The fat body transcriptomes of the yellow fever mosquito Aedes aegypti, pre- and post- blood meal.
ABSTRACT The fat body is the main organ of intermediary metabolism in insects and the principal source of hemolymph proteins. As part of our ongoing efforts to understand mosquito fat body physiology and to identify novel targets for insect control, we have conducted a transcriptome analysis of the fat body of Aedes aegypti before and in response to blood feeding.
We created two fat body non-normalized EST libraries, one from mosquito fat bodies non-blood fed (NBF) and another from mosquitoes 24 hrs post-blood meal (PBM). 454 pyrosequencing of the non-normalized libraries resulted in 204,578 useable reads from the NBF sample and 323,474 useable reads from the PBM sample. Alignment of reads to the existing reference Ae. aegypti transcript libraries for analysis of differential expression between NBF and PBM samples revealed 116,912 and 115,051 matches, respectively. De novo assembly of the reads from the NBF sample resulted in 15,456 contigs, and assembly of the reads from the PBM sample resulted in 15,010 contigs. Collectively, 123 novel transcripts were identified within these contigs. Prominently expressed transcripts in the NBF fat body library were represented by transcripts encoding ribosomal proteins. Thirty-five point four percent of all reads in the PBM library were represented by transcripts that encode yolk proteins. The most highly expressed were transcripts encoding members of the cathepsin b, vitellogenin, vitellogenic carboxypeptidase, and vitelline membrane protein families.
The two fat body transcriptomes were considerably different from each other in terms of transcript expression in terms of abundances of transcripts and genes expressed. They reflect the physiological shift of the pre-feeding fat body from a resting state to vitellogenic gene expression after feeding.
- SourceAvailable from: Dana Nayduch[Show abstract] [Hide abstract]
ABSTRACT: Unlike other important vectors such as mosquitoes and sandflies, genetic and genomic tools for Culicoides biting midges are lacking, despite the fact that they vector a large number of arboviruses and other pathogens impacting humans and domestic animals world-wide. In North America, female Culicoides sonorensis midges are important vectors of bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV), orbiviruses that cause significant disease in livestock and wildlife. Libraries of tissue-specific transcripts expressed in response to feeding and oral orbivirus challenge in C. sonorensis have previously been reported, but extensive genome-wide expression profiling in the midge has not. Here, we successfully used deep sequencing technologies to construct the first adult female C. sonorensis reference transcriptome, and utilized genome-wide expression profiling to elucidate the genetic response to blood and sucrose feeding over time. The adult female midge unigene consists of 19,041 genes, of which less than 7% are differentially expressed during the course of a sucrose meal, while up to 52% of the genes respond significantly in blood-fed midges, indicating hematophagy induces complex physiological processes. Many genes that were differentially expressed during blood feeding were associated with digestion (e.g. proteases, lipases), hematophagy (e.g., salivary proteins), and vitellogenesis, revealing many major metabolic and biological factors underlying these critical processes. Additionally, key genes in the vitellogenesis pathway were identified, which provides the first glimpse into the molecular basis of anautogeny for C. sonorensis. This is the first extensive transcriptome for this genus, which will serve as a framework for future expression studies, RNAi, and provide a rich dataset contributing to the ultimate goal of informing a reference genome assembly and annotation. Moreover, this study will serve as a foundation for subsequent studies of genome-wide expression analyses during early orbivirus infection and dissecting the molecular mechanisms behind vector competence in midges.PLoS ONE 01/2014; 9(5):e98123. · 3.73 Impact Factor
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ABSTRACT: The Culicine mosquito, Aedes aegypti, is both a major vector of arthropod-borne viruses (arboviruses) and a genetic model organism for arbovirus transmission. TALE nucleases (TALENs), a group of artificial enzymes capable of generating site-specific DNA lesions, consist of a non-specific FokI endonuclease cleavage domain fused to an engineered DNA binding domain specific to a target site. While TALENs have become an important tool for targeted gene disruption in a variety of organisms, application to the mosquito genome is a new approach. We recently described the use of TALENs to perform heritable genetic disruptions in Ae. aegypti. Here, we provide detailed methods that will allow other research laboratories to capitalize on the potential of this technology for understanding mosquito gene function. We describe target site selection, transient embryo-based assays to rapidly assess TALEN activity, embryonic microinjection and downstream screening steps to identify target site mutations.Methods 01/2014; · 3.64 Impact Factor
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ABSTRACT: The consumption of a vertebrate blood meal by adult female mosquitoes is necessary for their reproduction, but it also presents significant physiological challenges to mosquito osmoregulation and metabolism. The renal (Malpighian) tubules of mosquitoes play critical roles in the initial processing of the blood meal by excreting excess water and salts that are ingested. However, it is unclear how the tubules contribute to the metabolism and excretion of wastes (e.g., heme, ammonia) produced during the digestion of blood.PLoS neglected tropical diseases. 06/2014; 8(6):e2929.
