BACKGROUND
the genomes of eukaryotes typically comprise a high percentage of duplicated genes.
it has been proposed that gene duplication is the major mechanism for the origin of new gene function and that the refashioning of duplicate genes is a major contributor to the origin of adaptive evolutionary novelties  <cit> .
gene duplication may originate from unequal crossing over, retro-transposition, segmental duplication and chromosomal or genome duplication  <cit> .
force et al.
<cit>  proposed several theories to explain the retention of duplicate gene copies in the genome wherein the fate of each copy may depend on mutations that occur in both coding and regulatory regions.
the accumulation of mutations in one copy may eventually render the gene non-functional .
degenerative mutations may accumulate in the regulatory regions of gene copies that are separately capable of performing a distinct ancestral function  or one copy may retain the original function with the second copy acquiring a new function by the retention of beneficial mutations .
opsins initiate the photon-induced signaling cascade in vertebrates and invertebrates, and are members of the g protein-coupled receptor  family, characterized by seven trans-membrane domains .
insects typically possess three classes of visual opsins that are sensitive to ultraviolet , short  and long  wavelengths.
additionally, some insects have red  sensitive receptors  <cit> .
visual opsins are expressed in the rhabdomere of the ommatidia, the major structural unit of the arthropod compound eye.
non visual opsins have also been identified and include the apis mellifera  pteropsin identified in the bee brain, suggesting a possible function in extra-retinal detection of light and the regulation of circadian rhythm .
the functions of the drosophila melanogaster  opsin rh <dig>  <cit>  and the rgr-like and arthropsins identified in daphnia pulex  have not been determined.
mosquitoes  are one of the most important arthropod groups affecting human and animal health  <cit> .
the assembled genomes of the yellow fever mosquito aedes aegypti, the malaria mosquito anopheles gambiae and the southern house mosquito culex quinquefasciatus  provide an opportunity to investigate the molecular evolution of opsin genes in three mosquito taxa representing the lineages culicinae  and anophelinae .
it is predicted that the culicinae and anophelinae diverged approximately 145– <dig> million years ago   <cit>  or  <dig> my  <cit> .
species comprising these lineages transmit a variety of parasites and pathogens of medical and veterinary significance  <cit>  and exhibit markedly different behavioral periodicities.
aedes aegypti is a diurnal mosquito, while an.
gambiae and cx.
quinquefasciatus exhibit nocturnal and crepuscular behaviors  <cit> .
expansions in the genes coding for lw opsins have been noted in a number of invertebrates, including insects.
the fruitfly, d. melanogaster possesses three lw opsin genes  <cit>  and the butterflies papillio xuthus and papillio glaucus have three and four genes, respectively  <cit> .
examples of duplication in lw opsin genes have been observed in the aquatic crustacean, daphnia pulex, which has  <dig> lw opsin genes  <cit> , the stomatopods, gonodactylus smithii, neogonodactylus oerstedii, odontodactylus scyllarus, coronis scolopendra and squilla empusa, which each possess  <dig> lw opsin genes  <cit> , and the dragonflies anax parthenope, anotogaster sieboldii, asiagomphus melaenops, epiophlebia superstes, indolestes peregrinus, ischnura asiatica, macromia amphigena, mnais costalis, orthetrum albistylum, somatochlora uchidai, sympetrum frequens and tanypteryx pryeri which have 8– <dig> lw opsin genes  <cit> .
it has been proposed that the opsin expansion observed in daphnia and other aquatic lineages may be influenced by more complex light regimes associated with aquatic environments  <cit> .
the study of futahashi et al.
<cit>  also suggests an association of lw opsins with aquatic habitat.
previously, we identified  <dig> and  <dig> putative opsins in ae.
aegypti  <cit>  and an.
gambiae  <cit> , respectively, and an expansion of putative lw opsin genes in both species which possess six gene copies as compared to other insects that typically have between one to four lw opsins.
five an.
gambiae lw opsins  are tandemly arrayed within a 90 kb region on chromosome 2r and are separated from a sixth, and presumably more ancestral lw opsin , by approximately 3 mb  <cit> .
the density of genes and segmental duplications as well as the high gc content of 2r  is suggestive of a high recombination rate, a phenomenon typically associated with gene duplication  <cit> .
more recently,  <dig> opsin genes were found in the cx.
quinquefasciatus genome, although their wavelength sensitivity was not predicted  <cit> .
interest in novel strategies to control mosquitoes is high due to the emergence and re-emergence of arboviral diseases such as dengue  <cit> , yellow fever  <cit> , chikungunya and zika  <cit>  and the failure of traditional drug and insecticide control.
opsin-mediated processes could be targeted to disrupt mosquito mating, host finding and oviposition.
an improved understanding of these processes could also benefit the design of new mosquito traps, deterrent devices  and genetic control strategies.
here, we present detailed molecular evolutionary analyses of the lw opsins in ae.
aegypti, an.
gambiae and cx.
quinquefasciatus as a first step toward understanding the molecular basis of opsin-mediated visual processes that could be exploited for mosquito control.
phylogenetic analyses suggest that culicine and anopheline mosquitoes possess orthologs of invertebrate lw, sw and uv–sensitive opsins, extra-retinal pteropsins, and the d. melanogaster rh <dig>  we predicted six, six and eight putative lw opsins in ae.
aegypti, an.
gambiae, cx.
quinquefasciatus, respectively, indicative of an expansion of these genes in the culicidae relative to other insects.
molecular evolutionary analyses suggest that mosquito lw opsins originated from  <dig> or  <dig> duplication events that occurred between  <dig>  to < <dig> million years ago .
the genes for ten lw opsins  may have been retained through a combination of positive selection and coordinated regulation .
of the remaining mosquito opsin genes, five may have been retained through positive selection , and one via coordinated regulation .
four lw opsins lack evidence of either positive selection or coordinated regulation .
RESULTS
genes for  <dig>   <dig> and  <dig> opsins were manually annotated in the genomes of ae.
aegypti, an.
gambiae and cx.
quinquefasciatus, respectively and results are summarized in table  <dig>  opsin protein sequences are provided in the additional file  <dig> and are also available from vectorbase and genbank.
the protein sequences for all opsins  contain a predicted initiation methionine, a stop codon, three extracellular  and three intracellular loops , and seven tm domains.
excluding the incomplete models above, the mosquito opsins vary in length from  <dig> to  <dig> amino acids in ae.
aegypti , 370– <dig> in an.
gambiae , and 368– <dig> amino acids in cx.
quinquefasciatus .
we detected a single amino acid substitution between aagprop <dig>  and op <dig> .
of note, aggprop <dig> first reported in hill et al.
<cit>  may reflect an erroneous prediction based on an earlier version of the an.
gambiae assembly.
aagprop9_ <dig> is a truncated gene model identical to the first exon of aagprop9_ <dig> from nucleotides  <dig> to  <dig>  except for a non-synonymous c/a substitution at nucleotide  <dig>  and a synonymous t/c substitution at nucleotide  <dig>  we were unable to identify the second exon of aagprop9_ <dig>  the third intracellular loop  which is likely important for g protein interaction, is  <dig> amino acids in length in all mosquito opsins except for cqgprop <dig> and the presumably more ancestral opsins aagprop <dig>  aggprop <dig> and cqgprop <dig> where the loop is predicted to be  <dig>   <dig>   <dig> and  <dig> amino acids in length, respectively.
genea
ae.
aegypti
aagprop1
aael006498
aagprop2
aael006259
aagprop3
aael006484
aagprop4
aael005621
aagprop5
aael005625
aagprop7
aael007389
aagprop8
aael009615
aagprop9_1
aael003035
aagprop9_2
c
aael003035
aagprop10
aael005322
aagprop12
aael005373
an.
gambiae
aggprop1
agap013149
aggprop3
agap012985
aggprop4
agap012982
aggprop5
agap001162
aggprop6
agap001161
aggprop7
agap002462
aggprop8
agap006126
aggprop9
agap010089
aggprop10
agap007548
aggprop11
agap002443
aggprop12
agap002444
cx.
quinquefasciatus
cqgprop1
cpij004067
cqgprop2
cpij005000
cqgprop3
cpij009246
cqgprop4
cpij011419
cqgprop5
cpij012052
cqgprop6
cpij011571
cqgprop7
cpij011573
cqgprop8
cpij011574
cqgprop9
cpij011576
cqgprop10
c
cpij013056
cqgprop11
cpij013408
cqgprop12
cpij014334
cqgprop13
cpij020021
aannotations from hill et al.
<cit> , nene et al.
<cit>  and arensburger et al.
<cit> .
revised opsin annotations were produced using the aaegl <dig> , agamp <dig>  and cpip <dig>  gene sets from vectorbase; bexpression evidence for ae.
aegypti, an.
gambiae and cx.
quinquefasciatus obtained from pcr or rt-pcr data , ests , microarrays , rnaseq  <cit>  and mass spectrometry ; p, peptide evidence; t, transcript evidence; -, absence of transcript or peptide evidence; cindicates incomplete gene model; n.a.
not available
conservation was observed in the architecture between mosquito opsins.
the lw opsins have no or one intron, sw opsins have one intron , uv opsins have four introns , pteropsin-like genes have four introns, and orthologs of the d. melanogaster rh <dig> posses two introns  .
the ae.
aegypti lw opsin introns are approximately 5– <dig> times longer than those of an.
gambiae and cx.
quinquefasciatus , consistent with the gene architecture observed in this species  <cit> .
the putative coding sequences of the mosquito opsins were aligned to the rhodopsin from the squid todarodes pacificus  to identify amino acids and structural features, including those associated with functions in other organisms.
we identified  <dig> and  <dig> residues conserved among mosquito lw opsins and class a rhodopsin-like gpcrs and opsin visual pigments, respectively .
amino acids important for the interaction between the bovine rhodopsin and g-proteins are conserved in the mosquito opsins.
three classes of post-translational modifications characteristic of opsins  were predicted for the mosquito opsins based on comparison to the crystallized bovine rhodopsin and the squid rhodopsin  <cit> .
these include sites in the n-terminus  that undergo n-glycosylation during biosynthesis  <cit> , sites in the c-terminus  that undergo palmitoylation, the role of which in opsins remains undefined  <cit> , and several s and t residues located in the c-termini of the bovine and squid rhodopsins that are the potential targets for phosphorylation by rhodopsin kinase  <cit> .
the tmds of the mosquito opsins were predicted using a hidden markov model  using the tmhmm server v.  <dig>   <cit>  and aligned with muscle.
the tm domains of aagprop1- <dig> and  <dig>  aggprop <dig> and 3– <dig> and cqgprop <dig>  5– <dig> and  <dig> and  <dig>  share a minimum 62%,  <dig> and 68% amino acid identity , respectively.fig.
