BACKGROUND
micrornas  are approximately  <dig> nucleotide long, non-coding rnas that regulate the expression of cellular genes by binding to target mrnas for cleavage or translational repression .
thousands of mirnas have been reported in animals and plants  <cit> .
many mirnas are highly conserved across divergent species while others are specific to a particular evolutionary lineage .
lineage-specific mirnas can arise from non-mirna sequences, which has been observed in drosophila, or by modifications of existing mirnas  <cit> .
mirna genes can occur in intergenic regions and within introns  <cit> .
for some mirnas, their biogenesis starts with transcription of mirna genes, mostly by rna polymerase ii, which produces a primary mirna .
a given primary mirna can be either monocistronic, containing one mature mirna, or polycistronic, containing multiple mature mirnas.
in drosophila, the primary mirna is processed by a drosha-pasha complex to yield small stem-loop structures that are approximately  <dig> nucleotides in length called pre-mirnas  <cit> .
following export to the cytosol, the double-stranded pre-mirna is recognized by dicer- <dig>  which complexes with loqs and produces a ~ <dig> bp duplex with  <dig> nt 3' overhangs  <cit> .
the product is referred to as a mirna:mirna* duplex, which is further separated by a helicase and normally the mirna* strand is rapidly degraded  <cit> .
during "deep" sequencing studies, approximately  <dig> fold as many mirnas as mirna*s was observed for a given mirna except in cases where both strands could generate functional mirnas  <cit> .
a new category of pre-mirnas known as mirtrons are derived from introns and undergo drosha-independent processing  <cit> .
some researchers suggest that the presence of mirtrons indicates that rna sources may be less relevant for determining which rnas become mirnas than their structural characteristics  <cit> .
mature mirnas pair with target mrnas with the assistance of argonaute to achieve translational repression and/or mrna degradation .
micrornas play key regulatory roles during embryonic development, stem cell division, cancer development, neurogenesis, heart development, haematopoietic cell differentiation, and cell death .
they are also implicated in control of viral infection in vertebrates and in one recent report mirnas were linked to malaria infection in mosquitoes  <cit> .
similarly, mirnas are also linked to innate immunity .
there are reports describing anopheles gambiae mirnas on the basis of computational prediction or similarity to known mirnas from other species  <cit> .
direct experimental evidence for mosquito mirnas has been recently reported only in anopheline mosquitoes using small-scale sequencing  <cit> .
there is also a study on genes involved in small rna pathways in mosquitoes  <cit> .
using high throughput sequencing and bioinformatics approaches, we performed the first systematic analysis of mirnas in ae.
aegypti.
in the current study, we substantially expanded the list of mirnas for all mosquitoes and uncovered a number of novel and mosquito-specific mirnas.
northern and small rna sequencing revealed several mirnas that may play important roles during embryonic development and during blood feeding.
our analysis also offered insights into the evolution of conserved and lineage-specific mirnas in mosquitoes.
RESULTS
discovery of  <dig> distinct pre-mirna sequences in ae.
aegypti
as shown in table  <dig>  we have uncovered  <dig> different pre-mirnas in ae.
aegypti which could produce  <dig> distinct mirnas.
some of the  <dig> pre-mirna sequences produce identical mirnas and mirna*s. also included in table  <dig> are  <dig> 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 .
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  <cit> .
twenty-nine of the  <dig> pre-mirnas do not have small rna sequences in the embryo and midgut samples.
however, these  <dig> pre-mirnas all form hairpins and are conserved among ae.
aegypti, cx.
quinquefasciatus, and an.
gambiae, the three mosquito species with sequenced genomes.
notes:
<dig>  the first block  are novel mirnas that are discovered in this study.
we have not detected homology to any known mirnas or genomic sequences outside of mosquito species.
the second block  contains mirnas that are homologous to previously reported "mosquito-specific" mirnas.
the third block, comprised of the remaining mirnas, contains mirnas that have homologues outside of mosquitoes.
<dig>  naming in this column is temporary.
mirbase assigned names are shown in column  <dig>  which was received after the acceptance of the manuscript.
