miRNA Genes of an Invasive Vector Mosquito, Aedes albopictus
Citation: Gu J, Hu W, Wu J, Zheng P, Chen M, et al. (
miRNA Genes of an Invasive Vector Mosquito, Aedes albopictus
Jinbao Gu 0
Wanqi Hu 0
Jinya Wu 0
Peiming Zheng 0
Maoshan Chen 0
Anthony A. James 0
Xiaoguang Chen 0
Zhijian Tu 0
Kristin Michel, Kansas State University, United States of America
0 1 Key Laboratory of Prevention and Control of Emerging Infectious Diseases of Guangdong Higher Education Institutes, Department of Pathogen Biology, School of Public Health and Tropical Medicine, Southern Medical University , Guangzhou, Guangdong , P.R. China , 2 Department of Biochemistry, Virginia Tech, Blacksburg, Virginia, United States of America, 3 Beijing Genomics Institute , Beishan Road, Shenzhen, Guangdong , P.R. China , 4 Departments of Microbiology & Molecular Genetics and Molecular Biology & Biochemistry, University of California Irvine , Irvine, California , United States of America
Aedes albopictus, a vector of Dengue and Chikungunya viruses, is a robust invasive species in both tropical and temperate environments. MicroRNAs (miRNAs) regulate gene expression and biological processes including embryonic development, innate immunity and infection. While a number of miRNAs have been discovered in some mosquitoes, no comprehensive effort has been made to characterize them from different developmental stages from a single species. Systematic analysis of miRNAs in Ae. albopictus will improve our understanding of its basic biology and inform novel strategies to prevent virus transmission. Between 10-14 million Illumina sequencing reads per sample were obtained from embryos, larvae, pupae, adult males, sugar-fed and blood-fed adult females. A total of 119 miRNA genes represented by 215 miRNA or miRNA star (miRNA*) sequences were identified, 15 of which are novel. Eleven, two, and two of the newly-discovered miRNA genes appear specific to Aedes, Culicinae, and Culicidae, respectively. A number of miRNAs accumulate predominantly in one or two developmental stages and the large number that showed differences in abundance following a blood meal likely are important in blood-induced mosquito biology. Gene Ontology (GO) analysis of the targets of all Ae. albopictus miRNAs provides a useful starting point for the study of their functions in mosquitoes. This study is the first systematic analysis of miRNAs based on deep-sequencing of small RNA samples of all developmental stages of a mosquito species. A number of miRNAs are related to specific physiological states, most notably, pre- and post-blood feeding. The distribution of lineagespecific miRNAs is consistent with mosquito phylogeny and the presence of a number of Aedes-specific miRNAs likely reflects the divergence between the Aedes and Culex genera.
Funding: This work is supported in part by grants from the National Natural Science Foundation of China (U0832004) and GDUPS (2009) to Xiaoguang Chen, the
National Institute of Allergy and Infectious Diseases (AI070854) and NSFC (30828027) to Zhijian Tu and the National Institute of Allergy and Infectious Diseases
(U54AI065359) to Anthony A. James. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
MicroRNAs (miRNAs) are ,22 nucleotides (nt) in length and
modulate gene expression by targeting cognate mRNAs for
cleavage or translational repression. miRNAs are distributed
widely in metazoans and plants and are involved in the regulation
of many biological processes including apoptosis, cancer,
embryonic development, immunity and infection . miRNAs also
may have a role in mosquito responses to infection by malaria
parasite and arboviruses [4,5]. Many miRNAs are transcribed
from independent promoters to generate the primary miRNAs
(pri-miRNAs), which could contain one or more regions of
complementary bases that form secondary structures (hairpins).
These hairpins are recognized and liberated by the
Microprocessor complex to make precursor miRNAs (pre-miRNAs).
PremiRNAs are exported to the cytosol and processed further by
Dicer to make the ,22-nt miRNA:miRNA* duplex. The duplex is
separated by a helicase and the single-stranded miRNA is loaded
into the RISC complex., The miRNA* strand is normally rapidly
degraded . Other miRNAs reside in introns of genes and their
biogenesis is independent of the Microprocessor complex [7,8].
