs is described in the context of a multi-species survey in a later section. The nine conserved miRNAs include let-7, miR-1, -133, -14, -184, -210, -9a, -970, and -998. All nine miRNAs showed signals at ~20 nt by northern during at least one of the developmental stages. Shown in Figure are the expression patterns of five of the nine conserved miRNAs. The patterns of presence/absence of these miRNAs in embryo, larvae, pupa, and adult stages are similar to the patterns found in D. melanogaster
] and An. stephensi
Figure 3 Expression patterns of Ae. aegypti homologs of previously known miRNAs. Only Ae. aegypti RNA samples were examined. The top panels are northern results and the bottom panels are RNA gel images for verification of small ribosomal RNA and tRNA integrity (more ...)
Elevated levels of miRNAs after blood feeding in the midgut of Ae. aegypti
The numbers of small RNA sequences in the midgut samples from sugar-fed and blood-fed female Ae. aegypti
may not be high enough for quantitative comparison. Nonetheless, we decided to compare the relative miRNA levels for miRNAs that showed more than 25 hits in at least one of the midgut samples. We used either the total number of all miRNA hits [12
] or the total number of small RNA reads to normalize the data. As shown in the last two columns of Table , except for miR-989 and miR-281*, all miRNAs showed an increase after blood feeding. We then performed northern blots using miR-184 and miR-998 probes. It is clear that miR-998 level is higher in bloodfed samples than in sugar-fed samples. Although less obvious, miR-184 level also appears to be higher in blood-fed samples than in sugar-fed samples (Figure ). Thus, the northern results are largely consistent with the data shown in Table . We have previously analyzed the level of miR-989 in the midgut before and after blood feeding [27
]. The signal was too weak to confirm or rule out reduction of miR-989 after blood feeding. Some miRNAs that are expressed in the midgut samples are also found in large numbers in embryos in Ae. aegypti
Comparison of the number of miRNA sequences in sugar-fed and blood-fed midgut samples.
Figure 4 Higher levels of miRNAs are observed in the female Ae. aegypti midgut 24 hrs after blood feeding (Gut_BF) compared to sugar feeding (Gut_SF). Three-day old females were either fed on blood or sugar and dissected 24 hrs later. 10 μg of total RNA (more ...)
Blood feeding is critical for mosquito physiology and its ability to transmit disease pathogens. It is through feeding on an infected host mosquitoes acquire pathogens such as malaria parasites and dengue viruses. It is also through blood feeding by an infected mosquito these pathogens may spread to a different host. Midgut is the first barrier the pathogens have to cross before they establish infection in mosquitoes. Thus midgut is one of the most important links in the disease transmission cycle. Furthermore, blood-feeding triggers a cascade of gene regulatory events in multiple tissues including midgut through the interplay of endocrine signals and transcription factors and thus has great impact on mosquito biology [33
]. The correlation between blood feeding and miRNA levels in Ae. aegypti
midgut warrants further investigations, which may shed light on the possible roles of miRNAs in physiology related to blood feeding and perhaps in mosquito-pathogen interactions.
Multi-species survey of eight mosquito-specific miRNAs revealed conserved and lineage-specific miRNAs
Previously five miRNAs were reported to be only found in mosquitoes. These are miR-1174, miR-1175 [21
], miR-1889, miR-1890, and miR-1891 [27
]. As described above, we uncovered eight additional mosquito miRNAs, bringing the number of total "mosquito-specific" miRNAs to 13. We conducted a detailed multi-species expression analysis of eight of the 13 mosquito-specific miRNAs using northern blot. When appropriate, we examined the expression of these miRNAs across the life stages of four mosquito species, An. gambiae, An. stephensi, Ae. aegypti
, and T. amboinensis
Four of the eight miRNAs (miR-M1, -1175, -1890, and -1891) are detected in all of the above four species (Figure ). Furthermore, the expression patterns of these miRNAs are similar in the four species and expression is detected in multiple developmental stages in three of the four miRNAs. The exceptions are the relatively low embryonic expression of miR-1175 in Ae. aegypti
(Figure ) and the low or hardly detectable embryonic expression of miR-1891 in Ae. aegypti
and T. amboinensis
(Figure ), compared to the rest of the species studied here. Overall, this is consistent with the observation that conserved miRNAs tend to be widely expressed [12
]. On the other hand, miR-M1 is expressed only in the embryos in all four species.
