BACKGROUND
small rna-mediated gene silencing pathways are master regulators of critical cellular processes, from development to germ-line surveillance to anti-viral defense  <cit> .
although small rna regulatory pathways  operate using distinctly different mechanisms, they share several common features.
small regulatory rnas of  <dig> to  <dig> nucleotides  are used as guide strands for target substrate recognition by an rnase h type nuclease of the argonaute protein family.
target rnas are subsequently degraded or otherwise prevented from being translated into protein.
the three major pathways are the small interfering rna , microrna  and piwi small rna  pathways; the general functions of each are summarized in table  <dig>  due to the paucity of functional information from mosquitoes, we have relied on protein structure and functional information compiled from drosophila spp., caenorhabditis elegans, and mammals.
the argonaute protein family contains the argonaute and piwi protein subclasses.
all proteins in this family contain paz  and piwi domains  <cit> .
the paz domain is a small rna binding domain; the piwi domain is an rnase-h type domain which relies on divalent cation binding to facilitate dsrna-guided cleavage of ssrna .
argonaute  <dig>  and argonaute  <dig>  are in the argonaute protein subclass and are functionally distinct from piwis, in that they rely on dicer proteins to cleave long dsrnas into small rnas, which are then passed to the agos for further processing and recognition of target rnas.
these proteins physically interact with dicers at the piwi domain and use small rnas formed from double-stranded templates  <cit> .
ago <dig> and ago <dig> each participate in different gene silencing pathways.
mirna pathways of invertebrates have been best characterized in d. melanogaster and c. elegans .
in d. melanogaster, developmental and housekeeping gene expression is regulated by ago <dig> and the small rna subclass, mirnas   <cit> .
in general, ago <dig> acts with dicer- <dig>  micrornas  and accessory proteins to control gene expression of housekeeping genes   <cit> .
in an.
gambiae, the mirna pathway helps defend against plasmodium bergei infection  <cit> .
gene expression can be controlled by a variety of possible silencing mechanisms, including translational repression, decreased mrna stability or altered mrna localization  <cit> .
these small rnas are genome-encoded as primary transcript mirnas  up to  <dig> kilobases  in length and processed into mirna precursor  hairpin loops of about  <dig> nts by drosha and pasha   <cit> .
dicer- <dig> cleaves pre-mirnas to form mature mirnas, which are transferred to ago <dig> for silencing of target mrnas  <cit> .
in mammals, some mirnas are encoded in the introns of the genes they target  <cit> .
the sirna pathway provides defense against rna viruses in the degradation of double-stranded viral rnas  <cit> .
sirnas, generated from longer dsrna molecules by the dicer-2/r2d <dig> complex, serve as guides for ago <dig> to identify and degrade target rnas.
key proteins in the anti-viral rna interference  pathway, including ago <dig> and dicer- <dig>  are functional in vector mosquitoes and influence the ability of mosquitoes to serve as competent vectors of human virus pathogens  <cit> .
further, several recent reports of drosophila have shown that endogenous sirnas control retrotransposons and mrnas in somatic cells in an ago2/dicer- <dig> dependent manner  <cit> .
in drosophila melanogaster, the piwi protein subclass includes piwi, aubergine , and argonaute  <dig> .
these proteins contain the signature paz and piwi domains, but have remained enigmatic members of the argonaute family.
they have been implicated in a variety of functions specific to germ-line tissue in d. melanogaster, including the suppression of retrotransposon mobilization, maintenance of telomeres, prevention of double-stranded dna breaks during meiosis, and mrna silencing in germ-line cells  <cit> .
ago <dig>  aub, and piwi use piwi-associated rnas   in these pathways.
the mechanisms for production of this class of non-coding rnas are not well understood.
models have been proposed, suggesting they are processed from pirna cluster transcripts and amplified by a "ping pong" amplification loop between these transcripts and those derived from active transposons   <cit> .
three mosquito species, anopheles gambiae, aedes aegypti, and culex pipiens quinquefasciatus are important vectors of human and animal pathogens  <cit> .
the goal of this study is to increase understanding of the three small rna pathways in vector mosquitoes using comparative genomics, with special emphasis on the key catalytic enzymes of the argonaute protein family.
we performed phylogenetic analyses of argonaute family proteins and demonstrated expansion of ae.
aegypti and cx.
pipiens argonaute and piwi subfamilies.
in addition, we determined whether cis-acting regulatory elements upstream of srrp genes are conserved across mosquito species or among components of specific rna regulatory pathways, as has been successfully demonstrated in other invertebrates  <cit> .
to this end, previously validated mosquito transcription factor binding sites  and other elements in upstream control regions  of all suspected srrp components were identified.
further, we used motif discovery algorithms to identify novel upstream regulatory sequences.
the identification of common cis-acting regulatory features lends insight into species-specific differences in regulation of srrps and provides a basis for future studies of pathogen defense in vector mosquitoes.
RESULTS
the an.
gambiae, ae.
aegypti, and cx.
pipiens genomes encode components of the three major rna regulatory pathways previously identified in d. melanogaster   <cit> .
there are gene expansions in some categories; these are described below.
additional file 1a depicts, in gray highlight, the genes for which expression has been confirmed, either experimentally or in est libraries  <cit> .
importantly, key catalytic residues of the rnase-h domains are conserved in all mosquito ago1s, ago2s and piwi subfamily proteins   <cit> .
although a few studies have been done in ae.
aegypti and anopheline mosquitoes  <cit> , experimental verification will be required to confirm the roles played by most of these putative orthologs in rna regulatory pathways.
argonaute protein subfamily
ago <dig> is represented at a single genomic locus in an.
gambiae, cx.
pipiens and d. melanogaster but seems to be present in two copies in ae.
aegypti .
the flanking sequences of about  <dig>  kilobases  upstream and the introns were found to be unique.
ago <dig> of the human head louse, pediculus humanus was used as an outgroup for the analysis of argonaute subfamily proteins.
ago <dig> homologs from the silkworm, bombyx mori, and d. melanogaster were also included in the analysis.
in addition, orthologs from the important pathogen vectors, lutzomyia longipalpis, culex tarsalis, and ochlerotatus triseriatus were included.
based upon the jones-taylor-thornton probability model, the probability of amino acid substitutions among ago <dig> proteins was  <dig>   =  <dig> *  =  <dig> %) while among ago <dig> proteins the average probability was  <dig>     <cit> .
thus, the probability of amino acid substitutions among ago <dig> proteins is about 38-fold greater than among ago <dig> proteins.
because of this high rate of change among ago <dig> proteins, there was only weak bootstrap support  for the proteins from mosquitoes.