"-1", and "-2" suffixes refer to different hairpins that produce the same mature mirna.
"a", and "b" suffixes refer to different hairpins that produce similar but not identical mirnas.
<dig>  underlined sequences are cases where there are extra bases at the 5' or 3' ends and they are the majority in at least six small rna sequences.
italicized sequences are cases where such sequences are detected but they are either not the majority or there are less than six  <dig> sequence hits.
as poly-as are added to the small rnas, adenine at the 3' end cannot be confirmed by this sequencing approach.
<dig>  the contig, start, and end positions refer to the locations of the pre-mirna hairpins.
<dig>  the last three columns are the number of sequence hits in small rna libraries obtained by  <dig> sequencing.
the total small rna reads are  <dig>   <dig>   <dig>   <dig>  and  <dig>   <dig> in embryos, sugar-fed midguts , and blood-fed midguts , respectively.
the total number of hits for all ae.
aegypti mirnas are  <dig>   <dig>  and  <dig> in embryos, sugar-fed midguts , and blood-fed midguts , respectively.
there are a few cases where we did not distinguish the hits from nearly identical mirnas.
possible revisions at the ends of known mirnas and cases of abundant mirna* sequences
as shown in table  <dig>  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 .
to minimize the contribution of sequencing error, we only consider cases where there are at least six such sequences in the  <dig> database and these differences are the majority.
these mirnas include aae-mir-2a, aae-mir- <dig>  aae-mir-263b, aae-mir- <dig>  and aae-mir- <dig>  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  <dig> 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 .
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  <dig> clusters of mirnas that are defined as more than one mirna hairpin within  <dig> kb  <cit> .
twelve of these clusters have members that are separated by less than one kb.
all these clusters can be identified in table  <dig> and additional file  <dig> 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- <dig>  and mir- <dig>  we previously identified this cluster in both an.
gambiae and d. melanogaster and we thought mir- <dig> was missing in the ae.
aegypti cluster  <cit> .
however, small rna sequencing and closer analysis of the ae.
aegypti assembly suggest that mir- <dig> 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- <dig> shows  <dig> mismatches in the  <dig> bp overlap compared to aga-mir- <dig> and dme-mir- <dig>
the second cluster includes mir- <dig> and mir- <dig>  which flank either mir- <dig> in d. melanogaster or mir- <dig> in an.
gambiae  <cit> .
mir- <dig> has similarity to the reverse-complementary sequence of mir- <dig> but the difference is significant enough for mirbase to assign a unique name for it.
given that the d. melanogaster mir- <dig> and the an.
gambiae mir- <dig> are flanked by the same mirnas and that this mirna cluster is found in an intron of orthologous genes in the two species, mir- <dig> and mir- <dig> may have a common origin.
through small rna sequencing and a closer analysis of the genome assembly, we identified mir- <dig> in ae.
aegypti , which was previously thought to be missing in the ae.
aegypti cluster  <cit> .
the newly identified aae-mir- <dig> shows  <dig> mismatches in the  <dig> bp overlap with aga-mir- <dig>  the identification of aae-mir- <dig> further supports the strand orientation of the mosquito mir- <dig>  it is tempting to suggest that one of the two mirna hairpins, mir- <dig> or mir- <dig>  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  <dig> 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 .
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 , duplication was thought to be not important in drosophila  <cit> .
novel mirnas that are potentially specific to mosquitoes
nine of the  <dig> pre-mirna hairpins are novel and currently have only been found in mosquitoes.
these nine pre-mirnas produce seven distinct mature mirnas .
