SHORT COMMUNICATION
Cloning and Sequence Analysis of the Circadian Clock Genes period
and timeless in Aedes albopictus (Diptera: Culicidae)
KEITH SUMMA, JENNIFER M. URBANSKI, XIUMEI ZHAO, MONICA POELCHAU,
1
AND PETER ARMBRUSTER
Department of Biology, Reiss 406, Georgetown University, 37th and O Streets NW, Washington, DC 20057-1229
J. Med. Entomol. 49(3): 777Ð782 (2012); DOI: http://dx.doi.org/10.1603/ME11171
ABSTRACT The genes period (per) and timeless (tim) are core components of the circadian clock
that regulates a wide range of rhythmic biochemical, physiological, and behavioral processes in
prokaryotes and eukaryotes. We used degenerate polymerase chain reaction (PCR) and Rapid
AmpliÞcation of cDNA Ends (RACE) to clone and sequence the entire cDNAs of both the per and
tim genes in Aedes albopictus (Skuse). We also used the 5⬘ end of the Ae. albopictus per cDNA to
identify previously unannotated sequence coding for the N-terminal region of the PERIOD protein
in Aedes aegypti L. We sequenced genomic DNA of one mosquito from each of three geographically
distinct populations (New Jersey, Florida, and Brazil), and identiÞed three introns in the per gene and
eight introns in the tim gene. Phylogenetic analyses and comparison of functional domains support
the orthology of the newly identiÞed per and tim genes. Analysis of nonsynonymous to synonymous
substitution rates indicates that both the per and tim genes have evolved under strong selective
constraint subsequent to the divergence of Ae. albopictus and Ae. aegypti. Taken together, these results
provide resources that can be used to investigate the molecular genetics of circadian phenotypes in
Ae. albopictus and other culicids, to perform comparative analyses of insect circadian clock function,
and also to conduct phylogeographic analyses using single-copy nuclear introns.
KEY WORDS period, timeless, circadian clock, Aedes albopictus
Circadian rhythms are endogenous, self-sustaining biological rhythms with a period of ⬇24 h that are nearly
ubiquitous in nature (Dunlap et al. 2004). Circadian
rhythms provide an adaptive mechanism coordinating
biochemical, physiological, and behavioral activities
with the rhythmic patterns of light, temperature, and
other factors that ßuctuate periodically according to
the daily rotation of the Earth. These rhythms are
programmed by an internal biochemical oscillator
(i.e., “clock”) that consists of core molecular machinery that is well conserved among Metazoans (Harmer
et al. 2001).
The molecular underpinnings of the circadian clock
have been particularly well characterized in Drosophila melanogaster (Meigen) where the core clock consists of a set of proteins that form an autoregulatory
negative feedback loop with a period of ⬇24 h (Dunlap 1999, Young 2000, Panda et al. 2002, Allada and
Chung 2010). Brießy, the circadian proteins CLOCK
(CLK) and CYCLE (CYC) dimerize in the nucleus,
where they bind E-box elements in target gene promoters and activate transcription of a number of
downstream genes, including period (per) and timeless
(tim). The PER and TIM proteins then form a dimer
in the cytoplasm that translocates to the nucleus,
1
Corresponding author, e-mail: [email protected].
where it physically interacts with and inhibits CLKCYC, thereby repressing transcription. Temporal delays between these activation and repression steps, as
well as posttranslational modiÞcation of core clock
components, give rise to stable 24 h oscillations in gene
expression that characterize circadian rhythms and
underlie the generation of overt 24 h behavioral
rhythms, such as locomotor activity (Allada and
Chung 2010). Additionally, light-induced negative
regulation of TIMELESS by the photoreceptor CRYPTOCHROME (CRY) (Ceriani et al. 1999) provides a
mechanism for entrainment, coordinating the internal
circadian clock with the exogenous light:dark cycle.
Although many basic elements of the circadian clock
appear to be relatively well conserved among Metazoans, it is becoming increasingly clear that signiÞcant
evolutionary modiÞcations of the circadian clock have
taken place during the divergence of insects (Rubin et
al. 2006, Yuan et al. 2007, Sandrelli et al. 2008).
Recently, there has been increased interest regarding the molecular basis of circadian rhythms in vector
mosquitoes (Gentile et al. 2006, 2009; Das and Dimopoulos 2008; Rund et al. 2011; Ptitsyn et al. 2011).