<dig> amino acid alignment of the predicted trans-membrane domains  of ae.
aegypti putative lw opsins.
highlighted amino acids show positions conserved in lw opsins.
black shading, identical residues; gray shading, similar residues, based on the similarity matrix blosum <dig>  the asterisk  indicates the residue identified by fitmodel as under positive selection
detection of mosquito opsin transcripts
published est studies support the production of transcripts for aagprop1- <dig>  8-9_1; aggprop1- <dig>  8– <dig> and cqgprop <dig>  5– <dig> and  <dig> .
we reviewed published meta-analyses of whole body or individual organs to extract data regarding opsin temporal and spatial expression using the vectorbase expression tool  <cit>  .
microarray data support the production of transcripts for all ae.
aegypti and an.
gambiae opsins and cqgprop <dig>   <dig> and  <dig> .
pcr and rt-pcr studies support transcripts for all ae.
aegypti opsin genes  and all an.
gambiae opsin genes  , and rnaseq experiments support the production of transcripts for all ae.
aegypti and an.
gambiae opsins  <cit> .
peptide expression evidence was supported with mass spectrometry  for all an.
gambiae opsin proteins  .
collectively, these public data provide evidence for expression of  <dig> of the  <dig> mosquito opsins.
support for the production of mosquito transcripts was extended in the present study using rt-pcr analyses and includes transcripts corresponding to  <dig> ae.
aegypti and  <dig> an.
gambiae opsin genes.
transcripts were detected for all ae.
aegypti opsin genes by rt-pcr, except for aagprop <dig> in male and female adults and aagprop <dig> and  <dig> in larvae and pupae .
transcripts corresponding to opsin genes were not detected in ae.
aegypti embryos.
all pcr amplicons were sequenced to confirm coding regions.
transcripts for all an.
gambiae opsin genes  were detected by rt-pcr in male and female adults .
localization of mosquito opsin genes to chromosomes
genes for the an.
gambiae lw opsins  are co-localized on the distal portion of chromosome 2r, a region of the genome associated with multiple duplicated genes.
five genes  are located within a 90 kb region, and are separated from the sixth, aggprop <dig> by 3 mb.
localization of ae.
aegypti or cx.
quinquefasciatus opsins to chromosomes and syteny analyses were not possible due to the fragmented genome assemblies and the lack of more complete sequence and physical maps for these species.
phylogenetic analyses of mosquito opsins
two initial phylogenetic analyses were performed and employed a large survey of opsin protein  and nucleotide  sequences from diverse animals  and the trees were consistent with previous phylogenetic investigations of animal opsins  <cit> .
consistent with the phylogenies of feuda et al.
<cit> , the mosquito opsins were placed in one of five clades, namely the lw, sw, uv, rh7-like and pteropsin clades.
mosquitoes possess one or two putative genes for each of the uv, sw, rh7-like and pteropsin functional groups.
we identified six putative lw opsins in ae.
aegypti, six in an.
gambiae and eight in cx.
quinquefasciatus, and observed that the mosquito lw clades comprise an increased number of duplicate opsin gene lineages relative to opsin clades from insects.
to further investigate the retention of lw opsin genes in mosquitoes, phylogenetic analyses were conducted with  <dig> lw sensitive opsins from eight insect species .
the maximum likelihood tree  reflects the currently accepted hypothesis of insect phylogeny in which the hymenoptera were thought to derive from an early branch of the holometabolous insects  <cit> .
the r8s and beast results  are consistent with the estimated divergence dates  reported by misof et al.
<cit> , suggesting that the hemimetabolous and holometabolous insect lineages diverged approximately  <dig> – <dig>  my , and predict an approximate order of origin for hymenoptera of  <dig> – <dig>  my , lepidoptera of  <dig> – <dig>  my , coleoptera of  <dig> – <dig>  my  and diptera of  <dig> – <dig>  my .fig.
<dig> predicted divergence times of insect long-wavelength-sensitive  opsins, based on r8s and beast analyses.
estimates of time of divergence are shown in million years my with r8s  and beast  software.
color circles show the duplication events in the evolution of mosquito lw opsins: most recent common ancestor  duplication event ; an.
gambiae duplication event ; culicinae duplication event ; ae.
aegypti duplication event ; cx.
quinquefacitaus duplication event .
squares  show the most recent and the more ancestral mosquito lw duplication events.
the dagger shows the constrained nodes using fossils.
gray shading indicates opsins under positive selection.
species abbreviations: acyrthosiphon pisum , apis mellifera , bombyx mori , calliphora vicina , danaus plexippus , drosophila melanogaster , pediculus humanus , and tribolium castaneum .
a divergence times in my were estimated using a penalized likelihood  approach and calibrated with the sister group to culicidae .
b divergence times in my were estimated using a relaxed clock log normal model and calibrated with the following fossil calibrations  <cit> : westphalomerope maryvonneae  <dig>  my , triassoxyela foveolata  <dig>  my , parasabatinca aftimacrai  <dig>  my , grauvogelia arzvilleriana  <dig>  my .
the drops and flower represent the first predicted appearance of blood and nectar sources, namely reptiles , mammals , birds  and flowering plants   <cit>
the placement of the d. melanogaster dmrh <dig>  rh <dig> and rh <dig> and calliphora vicina cv <dig> lw opsin sequences was inconsistent between trees.
these sequences form a separate clade to the mosquito lw opsins in both the parsimony and maximum likelihood trees, but cluster with the mosquito lw opsins in the bayesian inference tree constructed with mrbayes.
attempts to resolve the placement of d. melanogaster and c. vicina sequences using additional amino acid and dna sequences from other insects and arthropods with and without manual improvement of the alignment and the deletion of 3′ and 5′ sequence, were unsuccessful.
molecular evolution of mosquito lw opsins
the mosquito lw opsins form four clades in the ml tree .
the most basal clade  comprises aggprop <dig>  aagprop <dig> and cqgprop1; these orthologs have  <dig> – <dig> % amino acid identity.
the next most derived clade  comprises aggprop <dig> and cqgprop7; these orthologs have  <dig> % amino acid identity.
clades  <dig> and  <dig> are sister clades.
clade three comprises aggprop <dig>  op <dig> and op <dig>  aagprop <dig> and op <dig>  and cqgprop <dig> and op6; opsin orthologs within these clades share between  <dig>  and  <dig> % amino acid identity, while paralogs share between  <dig>  and  <dig> % amino acid identity.
clade four comprises aagprop3– <dig>  aggprop <dig>  and cqgprop8– <dig> and op13; opsin orthologs in this clade share between  <dig>  and  <dig> % amino acid identity, while paralogs share between  <dig>  and 100% amino acid identity.
the details of these findings are presented in the following four sub-sections.
identification of amino acid residues in lw opsins under positive selection
fitmodel  <cit>  was used to test the hypothesis of adaptive changes during the evolution of duplicated lw opsins in mosquitoes .
based on the chi-squared analysis, m <dig> vs m <dig>  m <dig> vs m3 + s <dig> and m <dig> vs m3 + s <dig> were significant at p-value <  <dig> , with m3 + s <dig> the best-fit model .
we identified  <dig>  codons showing evidence of strong negative selection and  <dig> codons  under moderate negative selection.
the remaining codons  were under a combination of selection pressures where for the same position within the alignment, specific residues showed evidence of strong negative selection, moderate negative selection or positive selection.
specifically, nine sites among  <dig> lw opsins  belonging to clades  <dig> and  <dig>  showed evidence of adaptive evolution .
five residues are located in the n-termini, three are located in the c- termini and one is located in tmdiii .table  <dig> residues in mosquito long wavelength opsins predicted under positive selection
aagprop5
aagprop4
cqgprop10
cqgprop13
cqgprop8
cqgprop9
aagprop3
aggprop6
cqgprop6
cqgprop5
aagprop2
aagprop1
aggprop4
aggprop1
aggprop3
the asterisk  indicates residues under positive selection .
the phylogenetic relationship of genes under positive selection is shown in fig.
<dig>  the residue number was derived by numbering from the first amino acid of each sequence ; -, indicates a gap inserted in the alignment and thus, a residue is not available at this position
fig.