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  <dig> and  <dig> 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  produce the same mir-m <dig> in ae.
aegypti.
two physically linked pre-mirna hairpins produce similar but not identical mir-m4a and mir-m4b.
mir-m <dig>  -m <dig>  -m <dig>  -m4a and -m4b are found in all three available mosquito genome assemblies.
two physically linked pre-mirna hairpins  produce the same mir-n <dig> and they are also in close proximity to the mir-n <dig> hairpin in ae.
aegypti.
the three hairpins are in the first intron of a gene in ae.
aegypti  encoding a putative transcription factor with a basic leucine zipper domain.
sequence analysis suggests that mir-n <dig> is found in the orthologous gene in cx.
quinquefasciatus but not found in an.
gambiae.
mir-n <dig> is only found in ae.
aegypti.
mir-n <dig> 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-n <dig>  we name this mirna mir-n <dig> and it is only found in cx.
quinquefasciatus .
cqu-mir-n <dig> is not listed in table  <dig>  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  failed to identify any reliable homologues from mirbase or non-redundant genbank sequences.
we also performed oligomap comparisons  <cit>  of the "mosquito-specific" mirnas to all mirbase sequences using default parameters and did not identify any match in any other organism.
oligomap  <cit>  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  <cit>  to  <dig>  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- <dig>  mir- <dig>  - <dig>  - <dig>  - <dig>  - <dig>  -9a, - <dig>  and - <dig>  all nine mirnas showed signals at ~ <dig> nt by northern during at least one of the developmental stages.
shown in figure  <dig> 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  <cit>  and an.
stephensi  <cit> .
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  <dig> hits in at least one of the midgut samples.
we used either the total number of all mirna hits  <cit>  or the total number of small rna reads to normalize the data.
as shown in the last two columns of table  <dig>  except for mir- <dig> and mir-281*, all mirnas showed an increase after blood feeding.
we then performed northern blots using mir- <dig> and mir- <dig> probes.
it is clear that mir- <dig> level is higher in bloodfed samples than in sugar-fed samples.
although less obvious, mir- <dig> level also appears to be higher in blood-fed samples than in sugar-fed samples .
thus, the northern results are largely consistent with the data shown in table  <dig>  we have previously analyzed the level of mir- <dig> in the midgut before and after blood feeding  <cit> .
the signal was too weak to confirm or rule out reduction of mir- <dig> after blood feeding.
some mirnas that are expressed in the midgut samples are also found in large numbers in embryos in ae.
aegypti .
notes.
<dig>  only mirnas that showed  <dig> or more sequences in one of the gut samples are shown.
<dig>  mir- <dig> and mir- <dig> are physically linked, less than  <dig> bp apart.
<dig>  mir-13b and mir-2a/2b/2c are in a physically linked cluster.
mir-13b is less than  <dig> bp apart from mir-2b.
<dig>  it is difficult to distinguish between hits that match mir-2a, mir-2b, or mir-2c.
thus we refer to these hits mir-2a/2b/2c.
<dig>  mir- <dig> and mir-9b are physically linked, less than  <dig> bp apart.
<dig>  the second and third columns are raw numbers of sequence hits in sugar-fed  and blood-fed  midgut samples, respectively.
<dig>  column four is the fold difference of gut_bf over gut_sf, normalized by the total mirna hits within a sample, as suggested in reference  <cit> .
the total mirna hits are  <dig> and  <dig> in gut_sf and gut_bf samples, respectively.
thus, for each mirna, the fold change = /.
<dig>  column five is the fold difference of gut_bf over gut_sf, normalized by the total small rna reads within a sample.
the total small rna reads are  <dig>   <dig> and  <dig>   <dig> in gut_sf and gut_bf samples, respectively.
thus, for each mirna, the fold change = /.
<dig>  the numbers in columns  <dig> and  <dig> reflect the same trend, generally higher levels of these mirnas in blood-fed sample than in sugar-fed sample.
although the number of small rna sequences may not be sufficient for the analysis to be quantitative, overall trends suggest leads for further analysis.
<dig>  na, not applicable as the denominator is zero.
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  <cit> .
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- <dig>  mir- <dig>  <cit> , mir- <dig>  mir- <dig>  and mir- <dig>  <cit> .
as described above, we uncovered eight additional mosquito mirnas, bringing the number of total "mosquito-specific" mirnas to  <dig>  we conducted a detailed multi-species expression analysis of eight of the  <dig> 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  are detected in all of the above four species .