however, among the two genera of the tribe aedini it is interesting that between ochlerotatus triseriatus and ae.
aegypti the substitution probability is only  <dig> , while within and among culex species the probability is  <dig>  .
this pattern suggests that anti-viral pathway components are evolving more rapidly in culex spp.
than among subgenera of aedine mosquitoes.
an interesting parallel was found among drosophilid species, wherein key anti-viral rnai component genes, such as dmeago <dig> and dmedicer- <dig>  were found to be evolving at a higher rate than mirna pathway components or other immune response genes  <cit> .
synapomorphies that distinguish ago1s from ago2s can be found in the amino acids surrounding the first catalytic residue .
ago <dig> protein conservation indicates an evolutionary trend of purifying selection, probably due to conserved mechanisms in developmental pathways.
this hypothesis is supported in the conservation of some mirnas between drosophilids, mosquitoes, and humans  <cit> .
although many mosquito mirnas have not been experimentally verified, their presence suggests conservation of developmentally regulated gene silencing pathways.
alignment of ago <dig> proteins further illustrates the degree of conservation with overall amino acid identity of  <dig> to 89% among all mosquito orthologs and that of d. melanogaster .
this is in contrast to the  <dig> to 43% amino acid identity among all mosquito ago  <dig> proteins compared to dmeago <dig> .
ago <dig> is a single locus in ae.
aegypti, an.
gambiae, and d. melanogaster, but is present in two copies in cx.
pipiens .
fragments of two distinct mrnas have also been isolated from cx.
tritaeniorhyncus, suggesting that a gene duplication occurred early in the evolution of culex spp.
.
this first evidence of multiple ago <dig> loci in dipterans suggests that differential regulation of rnai anti-viral defense could occur in culex spp.
vectors.
although anti-viral rnai activity has not yet been reported for culex spp., this pathway has been established as an important facet of anti-viral defense in an.
gambiae and ae.
aegypti against alphaviruses and dengue viruses , respectively  <cit> .
piwi pathway gene expansions
a significant gene expansion has occurred in piwi subfamily proteins in both ae.
aegypti and cx.
pipiens   <cit> .
phylogenetic analyses of piwis used d. melanogaster aub and piwi as outgroups .
three well-supported clades are evident.
ago3s form a single clade with d. melanogaster.
in contrast, none of the mosquito piwis formed a clade with the drosophilid proteins, rather, two clades arise based on similarity to an.
gambiae ago <dig> or ago <dig>
the first clade has 100% support and contains the ago <dig> proteins.
ago3s can be readily distinguished from other argonaute family proteins by the amino acids surrounding the first conserved catalytic residue .
the average rate of change among these is high  <dig>  .
the ago <dig> proteins among the culicidae are monophyletic with  <dig> % support, and the average rate of change among these is also high  <dig>  .
the ago4-like clade has  <dig> % bootstrap support and contains agaago <dig>  cpipiwi <dig>  cpipiwi <dig>  cpipiwi <dig>  aaepiwi <dig>  aaepiwi <dig>  aaepiwi <dig> and aaepiwi <dig>  the amino acid substitution probability among these is low relative to the other  <dig> clades, .
members of the ago4-like group were distinguished by the synapomorphy "etgiqvlnlilrramnglnlqlvgrnly" within the first  <dig> amino acids .
this synapomorphy is maintained in dmepiwi, but not in dmeaub.
members of the ago5-like group have a variable sequence in the corresponding region.
agaago <dig> is basal to the ago5-like clade, which has  <dig> % bootstrap support and contains cpipiwi <dig>  cpipiwi <dig>  cpipiwi <dig>  aaepiwi <dig>  aaepiwi <dig> and aaepiwi <dig>  the amino acid substitution probability is high .
these analyses indicate that the expanded piwi protein subfamily comprises several different gene families with some resulting from recent gene duplication events .
expansion of argonaute family proteins has occurred in other organisms, as well.
one example is in c. elegans, in which a gene expansion has occurred, evidently to aid in sequential amplification of sirna signal.
a key difference between the c. elegans and mosquito argonaute family gene expansions is that the c. elegans secondary agos  lack the key metal binding residues required for rnase h catalytic activity and so are thought to play a secondary role in rnai  <cit> , whereas, these key catalytic residues are present in mosquito piwis .
in drosophilids, piwi pathway components have been implicated in the control of both retrotransposons and the long terminal repeats of telomeres  <cit> .
our analysis suggests the piwi subfamily gene expansion initially occurred in ancestral culicinae mosquitoes before the divergence of ae.
aegypti and cx.
pipiens ancestors  <cit> .
expansions in piwi pathway components may have been adaptive for controlling the increased burden of retrotransposons in ae.
aegypti and cx.
pipiens relative to an.
gambiae.
although retrotransposons are present in the an.
gambiae genome, the percentage of the genome bearing tes is much less than that of ae.
aegypti  <cit> .
about 47% of the ae.
aegypti genome harbors both class i and class ii transposable elements , whereas the an.
gambiae genome contains about 16% tes  <cit> .
tes are also present in culex spp.
genomes, however the percentage of the genome occupied has not yet been reported  <cit> .
retrotransposons, also known as class i tes, account for at least half of the te load in both anopheline and aedine genomes.
therefore, the aedine genome carries more than twice the retrotransposon load than anophelines carry.
in a related context, dmepiwi has also been implicated in maintenance of heterochromatin.
dmepiwi associates directly with chromatin and heterochromatin protein 1a   <cit> .
dmepiwi-hp1a interactions occur through conserved piwi protein motifs, pxvxv or pxvxm.
of the mosquito piwis, only cpipiwi4a, cpipiwi4b, aaepiwi <dig> and aaepiwi <dig> contain conserved pxvxv sites, and no proteins bear pxvxm motifs .
importantly, all four of these proteins are in the ago5-like protein class.
in contrast, neither agaago <dig> nor agaago <dig> carry these motifs.
accessory proteins
the rna helicases of drosophila and mosquitoes are clearly a large and complex family.
other than a genetic connection to anti-viral defense and retrotransposon maintenance in drosophilids, little is known about rm <dig> in dipterans.
using a high stringency search cut-off e value of e = 10- <dig>  multiple mosquito genes were identified that are homologous to dmerm <dig> rna helicase .
although reciprocal genome searches identified  <dig> homologous rna helicase genes in drosophila, only dmerm <dig> has been linked to rna interference  <cit> .
phylogenetic analysis demonstrated that there are independent orthologous groups of rm62-like proteins .
rm <dig> and armitage-like proteins carry predicted dead box atp-dependent helicase domains.