Deep-sequencing coupled with bioinformatic analyses has
produced comprehensive catalogs of miRNAs in model organisms.
For example, there are 171 miRNAs in Drosophila melanogaster
discovered in a large number of samples derived from different
developmental stages and tissues and from analysis of 12 Drosophila
genomes [9,10,11]. Deep-sequencing efforts to uncover miRNAs
in mosquitoes have been limited to specific developmental stages
or cell lines and the number discovered is relatively low compared
to D. melanogaster. A small number of miRNAs were verified
experimentally in two Anopheles mosquito species [4,12].
Ninetyeight miRNA genes that produce 86 distinct miRNAs were
discovered in Aedes aegypti, mostly by small-scale 454 sequencing of
embryo and midgut samples . A total of 65 and 77 miRNAs
were discovered following Illumina sequencing of small RNAs
from an Ae. albopictus cultured cell line and blood-fed Culex
quinquefasciatus females, respectively .While most of these
miRNAs are conserved across divergent species, 11 distinct
miRNA genes are found only in mosquitoes, some of which are
restricted to certain taxonomic groups [5,13].
Here we report discovery of 119 miRNA genes through
deepsequencing of small RNAs isolated from multiple developmental
stages of Ae. albopictus, of which 15 are novel and appear to be only
in mosquitoes. We chose Ae. albopictus for this study because of its
rapid expansion in world-wide distribution and its emerging
importance as a vector for Dengue and Chikungunya viruses .
Our analysis has doubled the number of known mosquito-specific
miRNAs, uncovered miRNAs showing stage-specific and
bloodmeal-regulated expression profiles, and provided the basis for the
investigations of the function and evolution of mosquito miRNAs.
Materials and Methods
All vertebrate animals were housed and handled in strict
accordance with the guidelines of the institutional and national
Committees of Animal Use and Protection. All experimental
procedures on mice were approved by the Committee on the
Ethics of Animal Experiments of Southern Medical University
(Permit Number: SCXK 2006-0015).
The CDC (Guangdong, China) strain of Aedes albopictus
originated in Guangzhou, Guangdong province, PRC, and was
established the laboratory in 1981. All mosquitoes were
maintained in humidified incubators at 2561uC on a 12 hour light:dark
Aedes albopictus Sample Preparation for Illumina
Embryos were collected 024 hours after egg deposition by
placing a damp collection cup within a cage. Larval samples were
collected at each instar and combined. Pupal samples were
collected from a pool of varied ages. Male and female adults were
collected five days post-emergence. Three- to five-day old adult
females were fed on mouse blood and collected at 1, 3 and 5 days
after feeding and pooled. Total RNA was isolated using Trizol
(Invitrogen). Approximately 20 mg of the total RNAs were
Figure 1. Size distribution of small RNAs derived from Illumina sequencing runs of six Aedes albopictus samples. Size distribution and
relative frequency in each sample are shown for the small RNAs derived from embryos, larvae, pupae, adult males, sugar-fed adult females, and
blood-fed adult females.
separated on a 15% denaturing polyacrylamide gel and small
RNAs ranging up to 30 nt in length were excised and sent to the
Beijing Genome Institute Inc. for sequencing and analysis. The
small RNAs were ligated sequentially to 59- and 39-end RNA
adapters. The small RNA molecules were amplified for 17 cycles
using the adaptor primers and fragments ,90 nt in length (small
RNA+adaptors) were isolated from agarose gels. The purified
DNA was used directly for cluster generation and sequencing
analysis using the Illumina Genome Analyzer (Illumina, San
Diego, CA, USA). Clean reads were processed for computational
analysis after removing adaptor sequences and contaminated
reads. ). All primary sequence read data have been submitted to
the National Center for Biotechnology Information (NCBI)
shortread archive (accession number SRA060684).