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- <dig> in ae.
aegypti  and the low or hardly detectable embryonic expression of mir- <dig> in ae.
aegypti and t. amboinensis , compared to the rest of the species studied here.
overall, this is consistent with the observation that conserved mirnas tend to be widely expressed  <cit> .
on the other hand, mir-m <dig> is expressed only in the embryos in all four species.
four other mirnas  are only detected in a subset of the four mosquitoes.
as shown in figure  <dig>  mir- <dig> is not found in t. amboinensis but strong signals were detected in the other three species.
mir- <dig> level in ae.
aegypti embryos is low or hardly detectable.
it is interesting to point out that mir- <dig> and mir- <dig> are in the same contig separated by only ~ <dig> bp.
the expression patterns of mir- <dig> and mir- <dig> are similar in all three blood-feeding mosquitoes, suggesting that they may be under the same transcriptional control.
mir- <dig> and mir- <dig> share some sequence similarity at the 5' end.
thus it is possible that mir- <dig> and mir- <dig> resulted from gene duplication and mir- <dig> may either have been lost in t. amboinensis or evolved beyond recognition by the mir- <dig> probe.
ae.
aegypti mir- <dig> and an.
gambiae mir- <dig> differ by one nt.
it is also possible that mir- <dig> simply was not duplicated in t. amboinensis.
as described earlier, mir-n <dig>  n <dig>  and n <dig> are from the same intronic cluster.
as shown in figure  <dig>  mir-n <dig> was abundant in both ae.
aegypti and cx.
quinquefasciatus embryos.
it was undetectable in an.
stephensi.
mir-n <dig> was abundant in ae.
aegypti embryos, but undetectable in the embryos of cx.
quinquefasciatus.
mir-n <dig> was also undetectable in an.
stephensi.
mir-n <dig> was found in cx.
quinquefasciatus embryos, but not in ae.
aegypti.
mir-n <dig> 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-n <dig>  n <dig>  n <dig> cluster.
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  <dig> 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  showed intense signals .
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 , 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- <dig> and mir-m1- <dig> 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-m <dig> is important during mosquito embryogenesis.
another interesting group of mirnas are the mir-n <dig>  -n <dig>  and -n <dig> cluster.
two mir-n <dig> and one mir-n <dig> hairpins are in the first intron of a gene in ae.
aegypti encoding a putative transcription factor.
two mir-n <dig> hairpins and a mir-n <dig> hairpin are found in the orthologous gene in cx.
quinquefasciatus.
none of these mirnas are found in an.
gambiae.
in addition, mir-n <dig> is only found in ae.
aegypti while mir-n <dig> exists only in cx.
quinquefasciatus.
these mirnas share the same 7- <dig> bp 5' sequences in the seed regions important for target recognition.
mir-n <dig>  n <dig>  and n <dig> 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/n <dig> 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.
CONCLUSIONS
we report the first systematic analysis of mirnas in ae.
aegypti during which  <dig> pre-mirnas were uncovered.
thus we substantially expanded the list of mirnas found in mosquitoes.
we also provided experimental evidence for  <dig> of the mirna and mirna* sequences.
we also uncovered highly abundant and conserved mirna* sequences, which is consistent with the suggestion that some mirna* are functional.
there are  <dig> mirna clusters and  <dig> cases where more than one pre-mirna hairpin produces the same or highly similar mature mirnas in ae.
aegypti .
these mirnas are a rich source for future comparative analysis to uncover the evolutionary patterns of mirna duplication and the process of creating new mirnas in mosquitoes.
perhaps most importantly, we identified eight novel mirnas that are potentially specific to mosquitoes.
this discovery expanded the list of mosquito-specific mirnas from five  <cit>  to  <dig>  four of the  <dig> mirnas are specific to certain lineages within mosquitoes.
the expression profiles of a few mirnas suggest stage-specific functions and functions related to embryonic development or blood feeding.
a better understanding of the functions of these mirnas will offer novel insights in mosquito biology and may lead to novel approaches to combat mosquito-borne infectious diseases.