dmearmitage is a transcriptional repressor of specific mrnas during male germ-line development and has been shown in a genetic screen to be involved in anti-viral defense  <cit> .
interestingly, the armitage gene duplication in aedes and culex has resulted in two different types of multi-domain helicases .
although both aedine armitage-like paralogs carry a type iii dna restriction enzyme domain, as does dmearmitage, only one of the two culex paralogs carries this domain .
instead, the second paralog is missing the restriction enzyme domain and carries a predicted dna helicase domain  .
in contrast, agaarmitage has two rna helicase domains, in addition to the dna restriction enzyme domain.
this interesting diversity in protein domains among mosquito species suggests that, if armitage participates in anti-viral defense as in d. melanogaster, it may be pivotal to species-specific differences.
upstream control regions
we took a conservative approach to determine whether cis-acting elements are conserved among srrp component upstream regions.
our goals were two-fold: 1) to identify previously validated mosquito tfbss and 2) novel upstream regulatory control elements in regions corresponding to - <dig> nts to + <dig> nts.
this region corresponds to the upstream genomic sequence, the 5' un-translated region of the transcript, the translation start site and some coding nucleotides in the first exon.
consensus sequences for the upstream motifs are listed in table  <dig>  we determined whether elements in each of these categories are conserved within each pathway or across mosquito species.
iupac consensus codes used, wherein "k" is t or g; "y" is t or c; "w" is t or a; "r" is a or g; "s" is c or g; "m" is c or a; "h" is a, t, or c; "v" is a, c or g; "b" is c, t, or g; "d" is a, t, or g.
within the last  <dig> million years, an.
gambiae and ae.
aegypti became distinct species, and more recently, cx.
pipiens diverged.
one might expect that among-species comparisons would yield fewer common elements than within-species elements.
however, non-coding dna is subject to the same stabilizing selection as protein coding regions, and, in drosophilids, non-coding dna is actually less polymorphic than protein coding regions at synonymous sites  <cit> .
if purifying selection has occurred in ucrs of mosquito srrp genes, common elements could be conserved across species, thus adding support to the functional significance of upstream regulatory elements, regardless of whether they were unique across the entire genome.
furthermore, selective pressures on non-coding dna can be significant and therefore exacerbate the search for tfbss  <cit> .
therefore, to maintain a conservative line of inquiry, we chose to examine only those tfbss that have been experimentally confirmed in mosquitoes.
the search for novel motifs was expected to yield conserved cis-acting regulatory elements.
transcription factor binding sites: nf-kappa b related sites
there is empirical evidence that rna viruses activate innate immunity pathways in dipterans.
the toll and jak-stat  pathways are activated in drosophilids upon infection with drosophila c virus or drosophila x virus  <cit> .
a search for stat binding motifs  among all pathway component ucrs yielded no matches, thus suggesting that the jak-stat pathway does not activate transcription of effectors of mosquito mirna, sirna, or piwi pathways.
mosquito nf kappa b-related proteins, rel <dig> and rel <dig>  participate in transcriptional induction of immune effectors during bacterial and fungal infections .
rel <dig> stimulates downstream effectors via the toll pathway, while rel <dig> stimulates downstream effectors via the immunodeficiency  pathway  <cit> .
during sindbis  infection of ae.
aegypti, rel <dig> transcripts are enriched early in infection  <cit> .
identification of rel tfbss in ucrs of srrp genes would suggest a link between small regulatory rna pathways and the innate immune response cascade.
consensus nf kappa b-related binding motifs in ae.
aegypti and an.
gambiae are "ggkgatyyac"  and "kgggawhmmm" , respectively.
a cross-species consensus is "kgkgawhhmm"   <cit> .
these rel tfbs consensus motifs could be bound by either aaerel <dig> or aaerel <dig>  search of all mosquito srrp components for nfkb <dig> yielded hits on only two rm62-like ucrs, and nfkb <dig> revealed few hits across all species .
the cross-species  <dig> nt consensus, nfkb <dig>  revealed more hits, some of which are redundant to those identified by nfkb <dig> .
to identify putative motifs under more relaxed conditions, a shortened version of the nfkb <dig> consensus, "gggawhm" , was used.
in this case, the relaxed search was followed by a more rigorous requirement of ≥  <dig> motifs per ucr.
proportions of ucrs containing these motifs are listed in table  <dig>
"^", see additional file  <dig> for details.
"*", for nfkb <dig> only, significance scores were based on the presence of  <dig> or more hits per ucr.
"#", enriched in an sirna pathway specific manner.
"+", enriched in an mirna pathway specific manner.
"bh", benjamini-hochberg test, cut-off =  <dig> .
"fet", fisher's exact test, cutoff =  <dig> .
"na", not applicable.
"aae", ae.
aegypti, "aga", an.
gambiae, "cpi", cx.
pipiens.
schematics showing distribution of nfkb <dig> motifs are shown in figures  <dig>   <dig> and  <dig>  hits were detected in key component ucrs of all three mosquito species and srrps.
for example, aaedrosha, agadrosha, aaetsn, cpitsn and agatsn ucrs share nfkb <dig> sites, and all three species have nfkb <dig> sites upstream of piwi pathway component ucrs.
although there were no significant pathway-specific or species-specific differences in the presence of nf kappa b-related binding sites, the presence of these motifs in key ucrs suggests a possible link to the innate immune response or development.
the drosophilid ortholog of tudor staphylococcal nuclease  is a component of the risc and has multiple functional attributes, such as transcriptional co-activation activity, non-specific ssrna cleavage, cleavage of hyper-edited dsrna substrates, and an un-defined role in anti-viral defense  <cit> .
microarray analysis of ae.
aegypti transcripts during a sinv infection, showed early enrichment of aaerel <dig> transcripts at  <dig> day post-infection , followed by stimulation of aaetsn at  <dig> dpi  <cit> .
analysis of anti-viral rnai component transcript levels in ae.
aegypti infected with alphaviruses or flaviviruses showed periodic enrichment of tsn transcripts in a virus-dependent manner  <cit> .
importantly, tsn ucrs from all three species contained nfkb <dig> motifs .
this evidence further suggests that a link exists between the anti-viral pathway and the innate immune response pathway of mosquitoes.
broad complex motifs
broad complex  transcription factors act during ecdysone-dependent regulation of gene expression in invertebrates  <cit> .
ecdysone, a steroid hormone, is of key importance in embryogenesis and development in insects.
of the four types of brc tfbss, brc_z <dig> is the most abundant in mirna and pirna pathway component ucrs, however, the enrichment is not statistically significant .