After removing low-quality sequences determined by inspection
of chromatographs, tags with lengths ranging from 1830 nt were
selected for further analysis. The subsequent procedures
performed with Solexa were summarizing data production, evaluating
sequencing quality, calculating length distribution of small RNA
reads and filtering reads contaminated by rRNA, tRNA, snRNA,
snoRNA, repeat, exon and intron sequences using the NCBI
Genbank database (http:www.ncbi.nlm.nih.gov/). Aedes aegypti, a
closely related mosquito in the same subgenus (Stegomyia) as Ae.
albopictus, provided a valuable reference genome to which clean
reads were aligned using SOAP . Sequences with a perfect
match or one mismatch were retained for further analyses. RNA
secondary structures were analyzed using 100 nt of genomic DNA
flanking each side of the sequence, and the secondary structures
predicted using RNAfold (http://rna.tbi.univie.ac.at/cgi-bin/
RNAfold.cgi) and analyzed by MIREAP (http://sourceforge.
net/projicts/mireap) at default settings. MIREAP is designed
specifically to identify genuine miRNAs from deeply-sequenced
small RNA libraries. It considers miRNA biogenesis, sequencing
depth and structural features to improve the sensitivity and
specificity of miRNA identification. Stem-loop hairpins were
considered typical only when they fulfilled three criteria: mature
miRNAs are present in one arm of the hairpin precursors, which
also lack large internal loops or mismatches; the secondary
structures of the hairpins are stable with free energies of
hybridization lower than -20 kcal/mol; and hairpins are located
in intergenic regions or introns. Those genes whose sequences and
structures satisfied all of these criteria were considered candidate
miRNA genes. Subsequently, the computational approach,
miRAlign, was adopted to predict new miRNA genes that are
paralogues or orthologues to known miRNAs sequences from
miRBase . The final step of miRNA confirmation was
performed by aligning Illumina small RNA reads to the predicted
pre-miRNA secondary structures to rule out any potential false
positives following the stringent criteria described in Berezikov
et al. . Aedes albopictus miRNAs were compared with the
miRBase version 17 using Blast searches with a low stringent cutoff
(e-value 10 and window size 7) to ensure that we do not miss any
potential homologues in miRBase (v.17). BLAST hits then were
subject to manual inspections. In addition, Mapmi pipeline 
was used to find homologues in the available genome assemblies of
other insect species including Ae. aegypti, C. quinquefasciatus, An.
gambiae, and four Drosophila species, D. melanogaster, D. ananassae, D.
pseudoobscura, and D. grimshawi . Two settings were used, one
allowing only one mismatch and the other allowing three
mismatches, and the results compared. We use 35 as the Mapmi
cutoff score for homologues . Potential homologues with a
score of less than 35 are subjected to further analysis by miRscan,
which takes into account evolutionary conservation . Read
counts of each miRNA in the six samples were obtained as
described previously . Either the mature miRNA or miRNA*
should have at least 15 reads in the six samples for a miRNA gene
to be considered expressed. All expression data were normalized
with the following formula: Normalized Expression of a miRNA =
(The count of the miRNA in a particular sample)/(Total miRNA
counts from this sample)61 million. A hierarchical clustering
analysis with MeV 4.8 using Pearson correlation with average
linkage was used to evaluate the expression pattern of individual
miRNAs [21,22]. Prediction of miRNA targets was performed
using miRanda (version 3.3a) . The 39-end UTRs retrieved
from Vectorbase (transcripts version L1.2) were used as an input
and the miRanda cut-off score was set at 150. The GO terms of
the predicted targets were retrieved from Vectorbase Biomart.
The GO terms of 6741 out of 9726 transcripts that have annotated
39-end UTR also were retrieved from Biomart and used as
reference in GO enrichment test. The GO enrichments for targets
of each miRNA cluster were performed by blast2go using Fishers
exact test under a false discovery rate of 0.01.