 micrornas are approximately nucleotide long, non-coding rnas that regulate the expression of cellular genes by binding to target mrnas for cleavage or translational repression. thousands of mirnas have been reported in animals and plants. many mirnas are highly conserved across divergent species while others are specific to a particular evolutionary lineage. lineage-specific mirnas can arise from non-mirna sequences, which has been observed in drosophila, or by modifications of existing mirnas. mirna genes can occur in intergenic regions and within introns. for some mirnas, their biogenesis starts with transcription of mirna genes, mostly by rna polymerase ii, which produces a primary mirna. a given primary mirna can be either monocistronic, containing one mature mirna, or polycistronic, containing multiple mature mirnas. in drosophila, the primary mirna is processed by a drosha-pasha complex to yield small stem-loop structures that are approximately nucleotides in length called pre-mirnas. following export to the cytosol, the double-stranded pre-mirna is recognized by dicer- which complexes with loqs and produces a ~ bp duplex with nt 3' overhangs. the product is referred to as a mirna:mirna* duplex, which is further separated by a helicase and normally the mirna* strand is rapidly degraded. during "deep" sequencing studies, approximately fold as many mirnas as mirna*s was observed for a given mirna except in cases where both strands could generate functional mirnas. a new category of pre-mirnas known as mirtrons are derived from introns and undergo drosha-independent processing. some researchers suggest that the presence of mirtrons indicates that rna sources may be less relevant for determining which rnas become mirnas than their structural characteristics. mature mirnas pair with target mrnas with the assistance of argonaute to achieve translational repression and/or mrna degradation. micrornas play key regulatory roles during embryonic development, stem cell division, cancer development, neurogenesis, heart development, haematopoietic cell differentiation, and cell death. they are also implicated in control of viral infection in vertebrates and in one recent report mirnas were linked to malaria infection in mosquitoes. similarly, mirnas are also linked to innate immunity. there are reports describing anopheles gambiae mirnas on the basis of computational prediction or similarity to known mirnas from other species. direct experimental evidence for mosquito mirnas has been recently reported only in anopheline mosquitoes using small-scale sequencing. there is also a study on genes involved in small rna pathways in mosquitoes. using high throughput sequencing and bioinformatics approaches, we performed the first systematic analysis of mirnas in ae. aegypti. in the current study, we substantially expanded the list of mirnas for all mosquitoes and uncovered a number of novel and mosquito-specific mirnas. northern and small rna sequencing revealed several mirnas that may play important roles during embryonic development and during blood feeding. our analysis also offered insights into the evolution of conserved and lineage-specific mirnas in mosquitoes.  discovery of distinct pre-mirna sequences in ae. aegypti as shown in table we have uncovered different pre-mirnas in ae. aegypti which could produce distinct mirnas. some of the pre-mirna sequences produce identical mirnas and mirna*s. also included in table are 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. 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. twenty-nine of the pre-mirnas do not have small rna sequences in the embryo and midgut samples. however, these pre-mirnas all form hairpins and are conserved among ae. aegypti, cx. quinquefasciatus, and an. gambiae, the three mosquito species with sequenced genomes. notes: the first block are novel mirnas that are discovered in this study. we have not detected homology to any known mirnas or genomic sequences outside of mosquito species. the second block contains mirnas that are homologous to previously reported "mosquito-specific" mirnas. the third block, comprised of the remaining mirnas, contains mirnas that have homologues outside of mosquitoes. naming in this column is temporary. mirbase assigned names are shown in column which was received after the acceptance of the manuscript. "-1", and "-2" suffixes refer to different hairpins that produce the same mature mirna. "a", and "b" suffixes refer to different hairpins that produce similar but not identical mirnas. underlined sequences are cases where there are extra bases at the 5' or 3' ends and they are the majority in at least six small rna sequences. italicized sequences are cases where such sequences are detected but they are either not the majority or there are less than six sequence hits. as poly-as are added to the small rnas, adenine at the 3' end cannot be confirmed by this sequencing approach. the contig, start, and end positions refer to the locations of the pre-mirna hairpins. the last three columns are the number of sequence hits in small rna libraries obtained by sequencing. the total small rna reads are and in embryos, sugar-fed midguts, and blood-fed midguts, respectively. the total number of hits for all ae. aegypti mirnas are and in embryos, sugar-fed midguts, and blood-fed midguts, respectively. there are a few cases where we did not distinguish the hits from nearly identical mirnas. 