<dig> conserved non-coding sequences  and transcription factor binding sites  in aggprop <dig> and aggprop <dig>  a
anopheles gambiae opsin genes showing 5′- and 3′-non coding region on chromosome  2r.
b alignment between the 5′ region of the aggprop <dig>  and of aggprop <dig>  was submitted to mvista using the lagan algorithm to identify cns.
conserved regions are shaded .
the minimum value on the y-axis is 50%, the minimum conservation identity is 70%, and the minimum length for a cns is 100 bp .
because vista calculates the percentage of conservation identity as the number of matches divided by the length of the reference sequence , the length in bp of the cns represents the reciprocal alignment value.
c sequences were submitted to rvista to identify conserved clusters of the  <dig> known tfbs in insects using transfac®.
vertical  lines indicate the position of the  <dig> conserved tfbs identified, which occur in the clusters  <dig> and 2
rates of evolution
evolutionary time estimates were generated for the lw opsins using the programs r8s  and beast .
both analyses suggest that insect lw opsins originated from an ancestral insect opsin approximately  <dig> – <dig>  my, following which paralog and ortholog genes duplicated several times, and as recently as  <dig> – <dig>  my.
in total,  <dig>  or  <dig>  possible duplication events produced the complement of lw opsins in ae.
aegypti, an.
gambiae, and cx.
quinquefasciatus.
six  or seven  duplication events occurred in the most recent common ancestor  of culicinae and anophelinae, representing the only section where topology differs between the trees.
in r8s, the four mosquito lw opsin clades are monophyletic, and there are six events between  <dig>  and  <dig>  my that occurred in the mrca.
in beast, the four mosquito lw opsin clades are not monophyletic; the d. melanogaster rh <dig> and  <dig> and c. vicina cv <dig> opsins form a sister clade to the mosquito clade  <dig> and there are two events a the base of the mosquito-fly taxa between  <dig>  to  <dig>  my and four events in mosquito clades  <dig>   <dig> and  <dig> between  <dig>  and  <dig>  my that occurred in the mrca of culicinae and anophelinae.
two events are specific to an.
gambiae and gave rise to aggprop <dig>   <dig> and  <dig>  with predicted separation from the lineage at  <dig> – <dig>  my, and subsequent divergence of op <dig> and op <dig> at  <dig> – <dig>  my.
six events are specific to the culicinae.
r8s analyses predicted these events at  <dig>  my ,  <dig>  my ,  <dig>  my , and  <dig>  my .
beast analyses predicted these events at  <dig>  my ,  <dig>  my ,  <dig>  my , and  <dig>  my .
one event between  <dig>  and  <dig>  my is specific to ae.
aegypti and gave rise to aagprop <dig> and op <dig>  three events between  <dig> – <dig> ,  <dig> – <dig> , and  <dig> – <dig>  my are specific to cx.
quinquefasciatus and gave rise to cqgprop <dig> and op <dig>  op <dig> and op <dig>  and op <dig> and op <dig>  respectively.
intron phase
the intron phases of the mosquito lw opsins were evaluated to further explore opsin evolution  where phase “0” introns are considered more ancient than phase “1” and “2”  <cit> .
the mosquito lw opsins are either single-exon genes or possess a single intron that is consistently in phase “0”.
the mosquito pteropsin-like genes have four introns, each in different phases.
the phase of the individual intron identified for the presumably more recently derived aagprop <dig> and aggprop <dig> is also “0”.
conserved non-coding sequences  and transcription factor binding sites
to begin to evaluate the role of regulatory sequences in the preservation of the mosquito lw opsins, we analyzed the non-coding regions of aagprop1- <dig>   <dig>  aggprop <dig>  3– <dig> and cqgprop <dig>  5– <dig>   <dig> for putative cns.
cns were identified in the up-stream regions of  <dig> of  <dig> lw opsin gene pair alignments  .
the an.
gambiae, ae.
aegypti and cx.
quinquefasciatus genome assemblies enabled comparative analyses of conserved tfbs across these three species .
an average of  <dig>  insect specific tfbs were identified for each of the seven gene pairs  when all  <dig> possible gene pairs where aligned.
the tfbs, abdb, brcz <dig>  brcz <dig>  byn, cf <dig>  cf1a, cf2ii, hsf, kr, tcf, zen tfbs were identified in all seven gene pairs.
two tfbs, croc and grh, were present in only one pair .
tfbs were identified only between paralog pairs and not between genes belonging to the presumably more ancestral clade  <dig>  or between orthologous lw opsins identified to other clades.
tfbs were clustered in genome regions with each cluster containing multiple binding sites for multiple transcription factors as shown in fig.
3c for the genes aggprop <dig> and op <dig>  the lack of conservation between cns regions and tfbs could reflect differential expression of the duplicated lw genes.
discussion
we describe the first detailed molecular and evolutionary analyses of the opsin gene family in the culicine mosquitoes ae.
aegypti and cx.
quinquefasciatus and the anopheline mosquito an.
gambiae.
these species exhibit different behavioral periodicities; ae.
aegypti is a diurnally active mosquito, while an.
gambiae and cx.
quinquefasciatus exhibit nocturnal and crepuscular behaviors  <cit> .
we report revised annotations for the published ae.
aegypti , an.
gambiae  and cx.
quinquefasciatus  opsin gene models.
transcript and amino acid sequence similarity data support the identification of functional visual and non-visual opsins in these mosquitoes.
transcripts were identified for each of the ae.
aegypti and an.
gambiae opsins and seven of the  <dig> cx.
quinquefasciatus genes.
the predicted protein sequences of the mosquito opsins contain amino acid residues conserved in other rhodopsin-like gpcrs, including residues important for opsin function, and involved in post-translational modifications for n-linked glycosylation, palmityolation and phosphorylation.
published microarray and rnaseq analyses provide further support for functionality of the mosquito opsins and insights into possible temporal expression.
aedes aegypti genes for four lw and one sw opsin  were each down regulated once at different points in a 96 h period following a blood meal, with the exception of op <dig> which was down regulated twice  <cit> .
aedes aegypti rnaseq data  <cit>  suggest two lw , the uv  and sw  opsin genes are up regulated in sugar fed females when compared to blood feed females.
these findings may help to explain why blood-fed females are largely unresponsive to external stimuli until ready to oviposit.
genes for the lw opsins aagprop <dig>   <dig> and op <dig>  uv  and sw  were up-regulated in male mosquitoes suggesting that some visual capacities may develop earlier in males than in females  <cit> .
in situ hybridization studies by hu et al.
<cit>  using anti-opsin antibodies provide evidence for expression of lw , uv  and sw  opsins in the photoreceptor cells  of adult ae.
aegypti.
mass spectrometry  studies  provide evidence for protein expression of an.
gambiae opsins in whole head, eye and brain.
little is currently known regarding the spectral sensitivity of the ae.
aegypti, an.
gambiae and cx.
quinquefasciatus opsins.
electroretinograms  and microspectrophotometry  sensitivity studies of larval eyes  and the adult compound eye have shown that ae.
aegypti exhibits two peaks of spectral sensitivity to uv  and lw  light , but equivalent erg and msp studies have not been published for an.
gambiae or cx.
quinquefasciatus.
landing studies have tested the response of ae.
aegypti and cx.
quinquefasciatus to color targets, and suggest that these mosquitoes prefer red and black, and black and brown, respectively  <cit> .
equivalent studies have not been performed for an.
gambiae, but this species exhibited an optomotor response  under visible and infrared wavelengths  <cit> .
phylogenetic analyses placed the mosquito opsin genes into five distinct “functional” clades, namely visual uv, sw and lw opsins, non-visual pteropsins, and orthologs of the d. melanogaster opsin rh <dig>  phylogenetic predictions for aagprop <dig> , op <dig>  and op <dig>  are supported by functional studies  <cit>  that report dual spectral sensitivity peaks of 500–550 nm and ~350 nm  for aagprop <dig>  and peaks of 350 nm  for op <dig> and 400–450 nm  for op <dig>  in apis mellifera  workers and drones, the non-visual pteropsin is expressed in the brain and is thought to function in regulation of circadian rhythm  <cit> .
the wavelength sensitivity of rh <dig> has not been determined and its role in d. melanogaster vision is not known  <cit> .
the mosquito orthologs of rh <dig> are located in a sister clade to the uv and sw clades, indicating that these receptors may respond to short wavelengths.
these results corroborate other phylogenetic studies and support hypotheses for the relative divergence times of the four major holometabolus orders, with hymenoptera as the more ancient  <cit>  and coleoptera, diptera and lepidoptera as more derived orders, as reviewed by grimaldi and engel  <cit> , and supported by the recent work of misof et al.
<cit> .
as suggested by cameron and mardulyn  <cit> , these findings highlight the utility of lw opsins for resolution of higher-level phylogenetic relationships.
expansions of lw opsins have been noted in insects and the aquatic invertebrates d. pulex, dragonflies and stomatopods.
we observed an expansion of putative lw opsins in ae.
aegypit , an.
gambiae  and cx.
quinquefasciatus  relative to other insects, which typically have between one to four lw opsins.
studies suggest that the order diptera  arose in the carboniferus approximately  <dig> my  <cit>  and that the divergence of the culicinae and anophelinae occurred in the permian, triassic or jurassic, possibly between  <dig> and  <dig> my  <cit>  or in the triassic period at approximately  <dig> my  <cit> .
our results suggest that the  <dig> lw opsins arose via multiple gene duplication events in the most recent common ancestor  of the anophelinae and culicinae lineage in the jurassic, approximately  <dig> – <dig>  my, following which independent duplications occurred at least once in each of the three mosquito lineages.
the mosquito putative lw opsins share significant amino acid identity  but limited nucleotide conservation in the non-coding regions between paralogs.
the studies of lynch and conery  <cit>  suggest that while the rate of origin of new gene duplicates may be high , the rate of duplicate preservation is low, and the authors predict the average half-life of a gene duplicate is approximately  <dig>  my  <cit> .
the retention of duplicated lw genes in mosquitoes over long evolutionary periods implies the functional importance of their gene products.
supporting the theory of duplication of lw opsins is the observation that five an.
gambiae lw opsins  are tandemly arrayed within a 90 kb region on chromosome 2r, and are separated from a sixth, presumably more ancestral lw opsin , by approximately 3 mb.
conservation of syntenic blocks has been observed between an.
gambiae, ae.
aegypti, and cx.
quinquefasciatus  <cit> .
the production of improved assemblies and physical maps for the latter two species will permit studies of the opsin synteny among these species and may similarly illuminate gene evolution in ae.
aegypti and cx.
quinquefasciatus.
the identification of multiple putative lw sensitive opsins in the three mosquitoes could reflect an adaptation to photic environments involving lw light, as suggested by futahashi et al.,  <cit> .
adult ae.
aegypti, an.
gambiae and cx.
quinquefasciatus exhibit differences in times of peak activity  but all are active at periods when long wavelength light predominates.
aedes aegypti oviposition peaks at sunset  <cit>  when longer wavelengths are more abundant.
studies have shown that ae.
aegypti larvae and adults are capable of responding to uv and visible light, with the highest peak of sensitivity between  <dig> and 523 nm – i.e., in the lw spectrum .
both an.
gambiae and cx.
quinquefasciatus exhibit activity peaks between 22: <dig> and 02:00 h  <cit>  and enter dwellings to blood feed usually when occupants are asleep  <cit> .
moonlight is long-wavelength-shifted and nocturnal mosquitoes are in general, more active during moonlit nights  <cit> .
additionally, the larvae and pupae of ae.
aegypti, an.
gambiae and cx.
quinquefasciatus are active in shallow aquatic environments that are typically associated with longer wavelengths due to the defraction of incident light  <cit> .
supporting this theory is the identification of extreme expansions in the lw opsins in the aquatic invertebrate, daphnia pulex , stomatopods   <cit>  and dragonflies .
lw opsin expansions have also been noted in cave fish  and the guppy, poecilia reticulate   <cit> .
the present study provides the first comparative genomics analyses of lw opsins in mosquitoes.
rt-pcr studies reported here support expression of all opsins except aagprop <dig>  and op <dig>  in ae.
aegypti 4th instar larvae and pupae, and a role in visual processes in immature mosquitoes.
little is known regarding opsin expression in mosquito larvae and pupae and the possible link between aquatic life-style and the duplication and retention of lw opsin deserves further investigation.
six  or seven  duplication events involving lw opsins are shared between the culicinae and anophelinae, suggesting retention following events at approximately  <dig> – <dig>  to  <dig> – <dig>  my in the mesozoic.
it is possible that lw opsins evolved following a variety of events such as asteroid impacts or volcanic activity toward the end of the mesozoic when sun- and moon-light would presumably have been reduced, or the appearance of diverse vertebrate hosts during the cenozoic period.
based on the morphology of mosquito ommatidia, kawada et al.