Unless noted otherwise, sample collection conditions for
northern blot analyses were identical to those described for
Table 1. Novel miRNAs discovered in Aedes albopictus.
1The nomenclature of the novel miRNAs is provisional pending further characterization. 1, and 2 suffixes refer to different pre-miRNA secondary structures that
produce the same mature miRNA. Mosquito-specific are those miRNAs found to date only in mosquitoes. Culicinae-specific are those miRNAs found to date only in
Aedes and Culex species. Aedes-specific are those miRNAs found to date only in Ae. albopictus and Ae. aegypti.
2Contig number in Aedes aegypti assembly version AaegL1.
3location in contig of first nucleotide.
4location in contig of last nucleotide.
55939 orientation in contig (+, positive strand; , negative strand).
6Shown are the most abundant variants and the longest variants in parentheses.
7* means star miRNA.
preparing samples for Illumina sequencing. Larvae were collected
at each instar and pooled to generate early (I and II instars) and
late (III and IV instars) larval samples. Female adults were fed on
mouse blood and sugar water, and blood-fed samples were
collected at 24hrs post-blood-meal. Samples were either
homogenized immediately for RNA isolation or flash frozen in liquid
nitrogen immediately following collection, then stored at 80uC.
Total RNA isolation was carried out using a mirVana miRNA
isolation kit (Ambion, Austin, TX). Northern blots were carried
out based on Mead et al. . Briefly, total RNA was loaded onto
15% denaturing polyacrylamide gels, and run with ssDNA
markers 19 and 23 nt in length. The RNA gels were transferred
to BrightStar-Plus nylon membranes (Ambion), cross linked with
UV, prehybridized, and then hybridized overnight in the
ULTRAhyb-Oligo Hybridization Buffer (Ambion) with the
appropriate DIG-labeled probe at 42uC. Wash conditions were
the same as described in Mead et al. . Antisense 59
digoxigenin-labeled miRCURY LNA probes were purchased
from Exiqon (Vedbaek, Denmark). Probe sequences were
complementary to those shown in Li et al. .
Results and Discussion
Small RNA Sequencing and Evidence for Transcription of
Small RNAs were sequenced from a number of developmental
stages to increase the likelihood of discovering the full complement
of Ae. albopictus miRNAs. Small RNA libraries were constructed
from embryos, larvae, pupae, adult males, sugar-fed adult females
and blood-fed adult females pooled at various time points after
feeding. Between 1014 million high-quality small RNA reads
were obtained from each sample after Illumina sequencing and
filtering out linker sequences and ambiguous reads (Figure 1).
More than 50% of the reads for all six samples were ,22 nt in
length as expected for insect miRNAs. There also is an elevated
population of small RNAs ,28 nt in length in embryos, larvae
and females, which may represent piwi-interacting RNAs (piRNAs).
The comparisons of these ,28 nt RNAs to the Ae. albopictus
genome sequence, when available, will help the confirmation of
their piRNA status and the identification of their sources. piRNAs
are known to derive from and suppress repetitive sequences
developmental stages were clustered. Stages are in columns and
miRNAs in rows. Red indicates that a gene is represented highly at the
stage, whereas green indicates the opposite. miRNAs with similar
expression patterns cluster together. There are five Clusters (15) with
variable numbers of sub-clusters. Abbreviations: E, embryos; L, larvae;
P, pupae; M, adult males; F, adult females and B, blood-fed adult
including transposable elements . Recently piRNAs have been
shown to be involved in suppression of virus infection in
mosquitoes [25,26]. Further investigation of the piRNA pathway
in Ae. albopictus will improve our understanding of how this
important vector species may defend against repetitive sequences
A total of 104 miRNA genes with sequence similarity to
previously-described miRNAs (miRBase v17) were identified in Ae.