The Fat Body Transcriptomes of the Yellow Fever
Mosquito Aedes aegypti, Pre- and Post- Blood Meal
David P. Price3, Vijayaraj Nagarajan5, Alexander Churbanov1,4, Peter Houde1,4, Brook Milligan1,4, Lisa L.
Drake1, John E. Gustafson1,3, Immo A. Hansen1,2,3*
1Department of Biology, New Mexico State University, Las Cruces, New Mexico, United States of America, 2The Institute of Applied Biosciences, New Mexico State
University, Las Cruces, New Mexico, United States of America, 3The Molecular Biology Program, New Mexico State University, Las Cruces, New Mexico, United States of
America, 4The Roadrunner Sequencing Lab, New Mexico State University, Las Cruces, New Mexico, United States of America, 5Bioinformatics and Computational
Biosciences Branch (BCBB), OCICB/OSMO/OD/NIAID/NIH, Bethesda, Maryland, United States of America
Background: The fat body is the main organ of intermediary metabolism in insects and the principal source of hemolymph
proteins. As part of our ongoing efforts to understand mosquito fat body physiology and to identify novel targets for insect
control, we have conducted a transcriptome analysis of the fat body of Aedes aegypti before and in response to blood
Results: We created two fat body non-normalized EST libraries, one from mosquito fat bodies non-blood fed (NBF) and
another from mosquitoes 24 hrs post-blood meal (PBM). 454 pyrosequencing of the non-normalized libraries resulted in
204,578 useable reads from the NBF sample and 323,474 useable reads from the PBM sample. Alignment of reads to the
existing reference Ae. aegypti transcript libraries for analysis of differential expression between NBF and PBM samples
revealed 116,912 and 115,051 matches, respectively. De novo assembly of the reads from the NBF sample resulted in 15,456
contigs, and assembly of the reads from the PBM sample resulted in 15,010 contigs. Collectively, 123 novel transcripts were
identified within these contigs. Prominently expressed transcripts in the NBF fat body library were represented by
transcripts encoding ribosomal proteins. Thirty-five point four percent of all reads in the PBM library were represented by
transcripts that encode yolk proteins. The most highly expressed were transcripts encoding members of the cathepsin b,
vitellogenin, vitellogenic carboxypeptidase, and vitelline membrane protein families.
Conclusion: The two fat body transcriptomes were considerably different from each other in terms of transcript expression
in terms of abundances of transcripts and genes expressed. They reflect the physiological shift of the pre-feeding fat body
from a resting state to vitellogenic gene expression after feeding.
Citation: Price DP, Nagarajan V, Churbanov A, Houde P, Milligan B, et al. (2011) The Fat Body Transcriptomes of the Yellow Fever Mosquito Aedes aegypti, Pre- and
Post- Blood Meal. PLoS ONE 6(7): e22573. doi:10.1371/journal.pone.0022573
Editor: Pedro Lagerblad Oliveira, Universidade Federal do Rio de Janeiro, Brazil
Received April 28, 2011; Accepted June 24, 2011; Published July 27, 2011
Copyright: ? 2011 Price et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: NIH/NIGMS 1SC2GM092300-01 (IAH). NSF DBI-0821806 (PH). 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.
* E-mail: firstname.lastname@example.org
The yellow fever mosquito, Aedes aegypti, is the primary vector for
dengue fever, several encephalitis viruses, yellow fever, and several
types of filariasis . Due to its ability to transmit diseases and its
widespread range, this disease vector continually affects the health
of millions of people around the globe .
The fat body is the principal organ of intermediary metabolism,
functioning as a storage unit for lipids, carbohydrates, and proteins
in both mosquitoes and insects in general. The principal cell type
of the fat body is a large polyploid cell referred to as the
trophocyte, which is capable of synthesizing large amounts of
protein and contains many ribosomes and oil droplets . The fat
body also acts as the main source of hemolymph proteins in all
developmental stages of holo-, hemi-, and a-metabolic insects .
Metabolic capabilities that differ among regions within the fat
body have been reported for several lepidopteran[5,6] and one
dipteran species , but these regional metabolic differences are
generally not well understood.
During the mosquito aquatic larval stage, the fat body
accumulates and stores nutrients for use in the adult stage.
Hexameric storage proteins of the arylphorin family are the major
amino acid stores in mosquito larvae and are synthesized by the fat
body and secreted into the hemolymph. These hexamerins are
then reabsorbed later by the larval fat body via receptor-mediated
endocytosis, stored in protein granula, and afterward hydrolyzed
to deliver energy and building blocks required to drive
metamorphosis into the adult insect [8,9].
In the adult stage the fat body continues to play a prominent
role in energy metabolism by providing precursors for flight and
yolk protein synthesis which is required by females for reproduc-
tion. It also assists in metabolizing potentially fatal amounts of
excess ammonia after the female takes a blood meal . In
addition, the fat body plays an important role in several immune
PLoS ONE | www.plosone.org1 July 2011 | Volume 6 | Issue 7 | e22573
pathways and antimicrobial peptide production which control and
prevent infection by microbial and protozoan pathogens . It
has been demonstrated several times that altering mosquito fat
body immune function via transgenic interventions drastically
alters vectorial capacity by resulting in overly pathogen-susceptible
or -resistant mosquitoes .