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. to minimize the contribution of sequencing error, we only consider cases where there are at least six such sequences in the database and these differences are the majority. these mirnas include aae-mir-2a, aae-mir- aae-mir-263b, aae-mir- and aae-mir- 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 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. 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 clusters of mirnas that are defined as more than one mirna hairpin within kb. 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 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- and mir- we previously identified this cluster in both an. gambiae and d. melanogaster and we thought mir- was missing in the ae. aegypti cluster. however, small rna sequencing and closer analysis of the ae. aegypti assembly suggest that mir- 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- shows mismatches in the bp overlap compared to aga-mir- and dme-mir- the second cluster includes mir- and mir- which flank either mir- in d. melanogaster or mir- in an. gambiae. mir- has similarity to the reverse-complementary sequence of mir- but the difference is significant enough for mirbase to assign a unique name for it. given that the d. melanogaster mir- and the an. gambiae mir- are flanked by the same mirnas and that this mirna cluster is found in an intron of orthologous genes in the two species, mir- and mir- may have a common origin. through small rna sequencing and a closer analysis of the genome assembly, we identified mir- in ae. aegypti, which was previously thought to be missing in the ae. aegypti cluster. the newly identified aae-mir- shows mismatches in the bp overlap with aga-mir- the identification of aae-mir- further supports the strand orientation of the mosquito mir- it is tempting to suggest that one of the two mirna hairpins, mir- or mir- 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 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. 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, duplication was thought to be not important in drosophila. novel mirnas that are potentially specific to mosquitoes nine of the pre-mirna hairpins are novel and currently have only been found in mosquitoes. these nine pre-mirnas produce seven distinct mature mirnas. 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 produce the same mir-m in ae. aegypti. two physically linked pre-mirna hairpins produce similar but not identical mir-m4a and mir-m4b. mir-m -m -m -m4a and -m4b are found in all three available mosquito genome assemblies. two physically linked pre-mirna hairpins produce the same mir-n and they are also in close proximity to the mir-n hairpin in ae. aegypti. the three hairpins are in the first intron of a gene in ae. aegypti encoding a putative transcription factor with a basic leucine zipper domain. sequence analysis suggests that mir-n is found in the orthologous gene in cx. quinquefasciatus but not found in an. gambiae. mir-n is only found in ae. aegypti. mir-n 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-n we name this mirna mir-n and it is only found in cx. quinquefasciatus. cqu-mir-n 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 failed to identify any reliable homologues from mirbase or non-redundant genbank sequences. we also performed oligomap comparisons of the "mosquito-specific" mirnas to all mirbase sequences using default parameters and did not identify any match in any other organism. oligomap 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 to 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- mir- - - - - -9a, - and - all nine mirnas showed signals at ~ 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. 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 hits in at least one of the midgut samples. we used either the total number of all mirna hits or the total number of small rna reads to normalize the data. as shown in the last two columns of table except for mir- and mir-281*, all mirnas showed an increase after blood feeding. we then performed northern blots using mir- and mir- probes. it is clear that mir- level is higher in bloodfed samples than in sugar-fed samples. although less obvious, mir- level also appears to be higher in blood-fed samples than in sugar-fed samples. thus, the northern results are largely consistent with the data shown in table we have previously analyzed the level of mir- in the midgut before and after blood feeding. the signal was too weak to confirm or rule out reduction of mir- after blood feeding. some mirnas that are expressed in the midgut samples are also found in large numbers in embryos in ae. aegypti. notes. only mirnas that showed or more sequences in one of the gut samples are shown. mir- and mir- are physically linked, less than bp apart. mir-13b and mir-2a/2b/2c are in a physically linked cluster. mir-13b is less than bp apart from mir-2b. it is difficult to distinguish between hits that match mir-2a, mir-2b, or mir-2c. thus we refer to these hits mir-2a/2b/2c. mir- and mir-9b are physically linked, less than bp apart. the second and third columns are raw numbers of sequence hits in sugar-fed