<cit>  proposed that “crepuscular behavior of mosquitoes is a transitional behavior in the course of evolution of nocturnal behavior to diurnal behavior”, explained as a consequence of either environmental changes and/or behavioral changes in vertebrate hosts.
diversification of lw opsin function  has not been established in mosquitoes, but could facilitate crepuscular and diurnal behavior.
interestingly, it has also been proposed that inversion complexes on the an.
gambiae chromosome arm 2r  are associated with ecological adaptations that increase the fitness of the carriers  <cit> .
analyses of synteny among an.
gambiae, ae.
aegypti and cx.
quinquefasciatus will permit further investigation of this hypothesis.
two lw opsin duplication events are specific to the anopheline and six to the culicine and may reflect lineage and species-specific light detection capabilities.
this hypothesis is supported by the observation of key morphological differences between the eyes of aedes, anopheles, and culex species.
the ae.
aegypti rhabdom is longer and cylindrical and the lens of the ommatidium is smaller and less hemispheric in comparison to that of an.
gambiae  <cit> .
key morphological differences in the size and form of the facet lenses, rhabdom and the interommatidial angle consistently group ae.
aegypti with other diurnal mosquitoes, and an.
gambiae and cx.
quinquefasciatus with other nocturnal mosquitoes .
thus, ommatidial structures may vary depending on the photo-environment in which mosquitoes are active and may be of limited application as a taxonomic character.
further studies are required to tease apart the contribution of lw opsins to the visual capacities of diurnal versus nocturnal mosquitoes.
the amino acid identity of the lw opsins observed both within and between mosquito taxa  is notable considering time of divergence predictions for these duplicates.
these data raise intriguing questions about the retention and conservation of lw opsin genes in the culicinae and anophelinae.
pseudogenization would appear unlikely and the contribution of both sub- and neofunctionalization deserves attention.
in this study, we explored two possible underlying molecular mechanisms - gene selection and differential expression – that could contribute to the retention of duplicated lw opsins in mosquitoes.
several studies have proposed opsin functional diversification  as an explanation for gene retention, and examples of this phenomenon have been described in insects.
the three d. melanogaster lw opsins rh <dig>  rh <dig> and rh <dig>  exhibit different spectral sensitivities  and are expressed in different ommatidial cells and elsewhere in the body.
rh <dig> is the major pigment in prcs 1– <dig> and is also involved in temperature discrimination by larvae  <cit> .
rh <dig> is expressed in both ocelli and testis, and rh <dig> is expressed in prc  <dig> and extra-retinal tissue associated with auditory processes  <cit> .
opsin transcripts have been identified in diverse tissues, including the rostrum, leg, abdomen, antenna, maxillary palp, proboscis and ovary of ae.
aegypti, and the maxillary palp and antenna of an.
gambiae  <cit>  and opsins have been identified in the antenna, maxillary palp and proboscis of an.
gambiae  <cit> .
based on several lines of evidence, including work in d. melanogaster, bohbot et al.
<cit>  speculated that aagprop <dig> and op <dig>  expressed in the maxillary palps of adult mosquitoes, may be involved in heat sensing.
the molecular role of mosquito opsins in a variety of sensory and reproductive tissues remains unclear and detailed studies are required to explore the possibility of functional diversification.
the p. xuthus and p. glaucus lw opsins, pxrh and pgrh have four amino acid substitutions in tm domain i  and tm domain iii  that are predicted to “shift” the absorption spectra from green  to red   <cit> .
it has also been proposed that the lw genes of d. pulex have a role in the adaptation of the water flea to a more complex light regime in aquatic environments  <cit>  and that the lw opsins in stomatopods diverged with respect to spectral tuning  and signal transduction   <cit> .
the immature and adult stages of species of culicine and anopheline mosquitoes are active at low light intensities.
we propose that a “suite” of opsins with different wavelength sensitivities may enable maximal capture of photons across the lw spectrum and improve visual acuity.
this hypothesis is supported by the studies of hu et al.
<cit>  who also proposed similar roles for these receptors.
physiological studies are required to explore the spectral sensitivities of mosquito lw opsins and the contributions of each opsin to light capture in mosquitoes.
using fitmodel, we identified nine residues in  <dig> lw opsins  that are possibly experiencing positive selection.
in preliminary analyses using paml software , we identified multiple candidate residues, including multiple residues in tmdiii possibly under positive selection.
the modest number of residues identified only in the most recently duplicated lw opsins likely reflects the stringency of fitmodel .
these residues are located in the 5′ extracellular and 3′ intracellular regions and tmdiii.
while further work such as site-directed mutagenesis studies are required to determine the significance of this finding, and to evaluate a possible association between tmd residues  and opsin spectral tuning, these analyses suggest that functional diversification may play a role in retention of at least some lw opsins in mosquitoes.
the prediction that the c <dig> residue in cqgprop <dig>   <dig> and aagprop <dig> is under positive selection is significant as previous studies involving species of the chelicerata, crustacea and insecta have shown that this region is important for tertiary structures  associated with opsin function  <cit> .
this region may play a crucial role in the functional diversification of arthropod opsins.
interestingly, studies in bees   <cit>  have shown that mutations in the positively selected lw opsins may enable spectral tuning to maximize visual capabilities for foraging in dim-light in ancestral bees, thought to be diurnal.
the authors identified  <dig> positively selected codons one of which aligns adjacent c <dig> in the positively selected aagprop <dig>  cqgprop <dig> and  <dig>  this suggests a case of possible convergent evolution, as it has been proposed that mosquitoes were also diurnal feeders that developed crepuscular and night feeding behaviors.
our discovery that nine out of a possible  <dig> sites may be under positive selection suggests that other mechanisms are likely responsible for the retention of the duplicated lw opsins over a timeframe of more than  <dig> my.
to evaluate adaptive changes associated with the non-coding regions of lw opsins, we examined  <dig> kb of upstream sequence for the presence of conserved non-coding sequences  and transcription factor binding sites .
binding sites for transcription factors that regulate the spatial and temporal patterns of gene expression are located in the non-coding sequence  <cit> .
accumulation of null mutations in the regulatory regions of recently duplicated genes that could contribute to differential gene regulation has been proposed as a mechanism for retention of duplicates  <cit> .
nucleotide conservation was observed in seven out of  <dig> alignments representing  <dig> lw mosquito opsins, suggesting some level of coordinated expression between at least seven paralog pairs.
this also suggests the deterioration of sequence similarity across the subfamily through accumulation of mutations.
the lack of dna sequence similarity in the 5′ and 3′ non-coding regions between opsin orthologs and paralogs, and the apparent lack of common transcription units, suggests the possibility of differential opsin expression in ae.
aegypti, an.
gambiae and cx.
quinquefasciatus.
our analysis revealed multiple copies of  <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> different tfbs in the conserved regions between aggprop <dig> and op <dig>  cqgprop <dig> and op <dig>  aagprop <dig> and op <dig>  aagprop <dig> and op <dig>  aagprop <dig> and op <dig>  cqgprop <dig> and op <dig>  aagprop <dig> and op <dig>  cqgprop <dig> and op <dig>  respectively.
these tfbs are insect specific and the possibility of additional, novel tfbs in these regions can’t be ruled out.
for context, shared tfbs are associated with less than 2% of genes in d. melanogaster   <cit> .
no tfbs were identified between aagprop <dig>  aggprop <dig> and cqgprop <dig> , supporting the hypothesis of greater time since divergence from the mrca.
presumably, extensive accumulation of mutations in the cns regions of these genes masks the identification of tfbs and suggests potential involvement of different regulatory units in expression.
interestingly, preliminary rt-pcr analyses detected transcripts for all opsins  in all stages and sexes of ae.
aegypti, and in an.
gambiae male and female adults .
the studies of rund et al.
<cit>  suggest a more complex picture, with rhythmic expression of  <dig> phototransduction pathway genes in an.
gambiae under light–dark  and/or dark-dark  conditions, including aggprop <dig> .
analyses of the rund et al.
<cit>  data identified rhythmic expression for six opsin genes under ld  and the two pteropsins .
for the dd regime, we identified rhythmic expression for three opsin genes, two lw  and the rh <dig> ortholog .
the studies of dissanayake et al.
<cit>  also support differential opsin transcript levels in ae.
aegypti males and females, and hu et al.
<cit>  showed differential expression of genes for aagprop <dig>  op <dig> and op <dig> in the retina, dorsal, central, and ventral regions of the adult compound eye.
collectively, these studies suggest a complex pattern of differential gene expression in mosquitoes.
further studies are required to examine the differential expression of lw opsin genes and their possible roles in adaptive visual sensitivity across mosquito taxa.