Indeed, a variety of crucial behavioral traits related to
disease transmission such as ßight activity, host seeking, and blood feeding have been shown to have a
circadian basis (Clements 1999, Das and Dimopoulos
0022-2585/12/0777Ð0782$04.00/0 䉷 2012 Entomological Society of America
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JOURNAL OF MEDICAL ENTOMOLOGY
Vol. 49, no. 3
Table 1. Percent identity of amino acid sequences in conserved functional domains of the inferred PERIOD and TIMELESS proteins
between Ae. albopictus and those of Ae. aegypti and D. melanogaster
PERIOD
NLS (%)
PAS-A (%)
PAS-B (%)
PAC (%)
CLD (%)
Ae. aegypti
D. melanogaster
93
84
97
69
97
58
95
64
98
69
TIMELESS
NLS (%)
32aa (%)
PER-1 (%)
PER-2 (%)
CLD (%)
AD (%)
Ae. aegypti
D. melanogaster
100
100
100
85
97
65
99
64
53
18
87
0
2008). For example, Das and Dimopolous (2008) demonstrated a circadian basis to blood feeding behavior
in Anopheles gambiae (Giles) and showed that RNAimediated knockdown of three clock genes signiÞcantly affected blood feeding activity. Gentile et al.
(2006, 2009) have examined the circadian expression
of eight clock genes in Aedes aegypti L. and Culex
quinquefasciatus (Say). Their results (Gentile et al.
2009) show that despite distinct locomotor activity
patterns, the circadian expression of these genes is
well-conserved, except for the clock gene Cry2, which
may play a role in mediating species-speciÞc differences in rhythmic behaviors. Ptitsyn et al. (2011) and
Rund et al. (2011) have identiÞed multiple molecular
pathways exhibiting circadian expression patterns, as
well as rhythmic expression of per and tim, in Ae.
aegypti and A. gambiae respectively. Further genetic
studies of circadian-controlled processes have the potential to yield important insights into the molecular
bases of key traits affecting both Þtness and disease
transmission of vector mosquito species.
Herein, we report on the sequence and comparative
analysis of the two core clock genes per and tim in the
Asian tiger mosquito, Ae. albopictus (Skuse). First, we
used degenerate polymerase chain reaction (PCR)
and Rapid AmpliÞcation of cDNA Ends (RACE) to
clone and sequence both genes. Next, these sequences
were used in BLAST searches to identify a previously
unannotated region of the per gene encoding the Nterminal region of the PER protein in Ae. aegypti.
Finally, we sequenced genomic DNA of a single mosquito from each of three geographically distinct populations (New Jersey, Florida, and Brazil) to identify
introns within the Ae. albopictus per and tim genes.
These results provide resources that can be used to
investigate the molecular genetics of circadian phenotypes in Ae. albopictus and other Culicids, to conduct comparative analyses of circadian clock function
in insects, and to perform phylogeographic analyses
using single-copy nuclear introns.
Materials and Methods
A lab F7 colony of Ae. albopictus from Vero Beach,
FL. was maintained in the laboratory under a 16:8 h
(light:dark) photoperiod at 21⬚C and near-optimal
conditions as described previously (Armbruster and
Hutchinson 2002, Armbruster and Conn 2006). Seven
to 14 d old adult males and females were collected at
16 h after light onset (Zeitgeiber time (ZT) 16) because studies in other Diptera indicated that clock
gene expression peaks around the time of light to dark
transition (Gvakharia et al. 2000; Warman et al. 2000;
Goto and Denlinger 2002; Gentile et al. 2006, 2009).
Adults were CO2 anesthetized, frozen at ⫺80⬚C and
⬇200 heads were removed from the adult bodies on
dry ice and returned to ⫺80⬚C before RNA extraction.
RNA was extracted from adult heads using TRI Reagent (Sigma-Aldrich, St. Louis, MO) followed by
isopropanol precipitation (Sambrook and Russell
2001). The RNA was then resuspended in nuclease
free water at a concentration of 50 ng/␮l and stored
at ⫺80⬚C.
cDNA synthesis was performed with the SMART
PCR cDNA Synthesis Kit (Clontech, Mountain View,
CA) and oligo-dT priming according to the supplierÕs
instructions. Reverse transcription was conducted
with the following thermal proÞle: 42⬚C for 2 min, after
which 1 ␮l of reverse transcriptase was added, 42⬚C for
50 min, 70⬚C for 15 min, after which 1 ␮l of RNase H
was added, and 37⬚C for 20 min.