albopictus (Tables S1). All but one of the 88 known Ae. aegypti
premiRNA genes were recovered (Table S1; Figure 2; Li et al. ;
miRBase version17). aae-miR-1174, the only Ae. aegypti miRNA that
was not represented in any of the Ae. albopictus libraries, also is
undetectable by northern blot analysis of samples from all
developmental stages . However, small RNAs matching
aaemiR-1174* are found, and the putative Ae. albopictus orthologue,
aal-miR-1174*, shares 21 nt identity with the mature cqu-miR-1174
from Cu. quinquefasciatus. Thus, members of the miR-1174 family
may have undergone an arm switch  with respect to its mature
and star sequences in different mosquito species. All orthologues
miRNAs that could be recovered according to published data were
found in this study, supporting the conclusion that the approach
used here is comprehensive.
A total of 15 novel miRNAs were found in Ae. albopictus by
matching small RNA reads with the Ae. aegypti genome sequence
and performing subsequent bioinformatic confirmation (Table 1;
Figure 3). The criteria set forth by Berezikov et al.  were used
to ensure the authenticity of the novel miRNAs. The reliance on
matching Ae. aegypti pre-miRNAs to confirm the authenticity of the
Ae. albopictus miRNAs results in finding only those that are
conserved between the two species. Aedes aegypti and Ae. albopictus
are relatively closely-related, both belonging to the same subgenus
Stegomya , thus it is likely that the number of albopictus- or
aegypti-specific miRNAs will be low. Indeed, all of the 88 reported
Ae. aegypti miRNA genes (miRBase v.17) were found in Ae.
albopictus. Small RNAs that do not map to the Ae. aegypti genome
were not investigated because genomic DNA sequencing flanking
them are needed to evaluate the folding of the pre-miRNA
hairpins. The presence of a pre-miRNA hairpin is a critical
requirement for authentic miRNAs . Such unmapped
miRNAs, if present, are likely albopictus-specific and will be
uncovered when the Ae. albopictus genome assembly becomes
Evolution and Expansion of Mosquito-specific miRNAs
While all mature miRNAs share sequence identity between the
two Aedes species, there are a few cases of variation in the miRNA*
sequences (Figures 2 and 3; Table S1).The Ae. albopictus miRNAs
that matched known Ae. aegypti pre-miRNAs showed identical
mature miRNA sequences although there are truncations at the
ends as observed in previous reports [12,13]. Three examples,
miR-375*, miR92b* and miR-2946*, show one nucleotide difference
between Ae. albopictus and Ae. aegypti, (Table S1, Figures 2 and 3).
The majority of the miRNA genes that have homologues in
nonmosquito species (Table S1) are conserved in all four mosquito
Figure 5. Northern blot analyses of representative miRNAs in Aedes albopictus. Nine representative miRNAs, miR-210 (A), miR-998 (B),
miR2941 (C), miR-133 (D), miR-2943 (E), miR-2946 (F), miR-1890 (G), miR-1891 (H) and miR-184 (I) were subjected to northern blot analyses. The top
panels are the northern results and the bottom panels are RNA gels for verification of small ribosomal and tRNA integrity and equal loading of total
RNA. ssDNA size markers (19 and 23 nts, not shown) also were visualized on the RNA gel for size estimation. Fifteen micrograms of total RNA were
loaded for each sample. Abbreviations: E, embryos; L12, mixed 1st and 2nd instar larvae; L34, mixed 3rd and 4th instar larvae; P, pupae; M, adult
males and F, adult females.
species analyzed, Ae. albopictus, Ae. aegypti, Cu. quinquefasciatus and
An. gambiae. The two exceptions are miR-282 and miR-927, which
are found in Ae. aegypti and An. gambiae but not in Cu.