CONCLUSIONS
evolutionary analyses of ae.
aegypti, an.
gambiae and cx.
quinquefasciatus lw opsins suggests gene retention in the lineages anophelinae and culicinae, and the functional importance of these genes.
similar expansions have been observed in other aquatic invertebrates and vertebrates, with the most extreme cases in invertebrates, suggesting a gene family that is prone to duplication.
time of divergence predictions suggest mosquito opsin gene duplication events occurred in the mesozoic and cenozoic.
positive selection and coordinated regulation represent two mechanisms for the retention of lw opsins in the three mosquitoes.
of the  <dig> genes, the retention of  <dig> can be explained through positive selection,  <dig> through coordinated regulation, and  <dig> through both mechanisms.
these genes were assigned to the more recent clades  <dig> and  <dig>  with the exception of cqgprop <dig>  which is a member of clade  <dig>  the retention of four lw opsins , members of the more ancestral clades  <dig> and  <dig>  was not explained by either mechanism.
research is needed to resolve the spectral sensitivity, spatio-temporal expression and function of mosquito lw opsins in visual/non-visual processes.
the potential connection of mosquito lw opsins to lifestyles associated with lw-dominated photo-environments  and crepuscular/nocturnal activity, deserves further investigation.
our data suggest involvement of lw opsins in lineage- and species-specific processes and provide an important foundation for future efforts directed at identifying opsin-mediated behaviors that could be exploited to achieve vector control.

 the genomes of eukaryotes typically comprise a high percentage of duplicated genes. it has been proposed that gene duplication is the major mechanism for the origin of new gene function and that the refashioning of duplicate genes is a major contributor to the origin of adaptive evolutionary novelties. gene duplication may originate from unequal crossing over, retro-transposition, segmental duplication and chromosomal or genome duplication. force et al. proposed several theories to explain the retention of duplicate gene copies in the genome wherein the fate of each copy may depend on mutations that occur in both coding and regulatory regions. the accumulation of mutations in one copy may eventually render the gene non-functional. degenerative mutations may accumulate in the regulatory regions of gene copies that are separately capable of performing a distinct ancestral function or one copy may retain the original function with the second copy acquiring a new function by the retention of beneficial mutations. opsins initiate the photon-induced signaling cascade in vertebrates and invertebrates, and are members of the g protein-coupled receptor family, characterized by seven trans-membrane domains. insects typically possess three classes of visual opsins that are sensitive to ultraviolet, short and long wavelengths. additionally, some insects have red sensitive receptors. visual opsins are expressed in the rhabdomere of the ommatidia, the major structural unit of the arthropod compound eye. non visual opsins have also been identified and include the apis mellifera pteropsin identified in the bee brain, suggesting a possible function in extra-retinal detection of light and the regulation of circadian rhythm. the functions of the drosophila melanogaster opsin rh and the rgr-like and arthropsins identified in daphnia pulex have not been determined. mosquitoes are one of the most important arthropod groups affecting human and animal health. the assembled genomes of the yellow fever mosquito aedes aegypti, the malaria mosquito anopheles gambiae and the southern house mosquito culex quinquefasciatus provide an opportunity to investigate the molecular evolution of opsin genes in three mosquito taxa representing the lineages culicinae and anophelinae. it is predicted that the culicinae and anophelinae diverged approximately 145– million years ago or my. species comprising these lineages transmit a variety of parasites and pathogens of medical and veterinary significance and exhibit markedly different behavioral periodicities. aedes aegypti is a diurnal mosquito, while an. gambiae and cx. quinquefasciatus exhibit nocturnal and crepuscular behaviors. expansions in the genes coding for lw opsins have been noted in a number of invertebrates, including insects. the fruitfly, d. melanogaster possesses three lw opsin genes and the butterflies papillio xuthus and papillio glaucus have three and four genes, respectively. examples of duplication in lw opsin genes have been observed in the aquatic crustacean, daphnia pulex, which has lw opsin genes, the stomatopods, gonodactylus smithii, neogonodactylus oerstedii, odontodactylus scyllarus, coronis scolopendra and squilla empusa, which each possess lw opsin genes, and the dragonflies anax parthenope, anotogaster sieboldii, asiagomphus melaenops, epiophlebia superstes, indolestes peregrinus, ischnura asiatica, macromia amphigena, mnais costalis, orthetrum albistylum, somatochlora uchidai, sympetrum frequens and tanypteryx pryeri which have 8– lw opsin genes. it has been proposed that the opsin expansion observed in daphnia and other aquatic lineages may be influenced by more complex light regimes associated with aquatic environments. the study of futahashi et al. also suggests an association of lw opsins with aquatic habitat. previously, we identified and putative opsins in ae. aegypti and an. gambiae, respectively, and an expansion of putative lw opsin genes in both species which possess six gene copies as compared to other insects that typically have between one to four lw opsins. five an. gambiae lw opsins are tandemly arrayed within a 90 kb region on chromosome 2r and are separated from a sixth, and presumably more ancestral lw opsin, by approximately 3 mb. the density of genes and segmental duplications as well as the high gc content of 2r is suggestive of a high recombination rate, a phenomenon typically associated with gene duplication. more recently, opsin genes were found in the cx. quinquefasciatus genome, although their wavelength sensitivity was not predicted. interest in novel strategies to control mosquitoes is high due to the emergence and re-emergence of arboviral diseases such as dengue, yellow fever, chikungunya and zika and the failure of traditional drug and insecticide control. opsin-mediated processes could be targeted to disrupt mosquito mating, host finding and oviposition. an improved understanding of these processes could also benefit the design of new mosquito traps, deterrent devices and genetic control strategies. here, we present detailed molecular evolutionary analyses of the lw opsins in ae. aegypti, an. gambiae and cx. quinquefasciatus as a first step toward understanding the molecular basis of opsin-mediated visual processes that could be exploited for mosquito control. phylogenetic analyses suggest that culicine and anopheline mosquitoes possess orthologs of invertebrate lw, sw and uv–sensitive opsins, extra-retinal pteropsins, and the d. melanogaster rh we predicted six, six and eight putative lw opsins in ae. aegypti, an. gambiae, cx. quinquefasciatus, respectively, indicative of an expansion of these genes in the culicidae relative to other insects. molecular evolutionary analyses suggest that mosquito lw opsins originated from or duplication events that occurred between to < million years ago. the genes for ten lw opsins may have been retained through a combination of positive selection and coordinated regulation. of the remaining mosquito opsin genes, five may have been retained through positive selection, and one via coordinated regulation. four lw opsins lack evidence of either positive selection or coordinated regulation.  genes for and opsins were manually annotated in the genomes of ae. aegypti, an. gambiae and cx. quinquefasciatus, respectively and results are summarized in table  opsin protein sequences are provided in the additional file and are also available from vectorbase and genbank. the protein sequences for all opsins contain a predicted initiation methionine, a stop codon, three extracellular and three intracellular loops, and seven tm domains. excluding the incomplete models above, the mosquito opsins vary in length from to amino acids in ae. aegypti, 370– in an. gambiae, and 368– amino acids in cx. quinquefasciatus. we detected a single amino acid substitution between aagprop and op. of note, aggprop first reported in hill et al. may reflect an erroneous prediction based on an earlier version of the an. gambiae assembly. aagprop9_ is a truncated gene model identical to the first exon of aagprop9_ from nucleotides to except for a non-synonymous c/a substitution at nucleotide and a synonymous t/c substitution at nucleotide we were unable to identify the second exon of aagprop9_ the third intracellular loop which is likely important for g protein interaction, is amino acids in length in all mosquito opsins except for cqgprop and the presumably more ancestral opsins aagprop aggprop and cqgprop where the loop is predicted to be and amino acids in length, respectively. genea ae. aegypti aagprop1 aael006498 aagprop2 aael006259 aagprop3 aael006484 aagprop4 aael005621 aagprop5 aael005625 aagprop7 aael007389 aagprop8 aael009615 aagprop9_1 aael003035 aagprop9_2 c aael003035 aagprop10 aael005322 aagprop12 aael005373 an. gambiae aggprop1 agap013149 aggprop3 agap012985 aggprop4 agap012982 aggprop5 agap001162 aggprop6 agap001161 aggprop7 agap002462 aggprop8 agap006126 aggprop9 agap010089 aggprop10 agap007548 aggprop11 agap002443 aggprop12 agap002444 cx. quinquefasciatus cqgprop1 cpij004067 cqgprop2 cpij005000 cqgprop3 cpij009246 cqgprop4 cpij011419 cqgprop5 cpij012052 cqgprop6 cpij011571 cqgprop7 cpij011573 cqgprop8 cpij011574 cqgprop9 cpij011576 cqgprop10 c cpij013056 cqgprop11 cpij013408 cqgprop12 cpij014334 cqgprop13 cpij020021 aannotations from hill et al., nene et al. and arensburger et al.. revised opsin annotations were produced using the aaegl, agamp and cpip gene sets from vectorbase; bexpression evidence for ae. aegypti, an. gambiae and cx. quinquefasciatus obtained from pcr or rt-pcr data, ests, microarrays, rnaseq and mass spectrometry; p, peptide evidence; t, transcript evidence; -, absence of transcript or peptide evidence; cindicates incomplete gene model; n.a. not available conservation was observed in the architecture between mosquito opsins. the lw opsins have no or one intron, sw opsins have one intron, uv opsins have four introns, pteropsin-like genes have four introns, and orthologs of the d. melanogaster rh posses two introns. the ae. aegypti lw opsin introns are approximately 5– times longer than those of an. gambiae and cx. quinquefasciatus, consistent with the gene architecture observed in this species. the putative coding sequences of the mosquito opsins were aligned to the rhodopsin from the squid todarodes pacificus to identify amino acids and structural features, including those associated with functions in other organisms. we identified and residues conserved among mosquito lw opsins and class a rhodopsin-like gpcrs and opsin visual pigments, respectively. amino acids important for the interaction between the bovine rhodopsin and g-proteins are conserved in the mosquito opsins. three classes of post-translational modifications characteristic of opsins were predicted for the mosquito opsins based on comparison to the crystallized bovine rhodopsin and the squid rhodopsin. these include sites in the n-terminus that undergo n-glycosylation during biosynthesis, sites in the c-terminus that undergo palmitoylation, the role of which in opsins remains undefined, and several s and t residues located in the c-termini of the bovine and squid rhodopsins that are the potential targets for phosphorylation by rhodopsin kinase. the tmds of the mosquito opsins were predicted using a hidden markov model using the tmhmm server v. and aligned with muscle. the tm domains of