The degenerate primers PERC2FOR and
PERD2REV and TIMAFOR and TIMC2REV (Supp.
Appendix A [online only]) were designed based on
the published sequences of insect per and tim available
from NCBI. PCR ampliÞcation was performed using
the BD Advantage 2 PCR Enzyme System Kit (Clontech) with 1 ␮l of cDNA at ⬇1 ␮g/␮l, 0.5 ␮l BD
Advantage 2 Polymerase Mix, 0.5 ␮l 50X dNTP mix, 2.5
␮l BD Advantage 2 PCR Buffer, and 1 ␮l of both the
forward and reverse primer, each at a concentration of
10 ␮M. The temperature proÞle for both the per and
tim ampliÞcations consisted of 5 min at 94⬚C, followed
by 30 cycles of 1 min at 94⬚C, 1 min at 48⬚C, 1 min at
72⬚C, followed by 5 min at 72⬚C. Per and tim ampliÞcation products of the expected size were excised from
a 1% agarose gel and puriÞed using a NucleoTrap Gel
Extraction Kit (Clontech) according to the manufacturerÕs instructions. The products were then cloned
into a pCR 2.1-TOPO vector used to transform TOP10
One Shot cells (Invitrogen, Carlsbad, CA). Plasmids
were puriÞed using the WizardPlus Miniprep DNA
puriÞcation system (Promega, Madison, WI) and inserts were sequenced using modiÞed M13 primers
(Supp. Appendix A [online only]) and Big Dye terminator chemistry. Contig assembly was performed
with Sequencher v. 4.5 (Gene Codes Corporation,
Ann Arbor, MI).
To determine the complete cDNA sequence of the
putative Ae. albopictus per and tim genes we performed Rapid AmpliÞcation of cDNA Ends (RACE)
using the BD SMART RACE cDNA AmpliÞcation kit
May 2012
SUMMA ET AL.: period AND timeless GENES IN Aedes albopictus
(Clontech). Gene-speciÞc primers (Supp. Appendix
A [online only]) were used to PCR-amplify 5⬘ and 3⬘
RACE products that were gel puriÞed, cloned, and
sequenced as described above. Contig assembly was
performed as described above and the inferred amino
acid sequences were deduced by using ORF Finder
(http://www.ncbi.nlm.nih.gov/projects/gorf/) followed by BlastP searches to verify sequence identity.
To identify introns and assess sequence variation
across partial regions of both genes we sequenced an
⬇3.3 Kb fragment of the per gene and two fragments
of the tim gene, one ca. 2.2 Kb and the other ca. 1.9 Kb.
Each of these three regions was sequenced from
genomic DNA of individual mosquitoes collected
from each of three populations: 1) Burlington, NJ; 2)
Fort Lauderdale, FL; and c) Salvador, Bahia State,
Brazil. Genomic DNA was isolated using methods described by Severson (1997). PCR reactions consisted
of 21 ␮l sterile H2O, 1 ␮l of template genomic DNA,
1 ␮l each of forward and reverse primers (10 ␮M
concentration) and a single “ready-to-go” PCR bead
(Amersham Pharmacia, Buckinghamshire, United
Kingdom). The thermal proÞle consisted of 5 min at
98⬚C followed by 35 cycles of 30 s at 94⬚C, 1 min at
58 Ð 60⬚C, 1 min at 72⬚C and then a Þnal 10 min extension period at 72⬚C. PCR products were puriÞed using
Centri-Spin columns (Princeton Separations, Adelphia,
NJ) before sequencing and contig assembly as described
above. Genomic sequences were aligned against corresponding cDNA sequences in ClustalW and introns were
identiÞed by visual inspection as well as comparison to
the predicted translation sequence.