in mosquitoes, brc_z <dig> is thought to be a transcriptional repressor that ensures proper temporal gene expression control for the yolk protein precursor gene, vitellogenin  <cit> .
the presence of a brc_z <dig> motif upstream of nearly all of the mirna pathway components and many piwi pathway components suggests the requirement for temporal control of these pathways, as well.
brc_z <dig> is required for 20-hydroxyecdysone-mediated transcriptional activation  <cit> .
brc_z <dig> motifs were identified on key genes of all three pathways, sometimes in tandem with brc_z <dig> motifs .
gata factors
gata factors control tissue- and temporally-specific gene expression.
gata binding sites are upstream of a variety of genes in mosquitoes, including lysosomal protease, gut trypsin, and vitellogenin genes  <cit> .
expression of gut trypsins and vitellogenin genes are tightly linked to bloodfeeding.
gata factors or gata repressors bind these sites to either activate or repress transcription.
multiple gata sites were identified in all ucrs, indicating no obvious species-specific or pathway-specific variation .
therefore, this element was not analyzed further.
novel elements
novel ucr elements could serve as genomic structural elements, mrna stability elements or tfbss.
we identified features in upstream non-coding regions that may have been selectively maintained over the course of evolution.
to accomplish this, we compared ucrs across mosquito species and across small rna regulatory pathways for all three mosquito species.
this focused approach allowed us to use a subset of each mosquito genome as background.
the presence of cis upstream elements across all three mosquito species for a single pathway would suggest that they may be important regulatory features of that pathway.
although some components, such as those of the risc, may act in multiple pathways; for our analysis, we categorized the groups according to the outline in figure  <dig>  the sequence motif search programs meme , mdscan, and weeder were used  <cit> .
these programs identify over-represented sequence patterns or motifs in a given dataset compared to all other datasets.
the top two elements discovered for each species and pathway were then identified among all ucrs, and significant differences among pathways or species were noted.
selected motifs are listed in table  <dig>  along with the proportion of ucrs with at least one species-specific or pathway-specific hit, and full descriptions are in additional file  <dig>  fisher's exact test was used to determine whether the ucr elements or tfbs motifs were enriched in a pathway-specific or species-specific manner.
the benjamini-hochberg  multiple testing adjustment was used to correct for false positives, thus, for any bh score over  <dig> , the fisher's exact test score was considered insignificant.
in addition, we performed full genome searches for all novel elements and removed those that did not show enrichment in srrp ucrs over the rest of the genome .
together, these methods allowed identification of cis-elements that could be important regulatory features of srrp pathways.
identification of species-specific cis elements could provide an important foundation for future characterization of species-specific differences in regulation of pathogen defense pathways.
by convention, strong regulatory sites are those represented in tandem repeats   <cit> .
due to the experimental design, elements that were identified in the species-specific search might also be enriched in a pathway-specific manner.
for example, the an.
gambiae elements m <dig> and m <dig> are enriched over the remainder of the genome in an sirna pathway-specific manner for both an.
gambiae and ae.
aegypti .
in addition, elements m <dig> and m <dig> are enriched in cx.
pipiens in a mirna pathway-specific manner over the remainder of the genome, even though fisher's exact test did not indicate the cx.
pipiens has significantly more of these elements than ae.
aegypti or an.
gambiae .
in contrast, the m <dig> and m <dig> elements of aedes aegypti are strongly enriched in all srrps over the remainder of the aedine genome .
m <dig>  and m <dig>  were enriched in a species-specific manner over all other srrp ucrs according to fisher's exact test , however, they were not enriched in srrp upstream regions compared to the remainder of the genome.
therefore, they are not likely to be important to regulation of small rna metabolism.
pathway-specific upstream elements were also identified.
of the five elements identified, m <dig> was the most interesting, because, it was enriched among the sirna pathway ucrs across all three species.
conservation of within-pathway features across species supports the hypothesis of purifying selection in ucrs of small rna regulatory components.
implications for vector competence
of the argonaute protein subfamily members, ago <dig>  the anti-viral rnase h-type nuclease, is significantly more diverse among mosquito species than the mirna pathway nuclease, ago <dig>  this finding suggests that anti-viral defense effectors are evolving at a faster rate than those involved in housekeeping functions.
anti-viral defense systems likely evolved to protect against entomopathogenic viruses.
in turn, some entomopathogenic viruses probably evolved into arboviruses.
when considering these pathways, it is important to keep in mind that evolutionary pressure exists on both the arbovirus and the vector mosquito to modulate the immune response.
differences in ucr element motif patterns suggest that there are likely to be species-specific differences in transcriptional regulation of sirna pathway components that could affect arbovirus vector competence.
loosely categorized, an.
gambiae primarily transmits malaria parasites, cx.
pipiens transmits encephalitic arboviruses, and ae.
aegypti transmits important hemorrhagic arboviruses.
for a given arthropod, low vector competence could arise from an effective immune response that clears the pathogen and prevents pathogen escape to the salivary glands.
with this model in mind, we hypothesize that an.
gambiae has a more effective antiviral immune response than either cx.
pipiens or ae.
aegypti.
characterization of these putative regulatory differences awaits further exploration in each species.
the demonstrated ability of some mosquito species to serve as competent arbovirus vectors in spite of the presence of anti-viral rnai components, begs the question of what mechanisms are used by arboviruses to evade anti-viral rnai defense.
this report brings to bear both the importance of piwi family gene expansion in vector mosquitoes and the need for research focus in this area.
a few exploratory studies have found evidence that piwi proteins could be important in anti-viral defense.
transient silencing of ago <dig> by dsrna injection increased o'nyong-nyong infection of an.
gambiae  <cit> .
however, characterization of piwi pathway activity in mosquito anti-viral defense is clearly required before additional conclusions can be drawn.
in drosophila, an apparent paradox is found, wherein piwi pathway proteins are reportedly expressed only in ovaries and are restricted to germline maintenance, however, piwi pathway components dmeaub, dmearmitage and dmerm <dig> have been implicated in anti-viral processes  <cit> .
recent reports describing the presence of endogenous sirnas  are beginning to shed light in this area  <cit> .
small rnas were found to control retrotransposons in somatic tissue in d. melanogaster in a dicer2/ago <dig> dependent manner.
CONCLUSIONS
this genomics study provides important contextual information for both vector biologists and the rnai community and highlights important differences between vector mosquitoes and model organisms, such as drosophila.
together, these data suggest a need to further investigate the relationship between vector competence and specific small rna pathways across mosquito species, as well as potential arbovirus strategies for evasion of these defense mechanisms.
however, it is still unclear what aspects of mosquito biology are driving evolution and gene expansion of argonaute family genes.