aagprop1- and aggprop and 3– and cqgprop 5– and and share a minimum 62%, and 68% amino acid identity, respectively.fig. amino acid alignment of the predicted trans-membrane domains of ae. aegypti putative lw opsins. highlighted amino acids show positions conserved in lw opsins. black shading, identical residues; gray shading, similar residues, based on the similarity matrix blosum the asterisk indicates the residue identified by fitmodel as under positive selection detection of mosquito opsin transcripts published est studies support the production of transcripts for aagprop1- 8-9_1; aggprop1- 8– and cqgprop 5– and. we reviewed published meta-analyses of whole body or individual organs to extract data regarding opsin temporal and spatial expression using the vectorbase expression tool. microarray data support the production of transcripts for all ae. aegypti and an. gambiae opsins and cqgprop and. pcr and rt-pcr studies support transcripts for all ae. aegypti opsin genes and all an. gambiae opsin genes, and rnaseq experiments support the production of transcripts for all ae. aegypti and an. gambiae opsins. peptide expression evidence was supported with mass spectrometry for all an. gambiae opsin proteins. collectively, these public data provide evidence for expression of of the mosquito opsins. support for the production of mosquito transcripts was extended in the present study using rt-pcr analyses and includes transcripts corresponding to ae. aegypti and an. gambiae opsin genes. transcripts were detected for all ae. aegypti opsin genes by rt-pcr, except for aagprop in male and female adults and aagprop and in larvae and pupae. transcripts corresponding to opsin genes were not detected in ae. aegypti embryos. all pcr amplicons were sequenced to confirm coding regions. transcripts for all an. gambiae opsin genes were detected by rt-pcr in male and female adults. localization of mosquito opsin genes to chromosomes genes for the an. gambiae lw opsins are co-localized on the distal portion of chromosome 2r, a region of the genome associated with multiple duplicated genes. five genes are located within a 90 kb region, and are separated from the sixth, aggprop by 3 mb. localization of ae. aegypti or cx. quinquefasciatus opsins to chromosomes and syteny analyses were not possible due to the fragmented genome assemblies and the lack of more complete sequence and physical maps for these species. phylogenetic analyses of mosquito opsins two initial phylogenetic analyses were performed and employed a large survey of opsin protein and nucleotide sequences from diverse animals and the trees were consistent with previous phylogenetic investigations of animal opsins. consistent with the phylogenies of feuda et al., the mosquito opsins were placed in one of five clades, namely the lw, sw, uv, rh7-like and pteropsin clades. mosquitoes possess one or two putative genes for each of the uv, sw, rh7-like and pteropsin functional groups. we identified six putative lw opsins in ae. aegypti, six in an. gambiae and eight in cx. quinquefasciatus, and observed that the mosquito lw clades comprise an increased number of duplicate opsin gene lineages relative to opsin clades from insects. to further investigate the retention of lw opsin genes in mosquitoes, phylogenetic analyses were conducted with lw sensitive opsins from eight insect species. the maximum likelihood tree reflects the currently accepted hypothesis of insect phylogeny in which the hymenoptera were thought to derive from an early branch of the holometabolous insects. the r8s and beast results are consistent with the estimated divergence dates reported by misof et al., suggesting that the hemimetabolous and holometabolous insect lineages diverged approximately – my, and predict an approximate order of origin for hymenoptera of – my, lepidoptera of – my, coleoptera of – my and diptera of – my.fig. predicted divergence times of insect long-wavelength-sensitive opsins, based on r8s and beast analyses. estimates of time of divergence are shown in million years my with r8s and beast software. color circles show the duplication events in the evolution of mosquito lw opsins: most recent common ancestor duplication event; an. gambiae duplication event; culicinae duplication event; ae. aegypti duplication event; cx. quinquefacitaus duplication event. squares show the most recent and the more ancestral mosquito lw duplication events. the dagger shows the constrained nodes using fossils. gray shading indicates opsins under positive selection. species abbreviations: acyrthosiphon pisum, apis mellifera, bombyx mori, calliphora vicina, danaus plexippus, drosophila melanogaster, pediculus humanus, and tribolium castaneum. a divergence times in my were estimated using a penalized likelihood approach and calibrated with the sister group to culicidae. b divergence times in my were estimated using a relaxed clock log normal model and calibrated with the following fossil calibrations: westphalomerope maryvonneae my, triassoxyela foveolata my, parasabatinca aftimacrai my, grauvogelia arzvilleriana my. the drops and flower represent the first predicted appearance of blood and nectar sources, namely reptiles, mammals, birds and flowering plants the placement of the d. melanogaster dmrh rh and rh and calliphora vicina cv lw opsin sequences was inconsistent between trees. these sequences form a separate clade to the mosquito lw opsins in both the parsimony and maximum likelihood trees, but cluster with the mosquito lw opsins in the bayesian inference tree constructed with mrbayes. attempts to resolve the placement of d. melanogaster and c. vicina sequences using additional amino acid and dna sequences from other insects and arthropods with and without manual improvement of the alignment and the deletion of 3′ and 5′ sequence, were unsuccessful. molecular evolution of mosquito lw opsins the mosquito lw opsins form four clades in the ml tree. the most basal clade comprises aggprop aagprop and cqgprop1; these orthologs have – % amino acid identity. the next most derived clade comprises aggprop and cqgprop7; these orthologs have % amino acid identity. clades and are sister clades. clade three comprises aggprop op and op aagprop and op and cqgprop and op6; opsin orthologs within these clades share between and % amino acid identity, while paralogs share between and % amino acid identity. clade four comprises aagprop3– aggprop and cqgprop8– and op13; opsin orthologs in this clade share between and % amino acid identity, while paralogs share between and 100% amino acid identity. the details of these findings are presented in the following four sub-sections. identification of amino acid residues in lw opsins under positive selection fitmodel was used to test the hypothesis of adaptive changes during the evolution of duplicated lw opsins in mosquitoes. based on the chi-squared analysis, m vs m m vs m3 + s and m vs m3 + s were significant at p-value < , with m3 + s the best-fit model. we identified codons showing evidence of strong negative selection and codons under moderate negative selection. the remaining codons were under a combination of selection pressures where for the same position within the alignment, specific residues showed evidence of strong negative selection, moderate negative selection or positive selection. specifically, nine sites among lw opsins belonging to clades and showed evidence of adaptive evolution. five residues are located in the n-termini, three are located in the c- termini and one is located in tmdiii.table residues in mosquito long wavelength opsins predicted under positive selection aagprop5 aagprop4 cqgprop10 cqgprop13 cqgprop8 cqgprop9 aagprop3 aggprop6 cqgprop6 cqgprop5 aagprop2 aagprop1 aggprop4 aggprop1 aggprop3 the asterisk indicates residues under positive selection. the phylogenetic relationship of genes under positive selection is shown in fig.  the residue number was derived by numbering from the first amino acid of each sequence; -, indicates a gap inserted in the alignment and thus, a residue is not available at this position fig. conserved non-coding sequences and transcription factor binding sites in aggprop and aggprop a anopheles gambiae opsin genes showing 5′- and 3′-non coding region on chromosome 2r. b alignment between the 5′ region of the aggprop and of aggprop was submitted to mvista using the lagan algorithm to identify cns. conserved regions are shaded. the minimum value on the y-axis is 50%, the minimum conservation identity is 70%, and the minimum length for a cns is 100 bp. because vista calculates the percentage of conservation identity as the number of matches divided by the length of the reference sequence, the length in bp of the cns represents the reciprocal alignment value. c sequences were submitted to rvista to identify conserved clusters of the known tfbs in insects using transfac®. vertical lines indicate the position of the conserved tfbs identified, which occur in the clusters and 2 rates of evolution evolutionary time estimates were generated for the lw opsins using the programs r8s and beast. both analyses suggest that insect lw opsins originated from an ancestral insect opsin approximately – my, following which paralog and ortholog genes duplicated several times, and as recently as – my. in total, or possible duplication events produced the complement of lw opsins in ae. aegypti, an. gambiae, and cx. quinquefasciatus. six or seven duplication events occurred in the most recent common ancestor of culicinae and anophelinae, representing the only section where topology differs between the trees. in r8s, the four mosquito lw opsin clades are monophyletic, and there are six events between and my that occurred in the mrca. in beast, the four mosquito lw opsin clades are not monophyletic; the d. melanogaster rh and and c. vicina cv opsins form a sister clade to the mosquito clade and there are two events a the base of the mosquito-fly taxa between to my and four events in mosquito clades and between and my that occurred in the mrca of culicinae and anophelinae. two events are specific to an. gambiae and gave rise to aggprop and with predicted separation from the lineage at – my, and subsequent divergence of op and op at – my. six events are specific to the culicinae. r8s analyses predicted these events at my, my, my, and my. beast analyses predicted these events at my, my, my, and my. one event between and my is specific to ae. aegypti and gave rise to aagprop and op three events between –, –, and – my are specific to cx. quinquefasciatus and gave rise to cqgprop and op op and op and op and op respectively. intron phase the intron phases of the mosquito lw opsins were evaluated to further explore opsin evolution where phase “0” introns are considered more ancient than phase “1” and “2”. the mosquito lw opsins are either single-exon genes or possess a single intron that is consistently in phase “0”. the mosquito pteropsin-like genes have four introns, each in different phases. the phase of the individual intron identified for the presumably more recently derived aagprop and aggprop is also “0”. conserved non-coding sequences and transcription factor binding sites to begin to evaluate the role of regulatory sequences in the preservation of the mosquito lw opsins, we analyzed the non-coding regions of aagprop1- aggprop 3– and cqgprop 5– for putative cns. cns were identified in the up-stream regions of of lw opsin gene pair alignments. the an. gambiae, ae. aegypti and cx. quinquefasciatus genome assemblies enabled comparative analyses of conserved tfbs across these three species. an average of insect specific tfbs were identified for each of the seven gene pairs when all possible gene pairs where aligned. the tfbs, abdb, brcz brcz byn, cf cf1a, cf2ii, hsf, kr, tcf, zen tfbs were identified in all seven gene pairs. two tfbs, croc and grh, were present in only one pair. tfbs were identified only between paralog pairs and not between genes belonging to the presumably more ancestral clade or between orthologous