and blood-fed midgut samples, respectively. column four is the fold difference of gut_bf over gut_sf, normalized by the total mirna hits within a sample, as suggested in reference. the total mirna hits are and in gut_sf and gut_bf samples, respectively. thus, for each mirna, the fold change = /. column five is the fold difference of gut_bf over gut_sf, normalized by the total small rna reads within a sample. the total small rna reads are and in gut_sf and gut_bf samples, respectively. thus, for each mirna, the fold change = /. the numbers in columns and reflect the same trend, generally higher levels of these mirnas in blood-fed sample than in sugar-fed sample. although the number of small rna sequences may not be sufficient for the analysis to be quantitative, overall trends suggest leads for further analysis. na, not applicable as the denominator is zero. 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. 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- mir-, mir- mir- and mir-. as described above, we uncovered eight additional mosquito mirnas, bringing the number of total "mosquito-specific" mirnas to we conducted a detailed multi-species expression analysis of eight of the 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 are detected in all of the above four species. 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- in ae. aegypti and the low or hardly detectable embryonic expression of mir- in ae. aegypti and t. amboinensis, compared to the rest of the species studied here. overall, this is consistent with the observation that conserved mirnas tend to be widely expressed. on the other hand, mir-m is expressed only in the embryos in all four species. four other mirnas are only detected in a subset of the four mosquitoes. as shown in figure mir- is not found in t. amboinensis but strong signals were detected in the other three species. mir- level in ae. aegypti embryos is low or hardly detectable. it is interesting to point out that mir- and mir- are in the same contig separated by only ~ bp. the expression patterns of mir- and mir- are similar in all three blood-feeding mosquitoes, suggesting that they may be under the same transcriptional control. mir- and mir- share some sequence similarity at the 5' end. thus it is possible that mir- and mir- resulted from gene duplication and mir- may either have been lost in t. amboinensis or evolved beyond recognition by the mir- probe. ae. aegypti mir- and an. gambiae mir- differ by one nt. it is also possible that mir- simply was not duplicated in t. amboinensis. as described earlier, mir-n n and n are from the same intronic cluster. as shown in figure mir-n was abundant in both ae. aegypti and cx. quinquefasciatus embryos. it was undetectable in an. stephensi. mir-n was abundant in ae. aegypti embryos, but undetectable in the embryos of cx. quinquefasciatus. mir-n was also undetectable in an. stephensi. mir-n was found in cx. quinquefasciatus embryos, but not in ae. aegypti. mir-n 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-n n n cluster. 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 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 showed intense signals. 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, 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- and mir-m1- 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-m is important during mosquito embryogenesis. another interesting group of mirnas are the mir-n -n and -n cluster. two mir-n and one mir-n hairpins are in the first intron of a gene in ae. aegypti encoding a putative transcription factor. two mir-n hairpins and a mir-n hairpin are found in the orthologous gene in cx. quinquefasciatus. none of these mirnas are found in an. gambiae. in addition, mir-n is only found in ae. aegypti while mir-n exists only in cx. quinquefasciatus. these mirnas share the same 7- bp 5' sequences in the seed regions important for target recognition. mir-n n and n 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/n 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.  we report the first systematic analysis of mirnas in ae. aegypti during which pre-mirnas were uncovered. thus we substantially expanded the list of mirnas found in mosquitoes. we also provided experimental evidence for of the mirna and mirna* sequences. we also uncovered highly abundant and conserved mirna* sequences, which is consistent with the suggestion that some mirna* are functional. there are mirna clusters and cases where more than one pre-mirna hairpin produces the same or highly similar mature mirnas in ae. aegypti. these mirnas are a rich source for future comparative analysis to uncover the evolutionary patterns of mirna duplication and the process of creating new mirnas in mosquitoes. perhaps most importantly, we identified eight novel mirnas that are potentially specific to mosquitoes. this discovery expanded the list of mosquito-specific mirnas from five to four of the mirnas are specific to certain lineages within mosquitoes. the expression profiles of a few mirnas suggest stage-specific functions and functions related to embryonic development or blood feeding. a better understanding of the functions of these mirnas will offer novel insights in mosquito biology and may lead to novel approaches to combat mosquito-borne infectious diseases. 