 small rna-mediated gene silencing pathways are master regulators of critical cellular processes, from development to germ-line surveillance to anti-viral defense. although small rna regulatory pathways operate using distinctly different mechanisms, they share several common features. small regulatory rnas of to nucleotides are used as guide strands for target substrate recognition by an rnase h type nuclease of the argonaute protein family. target rnas are subsequently degraded or otherwise prevented from being translated into protein. the three major pathways are the small interfering rna, microrna and piwi small rna pathways; the general functions of each are summarized in table due to the paucity of functional information from mosquitoes, we have relied on protein structure and functional information compiled from drosophila spp., caenorhabditis elegans, and mammals. the argonaute protein family contains the argonaute and piwi protein subclasses. all proteins in this family contain paz and piwi domains. the paz domain is a small rna binding domain; the piwi domain is an rnase-h type domain which relies on divalent cation binding to facilitate dsrna-guided cleavage of ssrna. argonaute and argonaute are in the argonaute protein subclass and are functionally distinct from piwis, in that they rely on dicer proteins to cleave long dsrnas into small rnas, which are then passed to the agos for further processing and recognition of target rnas. these proteins physically interact with dicers at the piwi domain and use small rnas formed from double-stranded templates. ago and ago each participate in different gene silencing pathways. mirna pathways of invertebrates have been best characterized in d. melanogaster and c. elegans. in d. melanogaster, developmental and housekeeping gene expression is regulated by ago and the small rna subclass, mirnas. in general, ago acts with dicer- micrornas and accessory proteins to control gene expression of housekeeping genes. in an. gambiae, the mirna pathway helps defend against plasmodium bergei infection. gene expression can be controlled by a variety of possible silencing mechanisms, including translational repression, decreased mrna stability or altered mrna localization. these small rnas are genome-encoded as primary transcript mirnas up to kilobases in length and processed into mirna precursor hairpin loops of about nts by drosha and pasha. dicer- cleaves pre-mirnas to form mature mirnas, which are transferred to ago for silencing of target mrnas. in mammals, some mirnas are encoded in the introns of the genes they target. the sirna pathway provides defense against rna viruses in the degradation of double-stranded viral rnas. sirnas, generated from longer dsrna molecules by the dicer-2/r2d complex, serve as guides for ago to identify and degrade target rnas. key proteins in the anti-viral rna interference pathway, including ago and dicer- are functional in vector mosquitoes and influence the ability of mosquitoes to serve as competent vectors of human virus pathogens. further, several recent reports of drosophila have shown that endogenous sirnas control retrotransposons and mrnas in somatic cells in an ago2/dicer- dependent manner. in drosophila melanogaster, the piwi protein subclass includes piwi, aubergine, and argonaute. these proteins contain the signature paz and piwi domains, but have remained enigmatic members of the argonaute family. they have been implicated in a variety of functions specific to germ-line tissue in d. melanogaster, including the suppression of retrotransposon mobilization, maintenance of telomeres, prevention of double-stranded dna breaks during meiosis, and mrna silencing in germ-line cells. ago aub, and piwi use piwi-associated rnas in these pathways. the mechanisms for production of this class of non-coding rnas are not well understood. models have been proposed, suggesting they are processed from pirna cluster transcripts and amplified by a "ping pong" amplification loop between these transcripts and those derived from active transposons. three mosquito species, anopheles gambiae, aedes aegypti, and culex pipiens quinquefasciatus are important vectors of human and animal pathogens. the goal of this study is to increase understanding of the three small rna pathways in vector mosquitoes using comparative genomics, with special emphasis on the key catalytic enzymes of the argonaute protein family. we performed phylogenetic analyses of argonaute family proteins and demonstrated expansion of ae. aegypti and cx. pipiens argonaute and piwi subfamilies. in addition, we determined whether cis-acting regulatory elements upstream of srrp genes are conserved across mosquito species or among components of specific rna regulatory pathways, as has been successfully demonstrated in other invertebrates. to this end, previously validated mosquito transcription factor binding sites and other elements in upstream control regions of all suspected srrp components were identified. further, we used motif discovery algorithms to identify novel upstream regulatory sequences. the identification of common cis-acting regulatory features lends insight into species-specific differences in regulation of srrps and provides a basis for future studies of pathogen defense in vector mosquitoes.  the an. gambiae, ae. aegypti, and cx. pipiens genomes encode components of the three major rna regulatory pathways previously identified in d. melanogaster. there are gene expansions in some categories; these are described below. additional file 1a depicts, in gray highlight, the genes for which expression has been confirmed, either experimentally or in est libraries. importantly, key catalytic residues of the rnase-h domains are conserved in all mosquito ago1s, ago2s and piwi subfamily proteins. although a few studies have been done in ae. aegypti and anopheline mosquitoes, experimental verification will be required to confirm the roles played by most of these putative orthologs in rna regulatory pathways. argonaute protein subfamily ago is represented at a single genomic locus in an. gambiae, cx. pipiens and d. melanogaster but seems to be present in two copies in ae. aegypti. the flanking sequences of about kilobases upstream and the introns were found to be unique. ago of the human head louse, pediculus humanus was used as an outgroup for the analysis of argonaute subfamily proteins. ago homologs from the silkworm, bombyx mori, and d. melanogaster were also included in the analysis. in addition, orthologs from the important pathogen vectors, lutzomyia longipalpis, culex tarsalis, and ochlerotatus triseriatus were included. based upon the jones-taylor-thornton probability model, the probability of amino acid substitutions among ago proteins was = * = %) while among ago proteins the average probability was. thus, the probability of amino acid substitutions among ago proteins is about 38-fold greater than among ago proteins. because of this high rate of change among ago proteins, there was only weak bootstrap support for the proteins from mosquitoes. however, among the two genera of the tribe aedini it is interesting that between ochlerotatus triseriatus and ae. aegypti the substitution probability is only, while within and among culex species the probability is. this pattern suggests that anti-viral pathway components are evolving more rapidly in culex spp. than among subgenera of aedine mosquitoes. an interesting parallel was found among drosophilid species, wherein key anti-viral rnai component genes, such as dmeago and dmedicer- were found to be evolving at a higher rate than mirna pathway components or other immune response genes. synapomorphies that distinguish ago1s from ago2s can be found in the amino acids surrounding the first catalytic residue. ago protein conservation indicates an evolutionary trend of purifying selection, probably due to conserved mechanisms in developmental pathways. this hypothesis is supported in the conservation of some mirnas between drosophilids, mosquitoes, and humans. although