To conduct phylogenetic analyses of inferred dipteran PER and TIM proteins we obtained cDNA or
amino acid sequences of the following taxa from NCBI
or VectorBase (accession numbers for PER and TIM,
respectively, are indicted in parentheses): 1) Ae. aegypti (XP_001658976, AAY40757.1); 2) Cx. quinquefasciatus (XP_001849299.1, XP_001848611.1); 3) A.
gambiae (AGAP001856, AGAP008288); 4) Sarcophaga
bullata (BAB85488, BAB85487); 5) Chymomyza
costata (BAA28711, BAB91179); 6) D. melanogaster
(NP_525056.2, P49021.2); and 7) D. virilis (P12349,
AAB94930). We used Antheraea pernyi (Q17062,
AAF66996) as an outgroup in these analyses. The
current Ae. aegypti per annotation on VectorBase
(AaegL1.2) is incomplete because the predicted protein sequence begins with a glycine residue rather
than methionine and is missing 98 N-terminal amino
acids relative to the predicted Ae. albopictus PER protein. The DNA sequence encoding this missing N-terminal section in Ae. aegypti is located ⬇30 Kb upstream of the incomplete annotated sequence
between bases 494063Ð 494358 on supercontig 1.303.
The resulting sequence matches the corresponding
Ae. albopictus per sequence with 79% identity at the
amino acid level and 81% identity at the nucleotide
level. This missing inferred amino acid sequence was
joined to the annotated inferred Ae. aegypti protein
sequence for phylogenetic analysis described below.
Inferred amino acid sequences of both PER and
TIM were aligned in ClustalW with a gap opening
779
Table 2. Intron characteristics for Ae. albopictus period and
timeless genes based on genomic DNA sequence of one mosquito
from each of a New Jersey, Florida, and Brazil
period
Intron 1
Intron 2
Intron 3
timeless
Intron 1
Intron 2
Intron 3
Intron 4
Intron 5
Intron 6
Intron 7
Intron 8
Positiona
Lengthb
(bp)
⌸c
Indel
sites
2,964
3,250
3,663
96
106
86
0.14
0.07
0.26
0
0
34
872
1,122
1,568
1,711
1,999
2,222
2,420
2,847
61
51
69
98
96
156
67
61
0.13
0.00
0.02
0.08
0.08
0.10
0.12
0.04
2
9
0
1
0
2
1
5
a
Position no. relative to sequence as presented in Appendices B
(period) and C (timeless).
b
Based on sequence from Burlington, NJ.
c
Nucleotide diversity (Nei and Li 1979).
penalty of 15 and a gap extension penalty of 0.3. (Supp.
Appendices D and E [online only]). Neighbor joining
trees were constructed in MEGA4 (Tamura et al.
2007) with Poisson corrected amino acid distances and
1,000 bootstrap replicates. Conserved functional domains of both proteins were identiÞed based on visual
inspection of the protein alignments and comparison
with previous annotations in D. melanogaster (Myers
et al. 1995, Saez and Young 1996, Ousley et al. 1998),
Lucilla cuprina (Warman et al. 2000), and Ae. aegypti
(Gentile et al. 2006). Percent amino acid identity was
computed between functional domains of the Ae. albopictus, Ae. Aegypti, and D. melanogaster inferred
protein sequences using BlastP. We also computed the
ratio of nonsynonymous (dN) to synonymous (dS)
substitutions between orthologues of per and tim in Ae.
albopictus and Ae. aegypti to test for evidence of positive selection acting on each gene. Codon-based
alignments were constructed using PAL2NAL (Suyama et al. 2006) and dN/dS ratios were computed
using a sliding window analysis in DNAsp v.5 (Librado
and Rozas 2009). We also searched for short areas of
conserved sequence in the 5⬘ and 3⬘ UTRs of per and
tim in Ae. albopictus, An. gambiae, and D. melanogaster
(UTRs for these genes are not annotated in Ae. aegypti). We used the web-based program MEME (Multiple EM for Motif Elicitation; http://meme.sdsc.edu/
meme/cgi-bin/meme.cgi; Bailey and Elkan 1994) with
the following modiÞcation to the default settings: we
searched for 5Ð50 bp motifs, where each motif could
occur two to three times per UTR. For the per 3⬘ UTR,
we were only able to use sequence from Ae. albopictus
and D. melanogaster, as this UTR is not annotated in
An. gambiae. We report only motifs that have Þve or
more perfectly conserved consecutive bases across all
three species. Finally, we calculated the average nucleotide diversity, ⌸ (Nei and Li 1979), among
genomic DNA samples from three populations (New
Jersey, Florida, and Brazil) for three per and eight tim
intron sequences (Table 2).