lw opsins identified to other clades. tfbs were clustered in genome regions with each cluster containing multiple binding sites for multiple transcription factors as shown in fig. 3c for the genes aggprop and op the lack of conservation between cns regions and tfbs could reflect differential expression of the duplicated lw genes. discussion we describe the first detailed molecular and evolutionary analyses of the opsin gene family in the culicine mosquitoes ae. aegypti and cx. quinquefasciatus and the anopheline mosquito an. gambiae. these species exhibit different behavioral periodicities; ae. aegypti is a diurnally active mosquito, while an. gambiae and cx. quinquefasciatus exhibit nocturnal and crepuscular behaviors. we report revised annotations for the published ae. aegypti, an. gambiae and cx. quinquefasciatus opsin gene models. transcript and amino acid sequence similarity data support the identification of functional visual and non-visual opsins in these mosquitoes. transcripts were identified for each of the ae. aegypti and an. gambiae opsins and seven of the cx. quinquefasciatus genes. the predicted protein sequences of the mosquito opsins contain amino acid residues conserved in other rhodopsin-like gpcrs, including residues important for opsin function, and involved in post-translational modifications for n-linked glycosylation, palmityolation and phosphorylation. published microarray and rnaseq analyses provide further support for functionality of the mosquito opsins and insights into possible temporal expression. aedes aegypti genes for four lw and one sw opsin were each down regulated once at different points in a 96 h period following a blood meal, with the exception of op which was down regulated twice. aedes aegypti rnaseq data suggest two lw, the uv and sw opsin genes are up regulated in sugar fed females when compared to blood feed females. these findings may help to explain why blood-fed females are largely unresponsive to external stimuli until ready to oviposit. genes for the lw opsins aagprop and op uv and sw were up-regulated in male mosquitoes suggesting that some visual capacities may develop earlier in males than in females. in situ hybridization studies by hu et al. using anti-opsin antibodies provide evidence for expression of lw, uv and sw opsins in the photoreceptor cells of adult ae. aegypti. mass spectrometry studies provide evidence for protein expression of an. gambiae opsins in whole head, eye and brain. little is currently known regarding the spectral sensitivity of the ae. aegypti, an. gambiae and cx. quinquefasciatus opsins. electroretinograms and microspectrophotometry sensitivity studies of larval eyes and the adult compound eye have shown that ae. aegypti exhibits two peaks of spectral sensitivity to uv and lw light, but equivalent erg and msp studies have not been published for an. gambiae or cx. quinquefasciatus. landing studies have tested the response of ae. aegypti and cx. quinquefasciatus to color targets, and suggest that these mosquitoes prefer red and black, and black and brown, respectively. equivalent studies have not been performed for an. gambiae, but this species exhibited an optomotor response under visible and infrared wavelengths. phylogenetic analyses placed the mosquito opsin genes into five distinct “functional” clades, namely visual uv, sw and lw opsins, non-visual pteropsins, and orthologs of the d. melanogaster opsin rh phylogenetic predictions for aagprop, op and op are supported by functional studies that report dual spectral sensitivity peaks of 500–550 nm and ~350 nm for aagprop and peaks of 350 nm for op and 400–450 nm for op in apis mellifera workers and drones, the non-visual pteropsin is expressed in the brain and is thought to function in regulation of circadian rhythm. the wavelength sensitivity of rh has not been determined and its role in d. melanogaster vision is not known. the mosquito orthologs of rh are located in a sister clade to the uv and sw clades, indicating that these receptors may respond to short wavelengths. these results corroborate other phylogenetic studies and support hypotheses for the relative divergence times of the four major holometabolus orders, with hymenoptera as the more ancient and coleoptera, diptera and lepidoptera as more derived orders, as reviewed by grimaldi and engel, and supported by the recent work of misof et al.. as suggested by cameron and mardulyn, these findings highlight the utility of lw opsins for resolution of higher-level phylogenetic relationships. expansions of lw opsins have been noted in insects and the aquatic invertebrates d. pulex, dragonflies and stomatopods. we observed an expansion of putative lw opsins in ae. aegypit, an. gambiae and cx. quinquefasciatus relative to other insects, which typically have between one to four lw opsins. studies suggest that the order diptera arose in the carboniferus approximately my and that the divergence of the culicinae and anophelinae occurred in the permian, triassic or jurassic, possibly between and my or in the triassic period at approximately my. our results suggest that the lw opsins arose via multiple gene duplication events in the most recent common ancestor of the anophelinae and culicinae lineage in the jurassic, approximately – my, following which independent duplications occurred at least once in each of the three mosquito lineages. the mosquito putative lw opsins share significant amino acid identity but limited nucleotide conservation in the non-coding regions between paralogs. the studies of lynch and conery suggest that while the rate of origin of new gene duplicates may be high, the rate of duplicate preservation is low, and the authors predict the average half-life of a gene duplicate is approximately my. the retention of duplicated lw genes in mosquitoes over long evolutionary periods implies the functional importance of their gene products. supporting the theory of duplication of lw opsins is the observation that five an. gambiae lw opsins are tandemly arrayed within a 90 kb region on chromosome 2r, and are separated from a sixth, presumably more ancestral lw opsin, by approximately 3 mb. conservation of syntenic blocks has been observed between an. gambiae, ae. aegypti, and cx. quinquefasciatus. the production of improved assemblies and physical maps for the latter two species will permit studies of the opsin synteny among these species and may similarly illuminate gene evolution in ae. aegypti and cx. quinquefasciatus. the identification of multiple putative lw sensitive opsins in the three mosquitoes could reflect an adaptation to photic environments involving lw light, as suggested by futahashi et al.,. adult ae. aegypti, an. gambiae and cx. quinquefasciatus exhibit differences in times of peak activity but all are active at periods when long wavelength light predominates. aedes aegypti oviposition peaks at sunset when longer wavelengths are more abundant. studies have shown that ae. aegypti larvae and adults are capable of responding to uv and visible light, with the highest peak of sensitivity between and 523 nm – i.e., in the lw spectrum. both an. gambiae and cx. quinquefasciatus exhibit activity peaks between 22: and 02:00 h and enter dwellings to blood feed usually when occupants are asleep. moonlight is long-wavelength-shifted and nocturnal mosquitoes are in general, more active during moonlit nights. additionally, the larvae and pupae of ae. aegypti, an. gambiae and cx. quinquefasciatus are active in shallow aquatic environments that are typically associated with longer wavelengths due to the defraction of incident light. supporting this theory is the identification of extreme expansions in the lw opsins in the aquatic invertebrate, daphnia pulex, stomatopods and dragonflies. lw opsin expansions have also been noted in cave fish and the guppy, poecilia reticulate. the present study provides the first comparative genomics analyses of lw opsins in mosquitoes. rt-pcr studies reported here support expression of all opsins except aagprop and op in ae. aegypti 4th instar larvae and pupae, and a role in visual processes in immature mosquitoes. little is known regarding opsin expression in mosquito larvae and pupae and the possible link between aquatic life-style and the duplication and retention of lw opsin deserves further investigation. six or seven duplication events involving lw opsins are shared between the culicinae and anophelinae, suggesting retention following events at approximately – to – my in the mesozoic. it is possible that lw opsins evolved following a variety of events such as asteroid impacts or volcanic activity toward the end of the mesozoic when sun- and moon-light would presumably have been reduced, or the appearance of diverse vertebrate hosts during the cenozoic period. based on the morphology of mosquito ommatidia, kawada et al. proposed that “crepuscular behavior of mosquitoes is a transitional behavior in the course of evolution of nocturnal behavior to diurnal behavior”, explained as a consequence of either environmental changes and/or behavioral changes in vertebrate hosts. diversification of lw opsin function has not been established in mosquitoes, but could facilitate crepuscular and diurnal behavior. interestingly, it has also been proposed that inversion complexes on the an. gambiae chromosome arm 2r are associated with ecological adaptations that increase the fitness of the carriers. analyses of synteny among an. gambiae, ae. aegypti and cx. quinquefasciatus will permit further investigation of this hypothesis. two lw opsin duplication events are specific to the anopheline and six to the culicine and may reflect lineage and species-specific light detection capabilities. this hypothesis is supported by the observation of key morphological differences between the eyes of aedes, anopheles, and culex species. the ae. aegypti rhabdom is longer and cylindrical and the lens of the ommatidium is smaller and less hemispheric in comparison to that of an. gambiae. key morphological differences in the size and form of the facet lenses, rhabdom and the interommatidial angle consistently group ae. aegypti with other diurnal mosquitoes, and an. gambiae and cx. quinquefasciatus with other nocturnal mosquitoes. thus, ommatidial structures may vary depending on the photo-environment in which mosquitoes are active and may be of limited application as a taxonomic character. further studies are required to tease apart the contribution of lw opsins to the visual capacities of diurnal versus nocturnal mosquitoes. the amino acid identity of the lw opsins observed both within and between mosquito taxa is notable considering time of divergence predictions for these duplicates. these data raise intriguing questions about the retention and conservation of lw opsin genes in the culicinae and anophelinae. pseudogenization would appear unlikely and the contribution of both sub- and neofunctionalization deserves attention. in this study, we explored two possible underlying molecular mechanisms - gene selection and differential expression – that could contribute to the retention of duplicated lw opsins in mosquitoes. several studies have proposed opsin functional diversification as an explanation for gene retention, and examples of this phenomenon have been described in insects. the three d. melanogaster lw opsins rh rh and rh exhibit different spectral sensitivities and are expressed in different ommatidial cells and elsewhere in the body. rh is the major pigment in prcs 1– and is also involved in temperature discrimination by larvae. rh is expressed in both ocelli and testis, and rh is expressed in prc and extra-retinal tissue associated with auditory processes. opsin transcripts have been identified in diverse tissues, including the rostrum, leg, abdomen, antenna, maxillary palp, proboscis and ovary of ae. aegypti, and the maxillary palp and antenna of an. gambiae and opsins have been identified in the antenna, maxillary palp and proboscis of an. gambiae. based on several