many mosquito mirnas have not been experimentally verified, their presence suggests conservation of developmentally regulated gene silencing pathways. alignment of ago proteins further illustrates the degree of conservation with overall amino acid identity of to 89% among all mosquito orthologs and that of d. melanogaster. this is in contrast to the to 43% amino acid identity among all mosquito ago proteins compared to dmeago. ago is a single locus in ae. aegypti, an. gambiae, and d. melanogaster, but is present in two copies in cx. pipiens. fragments of two distinct mrnas have also been isolated from cx. tritaeniorhyncus, suggesting that a gene duplication occurred early in the evolution of culex spp.. this first evidence of multiple ago loci in dipterans suggests that differential regulation of rnai anti-viral defense could occur in culex spp. vectors. although anti-viral rnai activity has not yet been reported for culex spp., this pathway has been established as an important facet of anti-viral defense in an. gambiae and ae. aegypti against alphaviruses and dengue viruses, respectively. piwi pathway gene expansions a significant gene expansion has occurred in piwi subfamily proteins in both ae. aegypti and cx. pipiens. phylogenetic analyses of piwis used d. melanogaster aub and piwi as outgroups. three well-supported clades are evident. ago3s form a single clade with d. melanogaster. in contrast, none of the mosquito piwis formed a clade with the drosophilid proteins, rather, two clades arise based on similarity to an. gambiae ago or ago the first clade has 100% support and contains the ago proteins. ago3s can be readily distinguished from other argonaute family proteins by the amino acids surrounding the first conserved catalytic residue. the average rate of change among these is high. the ago proteins among the culicidae are monophyletic with % support, and the average rate of change among these is also high. the ago4-like clade has % bootstrap support and contains agaago cpipiwi cpipiwi cpipiwi aaepiwi aaepiwi aaepiwi and aaepiwi the amino acid substitution probability among these is low relative to the other clades,. members of the ago4-like group were distinguished by the synapomorphy "etgiqvlnlilrramnglnlqlvgrnly" within the first amino acids. this synapomorphy is maintained in dmepiwi, but not in dmeaub. members of the ago5-like group have a variable sequence in the corresponding region. agaago is basal to the ago5-like clade, which has % bootstrap support and contains cpipiwi cpipiwi cpipiwi aaepiwi aaepiwi and aaepiwi the amino acid substitution probability is high. these analyses indicate that the expanded piwi protein subfamily comprises several different gene families with some resulting from recent gene duplication events. expansion of argonaute family proteins has occurred in other organisms, as well. one example is in c. elegans, in which a gene expansion has occurred, evidently to aid in sequential amplification of sirna signal. a key difference between the c. elegans and mosquito argonaute family gene expansions is that the c. elegans secondary agos lack the key metal binding residues required for rnase h catalytic activity and so are thought to play a secondary role in rnai, whereas, these key catalytic residues are present in mosquito piwis. in drosophilids, piwi pathway components have been implicated in the control of both retrotransposons and the long terminal repeats of telomeres. our analysis suggests the piwi subfamily gene expansion initially occurred in ancestral culicinae mosquitoes before the divergence of ae. aegypti and cx. pipiens ancestors. expansions in piwi pathway components may have been adaptive for controlling the increased burden of retrotransposons in ae. aegypti and cx. pipiens relative to an. gambiae. although retrotransposons are present in the an. gambiae genome, the percentage of the genome bearing tes is much less than that of ae. aegypti. about 47% of the ae. aegypti genome harbors both class i and class ii transposable elements, whereas the an. gambiae genome contains about 16% tes. tes are also present in culex spp. genomes, however the percentage of the genome occupied has not yet been reported. retrotransposons, also known as class i tes, account for at least half of the te load in both anopheline and aedine genomes. therefore, the aedine genome carries more than twice the retrotransposon load than anophelines carry. in a related context, dmepiwi has also been implicated in maintenance of heterochromatin. dmepiwi associates directly with chromatin and heterochromatin protein 1a. dmepiwi-hp1a interactions occur through conserved piwi protein motifs, pxvxv or pxvxm. of the mosquito piwis, only cpipiwi4a, cpipiwi4b, aaepiwi and aaepiwi contain conserved pxvxv sites, and no proteins bear pxvxm motifs. importantly, all four of these proteins are in the ago5-like protein class. in contrast, neither agaago nor agaago carry these motifs. accessory proteins the rna helicases of drosophila and mosquitoes are clearly a large and complex family. other than a genetic connection to anti-viral defense and retrotransposon maintenance in drosophilids, little is known about rm in dipterans. using a high stringency search cut-off e value of e = 10- multiple mosquito genes were identified that are homologous to dmerm rna helicase. although reciprocal genome searches identified homologous rna helicase genes in drosophila, only dmerm has been linked to rna interference. phylogenetic analysis demonstrated that there are independent orthologous groups of rm62-like proteins. rm and armitage-like proteins carry predicted dead box atp-dependent helicase domains. dmearmitage is a transcriptional repressor of specific mrnas during male germ-line development and has been shown in a genetic screen to be involved in anti-viral defense. interestingly, the armitage gene duplication in aedes and culex has resulted in two different types of multi-domain helicases. although both aedine armitage-like paralogs carry a type iii dna restriction enzyme domain, as does dmearmitage, only one of the two culex paralogs carries this domain. instead, the second paralog is missing the restriction enzyme domain and carries a predicted dna helicase domain. in contrast, agaarmitage has two rna helicase domains, in addition to the dna restriction enzyme domain. this interesting diversity in protein domains among mosquito species suggests that, if armitage participates in anti-viral defense as in d. melanogaster, it may be pivotal to species-specific differences. upstream control regions we took a conservative approach to determine whether cis-acting elements are conserved among srrp component upstream regions. our goals were two-fold: 1) to identify previously validated mosquito tfbss and 2) novel upstream regulatory control elements in regions corresponding to - nts to + nts. this region corresponds to the upstream genomic sequence, the 5' un-translated region of the transcript, the translation start site and some coding nucleotides in the first exon. consensus sequences for the upstream motifs are listed in table we determined whether elements in each of these categories are conserved within each pathway or across mosquito species. iupac consensus codes used, wherein "k" is t or g; "y" is t or c; "w" is t or a; "r" is a or g; "s" is c or g; "m" is c or a; "h" is a, t, or c; "v" is a, c or g; "b" is c, t, or g; "d" is a, t, or g. within the last million years, an. gambiae and ae. aegypti became distinct species, and more recently, cx. pipiens diverged. one might expect that among-species comparisons would yield fewer common elements than within-species elements. however, non-coding dna is subject to the same stabilizing selection as protein coding regions, and, in drosophilids, non-coding dna is actually less polymorphic than protein coding regions at synonymous sites. if purifying selection has occurred in ucrs of mosquito srrp genes, common elements could be conserved across species, thus adding support to the functional significance of