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Vol. 49, no. 3
Fig. 1. Neighbor-joining tree of Dipteran (A) PERIOD and (B) TIMELESS inferred amino acid sequences based on
Poisson corrected distances with the Lepidopteran A. pernyi as the outgroup. Percent bootstrap support based on 1,000
replicates with only values ⬎50% shown on tree.
Results
Degenerate PCR using primers PERC2FOR and
PERD2REV (Supp. Appendix A [online only]) produced a single fragment of the expected size (ca. 500
bp). Subsequent sequencing of 3⬘ and 5⬘ RACE PCR
products yielded a cDNA sequence of 4,900 bp with
546 bp in the 5⬘ UTR, an open reading frame of 3,897
bp and 457 bp in the 3⬘ UTR (GenBank JN559769;
Supp. Appendix B [online only]). The presence of a
stop codon as well as polyadenylation signal
(AATAAA) and poly-A tail indicates that the entire 3⬘
end of the transcript was sequenced. The deduced
protein sequence contains 1298 amino acids, and a
BlastP search against the NCBI nonredundant protein
database indicated 39% amino acid identity (Evalue ⫽ 0.0) to the PER protein in D. melanogaster.
The deduced amino acid sequence of PER in Ae.
albopictus has particularly high similarity to PER in Ae.
aegypti in several putative functional domains, with
percent amino acid identities ranging from 93Ð98%
for the nuclear localization signal (NLS; Vosshall et
al. 1994), PER-ANRT-SIM (PAS-A, PAS-B; Zhulin et
al. 1997), PAS-associated C-terminal (PAC; Zhulin
et al. 1997), and cytoplasmic localization (CLD;
Saez and Young 1996) domains (Table 1A). Percent
amino acid identity between the Ae. albopictus and
D. melanogaster PER sequences is also high in these
functional domains, ranging from 58 to 84% (Table
1A). The D. melanogaster PER contains a threonineglycine (TG) repeat region that is thought to contribute to temperature compensation of the circadian clock (Sawyer et al. 1997), but there is only one
TG pair in Ae. albopictus (Supp. Appendix B [online
only]) and all other Culicids considered here.
Degenerate PCR using primers TIMAFOR and
TIMC2REV (Supp. Appendix A [online only]) also
produced a single fragment of the expected size (ca.
1,000 bp). Sequencing of 5⬘ and 3⬘ RACE products
yielded a cDNA of 6,269 bp with 1,243 bp in the 5⬘
UTR, an open reading frame of 3,756 bp, and 1,270 bp
in the 3⬘ UTR (GenBank JN559770; Supp. Appendix C
[online only]). The tim cDNA contains a stop codon
and poly-A tail but it does not have a canonical polyadenylation signal in the 3⬘ UTR. The deduced amino
acid sequence contains 1,251 amino acids and has a
high level of similarity (47% amino acid identity, Evalue 0.0) to the TIM protein in D. melanogaster.
Similarity between putative functional domains of
the inferred TIM proteins is also high between Ae.
albopictus and Ae. aegypti, ranging from 97Ð100% identity for the NLS (Saez and Young 1996), the 32 amino
acid region (32aa; Ousley et al. 1998), the PER interaction domains (PER-1 and PER-2; Saez and Young
1996), 87% for the acidic domain region (AD; Myers
et al. 1995), and 54% for the CLD (Saez and Young
1996) region (Table 1B). Percent identity for the NLS,
32aa, PER-1 and PER-2 regions between Ae. albopictus
and D. melanogaster is also high, ranging from 64 Ð100%
(Table 1B). However, the CLD region is far less conserved (18% identity) between Ae. albopictus and D.
melanogaster, and the AD region appears to have completely lost amino acid sequence similarity (% similarity ⫽ 0; Table 1B).