quinquefasciatus, indicating a loss or rapid change of these two
miRNAs in the latter species (Table S3). Among the
previouslyknown mosquito-specific miRNAs, miR-2941 is found only in Aedes
while miR-1889, miR-2940, and miR-2946 are conserved in the
Culicinae subfamily, which includes Aedes and Culex species (Table
S1). The rest of the known mosquito miRNAs are conserved in all
four mosquito species (Table S3). In contrast, only two of the 15
novel miRNAs are conserved in all four species in the three
mosquito genera (Table 1, Table S1). Two additional miRNA
genes are conserved in the Culicinae subfamily, and 11 are found
only in Aedes. Furthermore, all 15 novel miRNAs appear to be
unique to mosquitoes and have no sequence similarity to any
known miRNAs in the miRBase (v.17) and in the sequenced
genomes of Drosophila and other insects (data not shown). This may
reflect the fact that the accumulation of entries from an
everincreasing number of organisms will exhaust the list of conserved
miRNAs and newly-discovered miRNAs are likely to be
lineagespecific. The current study increased the number of putative
mosquito-specific miRNAs from 10 to 25 (Table S3).
Two observations support the hypothesis that the conservation
and lineage-specificity of miRNAs may contain useful information
to infer mosquito species phylogeny as was shown recently in other
taxonomic groups [30,31]. First, only two of the
broadlyconserved miRNAs (miR-282 and miR-927) were either lost or
mutated significantly in C. quinquefasciatus. This low rate of miRNA
loss makes them good phylogenetic markers at the sub-family and
genus levels. Second, the pattern of lineage-specific miRNAs is
consistent with their phylogenetic relationship. For example, there
are 14 Aedes-specific (11 novel plus 3 previously known, Table S3)
and three Culicinae-specific (miR-2941, miR-new3 and miR-new4)
miRNAs. However, there are no mosquito-specific miRNAs that
are shared by Aedes and Anopheles alone or Culex and Anopheles alone.
Given the significant involvement of miRNAs in development,
these analyses have the potential to link miRNAs to novel
evolutionary and developmental features associated with a
particular mosquito taxon.
Stage-specific miRNA Expression Profiles
The abundance of transcription from all 119 miRNA genes was
evaluated for both miRNAs and miRNA*s on the basis of
normalized read counts per miRNA (Table S2; Figure 4). Five
clusters (15) and additional sub-clusters were identified.
Hierarchical clustering showed that many miRNAs were embryo-specific
(sub-cluster 1.2) including three mosquito-specific miRNAs,
miR2941, miR-2943, and miR-2946. The high level of miRNA
accumulation in embryos is consistent with that found in Ae.
aegypti , supporting the hypothesis that they may be critical for
early development. miRNAs in cluster 4 are larval-specific while
miRNAs in cluster 3 are either pupal-specific or expressed highly
in both larval and pupal stages. A total of 71 miRNAs and
miRNA*s are found predominantly in adult males (cluster 2) while
13 miRNAs and miRNA*s are abundant only in adult females
(sub-cluster 5.1 and 5.2). The blood-fed female sample comprises
individuals harvested at various time points after the blood meal
and therefore it is likely that sensitivity in detecting dynamic
changes of miRNA levels is lost. However, three small clusters of
miRNAs within clusters 4 and 5 are expressed highly in sugar-fed
females but at reduced levels in blood-fed females. Some miRNAs,
including the novel miRNA-new6, showed a higher expression in
blood-fed females than in sugar-fed females.
The relative levels of six phylogenetically-conserved (let-7,
miR133, miR-184, miR-210,miR-9a, and miR-998) and six
mosquitospecific (miR-1890, miR-1891, miR-1175, miR-2941, miR-2943 and
miR- 946) miRNAs were confirmed by northern blot analyses
(Figure 5). All miRNAs tested showed accumulation in at least one
of the five developmental samples. The patterns for all except
miR9a are consistent with the Illumina results and it is not clear at this
time what caused the discrepancy with miR-9a. The Illumina
sequencing results also are consistent with Ae. albopictus northern
blot data reported in Zheng et al . For example, similar to
what was shown in Figure 4, the northern blot of let-7 indicated
that it accumulated mainly in developmental stages after pupation
(Figure 5; Zheng et al. ). let-7 also was shown to accumulate
abundantly in the later developmental stages in D. melanogaster, An.