Figure 5 Four mosquito-specific miRNAs that are expressed in all four species of three highly divergent genera. MiRNAs examined include miR-M1 (A), miR-1175 (B), miR-1890 (C), and miR-1891 (D). Expression was examined across the developmental stages of An. stephensi, (more ...)
Four other miRNAs (miR-1174, miR-N1, miR-N2, and miR-N3) are only detected in a subset of the four mosquitoes. As shown in Figure , miR-1174 is not found in T. amboinensis but strong signals were detected in the other three species. MiR-1174 level in Ae. aegypti embryos is low or hardly detectable. It is interesting to point out that miR-1174 and miR-1175 are in the same contig separated by only ~200 bp. The expression patterns of miR-1174 and miR-1175 are similar in all three blood-feeding mosquitoes, suggesting that they may be under the same transcriptional control. MiR-1174 and miR-1175 share some sequence similarity at the 5' end. Thus it is possible that miR-1174 and miR-1175 resulted from gene duplication and miR-1174 may either have been lost in T. amboinensis or evolved beyond recognition by the miR-1174 probe. Ae. aegypti miR-1174 and An. gambiae miR-1174 differ by one nt. It is also possible that miR-1174 simply was not duplicated in T. amboinensis.
Figure 6 MiR-1174 is expressed in An. stephensi, An. gambiae, and Ae. aegypti, but not in T. amboinensis. The top panels are northern results and the bottom panels are RNA gel images for verification of small ribosomal RNA and tRNA integrity and loading of total (more ...)
As described earlier, miR-N1, N2, and N3 are from the same intronic cluster. As shown in Figure , miR-N1 was abundant in both Ae. aegypti and Cx. quinquefasciatus embryos. It was undetectable in An. stephensi. MiR-N2 was abundant in Ae. aegypti embryos, but undetectable in the embryos of Cx. quinquefasciatus. MiR-N2 was also undetectable in An. stephensi. MiR-N3 was found in Cx. quinquefasciatus embryos, but not in Ae. aegypti. MiR-N3 was also undetectable in An. stephensi. The expression data are consistent with genomic sequence analysis, which is described in the previous section on the miR-N1, N2, N3 cluster.
Figure 7 MiR-N1, miR-N2, and miR-N3 expression is restricted in particular lineages in mosquitoes. A) miR-N1 is expressed in Ae. aegypti and Cx. quinquefasciatus, but not in An. stephensi nor T. amboinensis. Emb, pooled embryos between 0-36 hr after egg deposition; (more ...)
We performed northern blots using all of the above eight miRNA probes to see if any signal was detected in D. melanogaster. We used at least 5 μg of total RNA from different developmental stages or a specific stage expected for a particular miRNA. None of the eight probes produced any miRNA signal while the positive control (Ae. aegypti sample) showed intense signals (data not shown). This is consistent with these miRNAs being only found in mosquitoes.
Functions of "mosquito-specific" miRNAs
All of the eight tested mosquito-specific miRNAs showed embryonic expression in at least one mosquito species (Figures , , and ), suggesting that these miRNAs may play important roles in mosquito embryonic development. Two of these miRNA clusters are worth noting. The first is the miR-M1-1 and miR-M1-2 cluster, which is only expressed in embryos in all four genera of mosquitoes tested. The conserved expression pattern and sequence conservation across all major branches of Culicidae suggest that miR-M1 is important during mosquito embryogenesis. Another interesting group of miRNAs are the miR-N1, -N2, and -N3 cluster. Two miR-N1 and one miR-N2 hairpins are in the first intron of a gene in Ae. aegypti encoding a putative transcription factor. Two miR-N1 hairpins and a miR-N3 hairpin are found in the orthologous gene in Cx. quinquefasciatus. None of these miRNAs are found in An. gambiae. In addition, miR-N2 is only found in Ae. aegypti while miR-N3 exists only in Cx. quinquefasciatus. These miRNAs share the same 7-8 bp 5' sequences in the seed regions important for target recognition. MiR-N1, N2, and N3 are all expressed in the embryos. Thus it is possible that these miRNAs derive from duplication events and the duplicated miRNAs may evolve into new sequences that acquire new functions. We postulate that, given their abundance and their lineage specificity, the N1/N2/N3 cluster may play a role in determining important lineage specific traits in mosquitoes. It will be important to determine the targets of these miRNAs to truly understand their function. Currently, the annotation of the 3'-UTRs of Ae. aegypti genes is limited. As these annotations improve, miRNA target prediction will likely be fruitful.