Phylogenetic analysis of putative orthologues of
PER (Fig. 1A) and TIM (Fig. 1B) inferred amino acid
May 2012
SUMMA ET AL.: period AND timeless GENES IN Aedes albopictus
sequences from a diverse group of Diptera indicates a
well supported topology that is consistent with the
established taxonomic relationships among these species. A sliding-window analysis of dN/dS between the
per nucleotide sequences of Ae. albopictus and Ae.
aegypti indicates that dN/dS ranges from 0 to 0.55
across the length of the coding sequence with an
overall average of 0.09. These results imply that the per
locus has been under strong purifying selection during
the divergence of Ae. albopictus and Ae. aegypti. The
dN/dS for tim nucleotide sequences of Ae. albopictus
and Ae. aegypti shows one narrow peak with values of
ca. 1.5 around nucleotide position 1,000 of the open
reading frame. However, similar to per, dN/dS values
range from 0 to 0.5 throughout all other areas of the
sequence and the overall dN/dS ratio is 0.17. These
results imply that while there may be narrow regions
of the tim gene that are evolving in a neutral fashion
between Ae. albopictus and Ae. aegypti, the majority of
the gene sequence is evolving under strong selective
constraint, similar to the per gene. We identiÞed two
short areas of conserved sequence in the 5⬘ UTR of per,
four in the 3⬘ UTR of per, two in the 5⬘ UTR of tim, and
zero in the 3⬘ UTR of tim (Supp. Appendices B and C
[online only]). These sequences may be of interest for
future functional investigation.
Analysis of genomic DNA sequence fragments of
both per and tim from individual mosquitoes from each
of New Jersey, Florida, and Brazil revealed three introns in the per gene ranging from 86 to 106 bp, and
eight introns in the tim gene ranging from 51 to 156 bp.
Overall nucleotide diversity (⌸) ranged from 0.00 to
0.26 between intron sequences from the three geographically disparate samples (Table 2), suggesting
that these noncoding regions might provide useful
markers for phylogeographic analyses.
Discussion
The circadian clock is an endogenous oscillator
comprised of an autoregulatory feedback loop that
regulates a wide range of rhythmic biochemical, physiological, and behavioral processes in prokaryotes and
eukaryotes (Dunlap et al. 2004). Phylogenetic analyses based on per and tim inferred amino acid sequences from Ae. albopictus and a diverse group of
Diptera indicate a strongly supported topology that is
consistent with taxonomic classiÞcation of these species and is highly congruent between both the per and
tim trees (Fig. 1). Overall percent amino acid identity
between Ae. albopictus and Ae. aegypti was high for
both per (83%) and tim (84%) and in several functional domains identities ranged between 87Ð100%
(Table 1). These results strongly support the orthology of the newly identiÞed per and tim sequences in
Ae. albopictus to previously identiÞed per and tim
genes in Ae. aegypti and other Diptera. D. melanogaster
has a paralogue of the timeless gene called timeless2/
timeout (Benna et al. 2000, Dunlap et al. 2004), but
when this paralogue is included in the phylogenetic
analysis of inferred TIMELESS sequences from Fig. 1B
it is positioned externally to all other sequences, in-
781
dicating that none of the sequences we included in our
analysis were paralogous rather than orthologous gene
copies of tim.
In addition to the relevance to circadian biology, the
sequence data we present from introns of the per and
tim genes may provide useful molecular markers for
phylogeographic analysis of Ae. albopictus populations. Such markers would be particularly valuable for
Ae. albopictus, which is infected with two strains of the
endosymbiotic bacterial parasite Wolbachia (Armbruster et al. 2003). Wolbachia-induced cytoplasmic
incompatibility is well known to reduce mitochondrial
DNA variation (Turelli and Hoffmann 1991, 1995),
consistent with the low observed mitochondrial DNA
diversity within and among geographic populations of
Ae. albopictus (Birungi and Munstermann 2002, Usmani-Brown et al. 2009). Single copy microsatellite
markers have also proven difÞcult to isolate from Ae.
albopictus, further limiting variable molecular markers
available for phylogeographic analysis in this mosquito. Therefore, single-copy nuclear introns may be
valuable resources for population-level analyses of
genetic diversity. We found a wide range of nucleotide
diversity among introns from both the per and tim
genes as well varying levels of insertion/deletion (indel) diversity (Table 2). Interestingly, the per sequence from the Brazilian sample exhibited a 32 bp
deletion in intron three that did not occur in either the
New Jersey of Florida sample, suggesting this deletion
might be phylogeographically informative. Intron
three of the per gene also exhibited highly conserved
ßanking sequence, further suggesting that this intron
might be a useful genetic marker.
Acknowledgments
We thank L. Philip Lounibos for the Brazilian samples of
Ae. albopictus and Georgetown University for Þnancial support. Two anonymous reviewers provided helpful comments
on a previous version of this manuscript.
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Received 9 August 2011; accepted 17 January 2012.