upstream regulatory elements, regardless of whether they were unique across the entire genome. furthermore, selective pressures on non-coding dna can be significant and therefore exacerbate the search for tfbss. therefore, to maintain a conservative line of inquiry, we chose to examine only those tfbss that have been experimentally confirmed in mosquitoes. the search for novel motifs was expected to yield conserved cis-acting regulatory elements. transcription factor binding sites: nf-kappa b related sites there is empirical evidence that rna viruses activate innate immunity pathways in dipterans. the toll and jak-stat pathways are activated in drosophilids upon infection with drosophila c virus or drosophila x virus. a search for stat binding motifs among all pathway component ucrs yielded no matches, thus suggesting that the jak-stat pathway does not activate transcription of effectors of mosquito mirna, sirna, or piwi pathways. mosquito nf kappa b-related proteins, rel and rel participate in transcriptional induction of immune effectors during bacterial and fungal infections. rel stimulates downstream effectors via the toll pathway, while rel stimulates downstream effectors via the immunodeficiency pathway. during sindbis infection of ae. aegypti, rel transcripts are enriched early in infection. identification of rel tfbss in ucrs of srrp genes would suggest a link between small regulatory rna pathways and the innate immune response cascade. consensus nf kappa b-related binding motifs in ae. aegypti and an. gambiae are "ggkgatyyac" and "kgggawhmmm", respectively. a cross-species consensus is "kgkgawhhmm". these rel tfbs consensus motifs could be bound by either aaerel or aaerel search of all mosquito srrp components for nfkb yielded hits on only two rm62-like ucrs, and nfkb revealed few hits across all species. the cross-species nt consensus, nfkb revealed more hits, some of which are redundant to those identified by nfkb. to identify putative motifs under more relaxed conditions, a shortened version of the nfkb consensus, "gggawhm", was used. in this case, the relaxed search was followed by a more rigorous requirement of ≥ motifs per ucr. proportions of ucrs containing these motifs are listed in table "^", see additional file for details. "*", for nfkb only, significance scores were based on the presence of or more hits per ucr. "#", enriched in an sirna pathway specific manner. "+", enriched in an mirna pathway specific manner. "bh", benjamini-hochberg test, cut-off =. "fet", fisher's exact test, cutoff =. "na", not applicable. "aae", ae. aegypti, "aga", an. gambiae, "cpi", cx. pipiens. schematics showing distribution of nfkb motifs are shown in figures and hits were detected in key component ucrs of all three mosquito species and srrps. for example, aaedrosha, agadrosha, aaetsn, cpitsn and agatsn ucrs share nfkb sites, and all three species have nfkb sites upstream of piwi pathway component ucrs. although there were no significant pathway-specific or species-specific differences in the presence of nf kappa b-related binding sites, the presence of these motifs in key ucrs suggests a possible link to the innate immune response or development. the drosophilid ortholog of tudor staphylococcal nuclease is a component of the risc and has multiple functional attributes, such as transcriptional co-activation activity, non-specific ssrna cleavage, cleavage of hyper-edited dsrna substrates, and an un-defined role in anti-viral defense. microarray analysis of ae. aegypti transcripts during a sinv infection, showed early enrichment of aaerel transcripts at day post-infection, followed by stimulation of aaetsn at dpi. analysis of anti-viral rnai component transcript levels in ae. aegypti infected with alphaviruses or flaviviruses showed periodic enrichment of tsn transcripts in a virus-dependent manner. importantly, tsn ucrs from all three species contained nfkb motifs. this evidence further suggests that a link exists between the anti-viral pathway and the innate immune response pathway of mosquitoes. broad complex motifs broad complex transcription factors act during ecdysone-dependent regulation of gene expression in invertebrates. ecdysone, a steroid hormone, is of key importance in embryogenesis and development in insects. of the four types of brc tfbss, brc_z is the most abundant in mirna and pirna pathway component ucrs, however, the enrichment is not statistically significant. in mosquitoes, brc_z is thought to be a transcriptional repressor that ensures proper temporal gene expression control for the yolk protein precursor gene, vitellogenin. the presence of a brc_z motif upstream of nearly all of the mirna pathway components and many piwi pathway components suggests the requirement for temporal control of these pathways, as well. brc_z is required for 20-hydroxyecdysone-mediated transcriptional activation. brc_z motifs were identified on key genes of all three pathways, sometimes in tandem with brc_z motifs. gata factors gata factors control tissue- and temporally-specific gene expression. gata binding sites are upstream of a variety of genes in mosquitoes, including lysosomal protease, gut trypsin, and vitellogenin genes. expression of gut trypsins and vitellogenin genes are tightly linked to bloodfeeding. gata factors or gata repressors bind these sites to either activate or repress transcription. multiple gata sites were identified in all ucrs, indicating no obvious species-specific or pathway-specific variation. therefore, this element was not analyzed further. novel elements novel ucr elements could serve as genomic structural elements, mrna stability elements or tfbss. we identified features in upstream non-coding regions that may have been selectively maintained over the course of evolution. to accomplish this, we compared ucrs across mosquito species and across small rna regulatory pathways for all three mosquito species. this focused approach allowed us to use a subset of each mosquito genome as background. the presence of cis upstream elements across all three mosquito species for a single pathway would suggest that they may be important regulatory features of that pathway. although some components, such as those of the risc, may act in multiple pathways; for our analysis, we categorized the groups according to the outline in figure the sequence motif search programs meme, mdscan, and weeder were used. these programs identify over-represented sequence patterns or motifs in a given dataset compared to all other datasets. the top two elements discovered for each species and pathway were then identified among all ucrs, and significant differences among pathways or species were noted. selected motifs are listed in table along with the proportion of ucrs with at least one species-specific or pathway-specific hit, and full descriptions are in additional file fisher's exact test was used to determine whether the ucr elements or tfbs motifs were enriched in a pathway-specific or species-specific manner. the benjamini-hochberg multiple testing adjustment was used to correct for false positives, thus, for any bh score over, the fisher's exact test score was considered insignificant. in addition, we performed full genome searches for all novel elements and removed those that did not show enrichment in srrp ucrs over the rest of the genome. together, these methods allowed identification of cis-elements that could be important regulatory features of srrp pathways. identification of species-specific cis elements could provide an important foundation for future characterization of species-specific differences in regulation of pathogen defense pathways. by convention, strong regulatory sites are those represented in tandem repeats. due to the experimental design, elements that were identified in the species-specific search might also be enriched in a pathway-specific manner. for example, the an. gambiae elements m and m are enriched over the remainder of the genome in an sirna pathway-specific manner for both an. gambiae and ae. aegypti. in