lines of evidence, including work in d. melanogaster, bohbot et al. speculated that aagprop and op expressed in the maxillary palps of adult mosquitoes, may be involved in heat sensing. the molecular role of mosquito opsins in a variety of sensory and reproductive tissues remains unclear and detailed studies are required to explore the possibility of functional diversification. the p. xuthus and p. glaucus lw opsins, pxrh and pgrh have four amino acid substitutions in tm domain i and tm domain iii that are predicted to “shift” the absorption spectra from green to red. it has also been proposed that the lw genes of d. pulex have a role in the adaptation of the water flea to a more complex light regime in aquatic environments and that the lw opsins in stomatopods diverged with respect to spectral tuning and signal transduction. the immature and adult stages of species of culicine and anopheline mosquitoes are active at low light intensities. we propose that a “suite” of opsins with different wavelength sensitivities may enable maximal capture of photons across the lw spectrum and improve visual acuity. this hypothesis is supported by the studies of hu et al. who also proposed similar roles for these receptors. physiological studies are required to explore the spectral sensitivities of mosquito lw opsins and the contributions of each opsin to light capture in mosquitoes. using fitmodel, we identified nine residues in lw opsins that are possibly experiencing positive selection. in preliminary analyses using paml software, we identified multiple candidate residues, including multiple residues in tmdiii possibly under positive selection. the modest number of residues identified only in the most recently duplicated lw opsins likely reflects the stringency of fitmodel. these residues are located in the 5′ extracellular and 3′ intracellular regions and tmdiii. while further work such as site-directed mutagenesis studies are required to determine the significance of this finding, and to evaluate a possible association between tmd residues and opsin spectral tuning, these analyses suggest that functional diversification may play a role in retention of at least some lw opsins in mosquitoes. the prediction that the c residue in cqgprop and aagprop is under positive selection is significant as previous studies involving species of the chelicerata, crustacea and insecta have shown that this region is important for tertiary structures associated with opsin function. this region may play a crucial role in the functional diversification of arthropod opsins. interestingly, studies in bees have shown that mutations in the positively selected lw opsins may enable spectral tuning to maximize visual capabilities for foraging in dim-light in ancestral bees, thought to be diurnal. the authors identified positively selected codons one of which aligns adjacent c in the positively selected aagprop cqgprop and this suggests a case of possible convergent evolution, as it has been proposed that mosquitoes were also diurnal feeders that developed crepuscular and night feeding behaviors. our discovery that nine out of a possible sites may be under positive selection suggests that other mechanisms are likely responsible for the retention of the duplicated lw opsins over a timeframe of more than my. to evaluate adaptive changes associated with the non-coding regions of lw opsins, we examined kb of upstream sequence for the presence of conserved non-coding sequences and transcription factor binding sites. binding sites for transcription factors that regulate the spatial and temporal patterns of gene expression are located in the non-coding sequence. accumulation of null mutations in the regulatory regions of recently duplicated genes that could contribute to differential gene regulation has been proposed as a mechanism for retention of duplicates. nucleotide conservation was observed in seven out of alignments representing lw mosquito opsins, suggesting some level of coordinated expression between at least seven paralog pairs. this also suggests the deterioration of sequence similarity across the subfamily through accumulation of mutations. the lack of dna sequence similarity in the 5′ and 3′ non-coding regions between opsin orthologs and paralogs, and the apparent lack of common transcription units, suggests the possibility of differential opsin expression in ae. aegypti, an. gambiae and cx. quinquefasciatus. our analysis revealed multiple copies of and different tfbs in the conserved regions between aggprop and op cqgprop and op aagprop and op aagprop and op aagprop and op cqgprop and op aagprop and op cqgprop and op respectively. these tfbs are insect specific and the possibility of additional, novel tfbs in these regions can’t be ruled out. for context, shared tfbs are associated with less than 2% of genes in d. melanogaster. no tfbs were identified between aagprop aggprop and cqgprop, supporting the hypothesis of greater time since divergence from the mrca. presumably, extensive accumulation of mutations in the cns regions of these genes masks the identification of tfbs and suggests potential involvement of different regulatory units in expression. interestingly, preliminary rt-pcr analyses detected transcripts for all opsins in all stages and sexes of ae. aegypti, and in an. gambiae male and female adults. the studies of rund et al. suggest a more complex picture, with rhythmic expression of phototransduction pathway genes in an. gambiae under light–dark and/or dark-dark conditions, including aggprop. analyses of the rund et al. data identified rhythmic expression for six opsin genes under ld and the two pteropsins. for the dd regime, we identified rhythmic expression for three opsin genes, two lw and the rh ortholog. the studies of dissanayake et al. also support differential opsin transcript levels in ae. aegypti males and females, and hu et al. showed differential expression of genes for aagprop op and op in the retina, dorsal, central, and ventral regions of the adult compound eye. collectively, these studies suggest a complex pattern of differential gene expression in mosquitoes. further studies are required to examine the differential expression of lw opsin genes and their possible roles in adaptive visual sensitivity across mosquito taxa.  evolutionary analyses of ae. aegypti, an. gambiae and cx. quinquefasciatus lw opsins suggests gene retention in the lineages anophelinae and culicinae, and the functional importance of these genes. similar expansions have been observed in other aquatic invertebrates and vertebrates, with the most extreme cases in invertebrates, suggesting a gene family that is prone to duplication. time of divergence predictions suggest mosquito opsin gene duplication events occurred in the mesozoic and cenozoic. positive selection and coordinated regulation represent two mechanisms for the retention of lw opsins in the three mosquitoes. of the genes, the retention of can be explained through positive selection, through coordinated regulation, and through both mechanisms. these genes were assigned to the more recent clades and with the exception of cqgprop which is a member of clade the retention of four lw opsins, members of the more ancestral clades and was not explained by either mechanism. research is needed to resolve the spectral sensitivity, spatio-temporal expression and function of mosquito lw opsins in visual/non-visual processes. the potential connection of mosquito lw opsins to lifestyles associated with lw-dominated photo-environments and crepuscular/nocturnal activity, deserves further investigation. our data suggest involvement of lw opsins in lineage- and species-specific processes and provide an important foundation for future efforts directed at identifying opsin-mediated behaviors that could be exploited to achieve vector control. 
we explore two mechanisms - positive selection and differential expression mediated by regulatory units in cns – that may have contributed to the retention of lw opsin genes in culicinae and anophelinae. mosquitoopsinlong-wavelengthgene duplicationpositive selectiondifferential expressionconserved non-coding sequencesvisionhttp://dx.doi.org/ /100006377purdue universityinternal fundshill catherine a. issue-copyright-statement© the author 2017 alignment of 5′ non-coding regions revealed potential conserved non-coding sequences and transcription factor binding sites in seven pairs of lw opsin paralogs. of these, eight amino acids occur in the n and c termini and are shared among all three species, and one residue in tmiii was unique to culicine species. time of divergence estimates suggest that the mosquito lw opsins originated from or duplication events between / to / million years ago and that these likely occurred following the predicted divergence of the lineages anophelinae and culicinae 145– my. we discuss the evolution of mosquito lw opsins in the context of major earth events and possible adaptation of mosquitoes to lw-dominated photo environments, and implications for mosquito control strategies based on disrupting vision-mediated behaviors. fitmodel analyses identified nine amino acid residues in the lw opsins that may be under positive selection. here we report the first detailed molecular and evolutionary analyses of lw opsins in three mosquito vectors, with a goal to understanding the molecular basis of opsin-mediated visual processes that could be exploited for mosquito control. opsins are light sensitive receptors associated with visual processes. six putative lw-sensitive opsins predicted in the yellow fever mosquito, aedes aegypti and malaria mosquito, anopheles gambiae, and eight in the southern house mosquito, culex quinquefasciatus, suggest gene expansion in the family culicidae relative to other insects.
 opsins are light sensitive receptors associated with visual processes. insects typically possess opsins that are stimulated by ultraviolet, short and long wavelength radiation. six putative lw-sensitive opsins predicted in the yellow fever mosquito, aedes aegypti and malaria mosquito, anopheles gambiae, and eight in the southern house mosquito, culex quinquefasciatus, suggest gene expansion in the family culicidae relative to other insects. here we report the first detailed molecular and evolutionary analyses of lw opsins in three mosquito vectors, with a goal to understanding the molecular basis of opsin-mediated visual processes that could be exploited for mosquito control.  time of divergence estimates suggest that the mosquito lw opsins originated from or duplication events between / to / million years ago and that these likely occurred following the predicted divergence of the lineages anophelinae and culicinae 145– my. fitmodel analyses identified nine amino acid residues in the lw opsins that may be under positive selection. of these, eight amino acids occur in the n and c termini and are shared among all three species, and one residue in tmiii was unique to culicine species. alignment of 5′ non-coding regions revealed potential conserved non-coding sequences and transcription factor binding sites in seven pairs of lw opsin paralogs.  our analyses suggest opsin gene duplication and residues possibly associated with spectral tuning of lw-sensitive photoreceptors. we explore two mechanisms - positive selection and differential expression mediated by regulatory units in cns – that may have contributed to the retention of lw opsin genes in culicinae and anophelinae. we discuss the evolution of mosquito lw opsins in the context of major earth events and possible adaptation of mosquitoes to lw-dominated photo environments, and implications for mosquito control strategies based on disrupting vision-mediated behaviors. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords mosquitoopsinlong-wavelengthgene duplicationpositive selectiondifferential expressionconserved non-coding sequencesvisionhttp://dx.doi.org/ /100006377purdue universityinternal fundshill catherine a. issue-copyright-statement© the author 2017 