we also identified five potential revisions to previously annotated mirnas at the mirna termini, two cases of highly abundant mirna* sequences, mirna clusters, and cases where more than one pre-mirna hairpin produces the same or highly similar mature mirnas. a better understanding of the functions of these mirnas will offer new insights in mosquito biology and may lead to novel approaches to combat mosquito-borne infectious diseases. the mature mirnas play key regulatory roles during embryonic development as well as other cellular processes. direct experimental evidence for mosquito mirnas has been recently reported in anopheline mosquitoes based on small-scale cloning efforts. they are also implicated in control of viral infection as well as innate immunity. the combination of these approaches uncovered different pre-mirnas in ae. detailed expression analysis of eight of the mosquito-specific mirnas in four divergent mosquito genera identified cases of clearly conserved expression patterns and obvious differences. a number of mirnas showed higher levels in midgut from blood-fed female than that from sugar-fed female, which was confirmed by northern blots on two of these mirnas. northern blots also revealed several mirnas that showed stage-specific expression. the expression profiles of a few mirnas suggest stage-specific functions and functions related to embryonic development or blood feeding. new insights were gained on the evolution of conserved and lineage-specific mirnas in mosquitoes. a pre-mirna is processed to make a mirna:mirna* duplex, which is then separated to generate a mature mirna and a mirna*. we obtained approximately small rna sequences from the yellow fever mosquito, aedes aegypti, by sequencing of samples that were isolated from mixed-age embryos and midguts from sugar-fed and blood-fed females, respectively. this study provides the first systematic analysis of mirnas in ae. thirteen mirnas, including eight novel mirnas identified in this study, are currently only found in mosquitoes.
 micrornas are a novel class of gene regulators whose biogenesis involves hairpin structures called precursor mirnas, or pre-mirnas. a pre-mirna is processed to make a mirna:mirna* duplex, which is then separated to generate a mature mirna and a mirna*. the mature mirnas play key regulatory roles during embryonic development as well as other cellular processes. they are also implicated in control of viral infection as well as innate immunity. direct experimental evidence for mosquito mirnas has been recently reported in anopheline mosquitoes based on small-scale cloning efforts.  we obtained approximately small rna sequences from the yellow fever mosquito, aedes aegypti, by sequencing of samples that were isolated from mixed-age embryos and midguts from sugar-fed and blood-fed females, respectively. we also performed bioinformatics analysis on the ae. aegypti genome assembly to identify evidence for additional mirnas. the combination of these approaches uncovered different pre-mirnas in ae. aegypti which could produce distinct mirnas. thirteen mirnas, including eight novel mirnas identified in this study, are currently only found in mosquitoes. we also identified five potential revisions to previously annotated mirnas at the mirna termini, two cases of highly abundant mirna* sequences, mirna clusters, and cases where more than one pre-mirna hairpin produces the same or highly similar mature mirnas. a number of mirnas showed higher levels in midgut from blood-fed female than that from sugar-fed female, which was confirmed by northern blots on two of these mirnas. northern blots also revealed several mirnas that showed stage-specific expression. detailed expression analysis of eight of the mosquito-specific mirnas in four divergent mosquito genera identified cases of clearly conserved expression patterns and obvious differences. four of the mirnas are specific to certain lineage within mosquitoes.  this study provides the first systematic analysis of mirnas in ae. aegypti and offers a substantially expanded list of mirnas for all mosquitoes. new insights were gained on the evolution of conserved and lineage-specific mirnas in mosquitoes. the expression profiles of a few mirnas suggest stage-specific functions and functions related to embryonic development or blood feeding. a better understanding of the functions of these mirnas will offer new insights in mosquito biology and may lead to novel approaches to combat mosquito-borne infectious diseases. 