stephensi and Ae. aegypti [12,13,32]. Furthermore, let-7 plays a
critical developmental regulatory role in the worm, Caenorhabditis
elegans, by promoting the development of the 4th instar larva to
miRNAs and Mosquito Biology
Demonstrating the functions of the 25 mosquito-specific
miRNAs is expected to inform studies of mosquito biology and
mosquito-specific adaptations. Target predictions have been used
as clues to miRNA functions and such predictions for known
miRNAs are available already at (www.mirbase.org). Analyses of
all Ae. albopictus miRNAs including the 15 novel miRNAs were
performed using Miranda  and the annotated transcripts from
Ae. aegypti (Vectorbase). Each miRNA on average has .100
predicted targets at a stringent score of 150 (only the targets of the
novel miRNAs are shown in Table S4). Gene Ontology terms
associated with these targets were retrieved and enrichment
analyses were performed separately for the targets of miRNAs of
each of the five major expression clusters (Figure 4).The GO terms
of all Ae. aegypti transcripts that have annotated 39-end untranslated
regions (UTR) were used as the reference and the results are
shown in Figures S1 and S2. These analyses provide the basis for
further functional studies to confirm the miRNA-target
relationships and to investigate the functions of these miRNAs. For
example, targets of miRNAs that are expressed predominantly
during the embryonic stage showed significant GO term
enrichment in transcription regulation, signal transduction, and
cytoskeletal protein binding. The enrichment in these types of
genes is consistent with expected molecular processes during
embryonic development. The UTR and GO annotation is
currently incomplete for the Ae. aegypti genome. Potential mRNA
targets will not be identified unless their 39UTRs are annotated.
Nonetheless, GO terms of 6741 transcripts are available for
analysis. Therefore the incompleteness of UTR and GO
annotation should not significantly affect the general conclusion
of the GO analysis described above, unless there is a systemic bias
in the annotation of a certain class of genes.
Female mosquitoes feed on blood to acquire proteins necessary
for reproduction and nutrition. Transmission of vector-borne
pathogens requires blood feeding on an infected host and the
blood meal also triggers a cascade of endocrinological, molecular,
and physiological events that switch the adult female from
hostseeking to reproduction [34,35]. Therefore it is not unexpected to
detect increases or decreases in accumulation of the majority of
119 miRNAs post blood-feeding. These miRNAs are likely
important regulators of blood-meal-induced molecular changes.
For example, aae-miR-275 affects blood digestion, fluid secretion,
and egg development in Ae. Aegypti . Aedes albopictus has
undergone a rapid expansion in its world-wide distribution and it
is emerging as a vector for Dengue and Chikungunya viruses .
Understanding the function of a key class of gene regulators in this
important mosquito vector has biological and practical
Figure S1 Color-coded gene ontology (GO) graph
showing significantly-enriched GO terms describing
biological processes. Predicted targets of miRNAs within each cluster
were analyzed separately. A false discovery rate (FDR) of 0.01 was
used as the threshold. For each GO term, a brief description, GO
number, FDR and P value were shown.
Figure S2 Color-coded gene ontology (GO) graph
showing significantly enriched GO terms describing
molecular functions. Predicted targets of miRNAs within each cluster
(Figure 4) were analyzed separately. A false discovery rate (FDR)
of 0.01 was used as the threshold. For each GO term, a brief
description, GO number, FDR and P value were shown. No GO
term was enriched significantly for targets of miRNAs in cluster 3
at a FDR of 0.01.
Number of small RNA reads mapped to a miRNA/
We thank Xiaofang Jiang (Genetics, Bioinformatics, and Computational
Biology graduate program at Virginia Tech) for bioinformatics support.
Conceived and designed the experiments: XC ZT. Performed the
experiments: JG WH JW PZ. Analyzed the data: MC JG WH JW AAJ
XC ZT. Contributed reagents/materials/analysis tools: MC. Wrote the
paper: JG WH JW AAJ XC ZT.
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