addition, elements m and m are enriched in cx. pipiens in a mirna pathway-specific manner over the remainder of the genome, even though fisher's exact test did not indicate the cx. pipiens has significantly more of these elements than ae. aegypti or an. gambiae. in contrast, the m and m elements of aedes aegypti are strongly enriched in all srrps over the remainder of the aedine genome. m and m were enriched in a species-specific manner over all other srrp ucrs according to fisher's exact test, however, they were not enriched in srrp upstream regions compared to the remainder of the genome. therefore, they are not likely to be important to regulation of small rna metabolism. pathway-specific upstream elements were also identified. of the five elements identified, m was the most interesting, because, it was enriched among the sirna pathway ucrs across all three species. conservation of within-pathway features across species supports the hypothesis of purifying selection in ucrs of small rna regulatory components. implications for vector competence of the argonaute protein subfamily members, ago the anti-viral rnase h-type nuclease, is significantly more diverse among mosquito species than the mirna pathway nuclease, ago this finding suggests that anti-viral defense effectors are evolving at a faster rate than those involved in housekeeping functions. anti-viral defense systems likely evolved to protect against entomopathogenic viruses. in turn, some entomopathogenic viruses probably evolved into arboviruses. when considering these pathways, it is important to keep in mind that evolutionary pressure exists on both the arbovirus and the vector mosquito to modulate the immune response. differences in ucr element motif patterns suggest that there are likely to be species-specific differences in transcriptional regulation of sirna pathway components that could affect arbovirus vector competence. loosely categorized, an. gambiae primarily transmits malaria parasites, cx. pipiens transmits encephalitic arboviruses, and ae. aegypti transmits important hemorrhagic arboviruses. for a given arthropod, low vector competence could arise from an effective immune response that clears the pathogen and prevents pathogen escape to the salivary glands. with this model in mind, we hypothesize that an. gambiae has a more effective antiviral immune response than either cx. pipiens or ae. aegypti. characterization of these putative regulatory differences awaits further exploration in each species. the demonstrated ability of some mosquito species to serve as competent arbovirus vectors in spite of the presence of anti-viral rnai components, begs the question of what mechanisms are used by arboviruses to evade anti-viral rnai defense. this report brings to bear both the importance of piwi family gene expansion in vector mosquitoes and the need for research focus in this area. a few exploratory studies have found evidence that piwi proteins could be important in anti-viral defense. transient silencing of ago by dsrna injection increased o'nyong-nyong infection of an. gambiae. however, characterization of piwi pathway activity in mosquito anti-viral defense is clearly required before additional conclusions can be drawn. in drosophila, an apparent paradox is found, wherein piwi pathway proteins are reportedly expressed only in ovaries and are restricted to germline maintenance, however, piwi pathway components dmeaub, dmearmitage and dmerm have been implicated in anti-viral processes. recent reports describing the presence of endogenous sirnas are beginning to shed light in this area. small rnas were found to control retrotransposons in somatic tissue in d. melanogaster in a dicer2/ago dependent manner.  this genomics study provides important contextual information for both vector biologists and the rnai community and highlights important differences between vector mosquitoes and model organisms, such as drosophila. together, these data suggest a need to further investigate the relationship between vector competence and specific small rna pathways across mosquito species, as well as potential arbovirus strategies for evasion of these defense mechanisms. however, it is still unclear what aspects of mosquito biology are driving evolution and gene expansion of argonaute family genes. 
to do this, we compared the genomic structure of srrp components across three mosquito species and three major small rna pathways. in addition, sequence pattern recognition algorithms meme, mdscan and weeder were used to identify upstream regulatory motifs for all srrp components. common ucr elements among srrp components indicate that 1) key components from the mirna, sirna, and pirna pathways contain nf-kappab-related and broad complex transcription factor binding sites, 2) purifying selection has occurred to maintain common pathway-specific elements across mosquito species and 3) species-specific differences in upstream elements suggest that there may be differences in regulatory control among mosquito species. analysis of argonaute and piwi subfamily genes suggests that the small regulatory rna pathways of the major arbovirus vectors, ae. phylogenetic analyses were performed for these protein families. further, protein and genomic features suggest functional differences between subclasses of piwi proteins and provide a basis for future analyses. members of the argonaute family of catalytic enzymes degrade target rnas in each of these pathways. small rna regulatory pathways control key aspects of development and anti-viral defense in metazoans. increased knowledge of these pathways and functional components should increase understanding of the limitations of anti-viral defense in vector mosquitoes. implications for arbovirus vector competence in mosquitoes are discussed.
 small rna regulatory pathways control key aspects of development and anti-viral defense in metazoans. members of the argonaute family of catalytic enzymes degrade target rnas in each of these pathways. srrps include the microrna, small interfering rna and piwi-type gene silencing pathways. mosquitoes generate viral sirnas when infected with rna arboviruses. however, in some mosquitoes, arboviruses survive antiviral rna interference and are transmitted via mosquito bite to a subsequent host. increased knowledge of these pathways and functional components should increase understanding of the limitations of anti-viral defense in vector mosquitoes. to do this, we compared the genomic structure of srrp components across three mosquito species and three major small rna pathways.  the ae. aegypti, an. gambiae and cx. pipiens genomes encode putative orthologs for all major components of the mirna, sirna, and pirna pathways. ae. aegypti and cx. pipiens have undergone expansion of argonaute and piwi subfamily genes. phylogenetic analyses were performed for these protein families. in addition, sequence pattern recognition algorithms meme, mdscan and weeder were used to identify upstream regulatory motifs for all srrp components. statistical analyses confirmed enrichment of species-specific and pathway-specific cis-elements over the rest of the genome.  analysis of argonaute and piwi subfamily genes suggests that the small regulatory rna pathways of the major arbovirus vectors, ae. aegypti and cx. pipiens, are evolving faster than those of the malaria vector an. gambiae and d. melanogaster. further, protein and genomic features suggest functional differences between subclasses of piwi proteins and provide a basis for future analyses. common ucr elements among srrp components indicate that 1) key components from the mirna, sirna, and pirna pathways contain nf-kappab-related and broad complex transcription factor binding sites, 2) purifying selection has occurred to maintain common pathway-specific elements across mosquito species and 3) species-specific differences in upstream elements suggest that there may be differences in regulatory control among mosquito species. implications for arbovirus vector competence in mosquitoes are discussed. 