Before a female mosquito takes a blood meal, her fat body is in a
state-of-arrest with regards to the expression of genes involved in
reproduction. When a blood meal is taken, a sequence of signals
originating in other tissues affects the fat body, priming it for gene
expression alterations. These signals include raised amino acid
levels in the hemolymph, peptide hormones from the gut and the
central nervous system , and ecdysteroids from the ovaries
[14,15,16]. Additional signaling molecules present in the blood
meal itself (e.g., vertebrate insulin) have been shown to alter
mosquito fat body metabolism . Collectively, these signals can
activate the insulin and target of rapamycin signaling pathway
within the fat body, which leads to the production of yolk protein
precursors [14,16,18,19,20]. During vitellogenesis, yolk proteins
and lipids are secreted by the fat body trophocytes and transported
to the ovaries which deposit them into developing eggs .
The adult fat body is a key organ in mosquito reproduction and
immunity; therefore, it is important to understand fat body
physiology on a molecular level. We now report on transcriptional
alterations occurring in Ae. aegypti fat body tissue following a blood
meal. This was accomplished by comparing and contrasting read
and contig libraries, obtained by 454 pyrosequencing, derived
from fat body tissue of female non-blood fed (NBF) Ae. aegypti and
24 h post-blood meal (PBM) fat body tissue. This study describes a
window of transcriptional alterations appearing in the Ae. aegypti fat
body during the digestion of a blood meal and subsequent
accumulation and utilization of nutrients. In particular we used an
RNA-seq approach to observe transcript expression transitions
from a resting fat body prepared for translational activity to a fat
body producing transcripts involved in Ae. aegypti vitellogenesis.
Results and Discussion
Twenty four hours after a full blood meal, vitellogenesis, the
production, secretion, and reuptake of yolk proteins, is at a peak in
Ae. aegypti . Due to the relationship of this process with fat body
metabolism, in this study we choose to compare the fat body tissue
transcriptomes of NBF mosquitoes with fat body tissue of
mosquitoes 24 hrs after feeding.
For practical reasons we isolated abdominal body walls with
attached abdominal fat body tissue and processed them for library
construction. This is a standard preparation for studying mosquito
fat body physiology [14,18,23]. It must be noted that this fat body
preparation contains other cells and tissues besides fat body
trophocytes for example epidermis, tracheas, muscle, and ventral
nerve cord. However, the fat body is the dominant tissue in this
preparation and there is little doubt that the transcripts we discuss
below are expressed in the fat body.
Alterations in total RNA quantities in the fat body tissue
It has been shown previously that there is a large increase in
RNA synthesis and accumulation in the PBM fat body compared
to the NBF fat body . As expected, comparing total RNA
isolated from fat bodies (4 samples per treatment consisting of 5 fat
bodies each) in NBF and PBM mosquitoes revealed an average of
2.1 mg of RNA per NBF fat body and 4.7 mg per PBM fat body
(p,0.005, students t-test). These results indicate that the total
amount of RNA in the fat body doubles during the 24 hrs
following a blood meal. Based on previous findings , we
speculate that these differences in RNA accumulation may reflect
the increased transcription of ribosomal structural RNAs in the
PBM fat body.
454 pyrosequencing and contig assembly
The NBF and PBM fat body libraries were derived by 454
sequencing: a total of 40 and 55 megabases were read, consisting
of 204,578 and 323,474 individual reads averaging 194 and 171
bases in length, respectively. Sequence quality reported by the
sequencer for our NBF and PBM sequences using the phred
quality scale [25,26] revealed that the average quality was 32.9
and 33.1 (equivalent to .99.9% base call accuracy), respectively.
Therefore, over half a million high quality Ae. aegypti fat body reads
were generated in this study (for size distribution of library reads
see Figure S1 A & B). All sequences generated were submitted to
the sequence read archive  and accepted under accession
The individual reads were de novo assembled using Mira
assembly software . The assembly process produced 15,456
and 15,010 contigs from the NBF and PBM sample reads,
respectively (Table 1). These contigs were used in our analyses. Of
the total NBF and PBM contigs, 11,588 and 11,129 respectively,
qualified as ‘‘true contigs’’, which were assembled out of at least
two reads each. The average GC content of the NBF contigs is
47.5% and the PBM contig populations is 46.9% (See Figures S2
A and B). These numbers are comparable with other insect and
eukaryote sequencing projects, which have reported GC content
between 38.7% and 56.5% [29,30,31,32].
Gene Ontology (GO) analysis
The Blast2GO [33,34] program, which uses a pipeline of
BlastX followed by GO term assignment and annotation, was
utilized to analyze gene ontology (GO) and categorize fat body
expressed genes in the NBF and PBM contig samples. A sanity
check of the contigs was performed and, as expected, the
overwhelming majority of contigs with BlastX results for both
NBF and PBM libraries exhibited greatest similarity to protein
sequences from Ae. aegypti or other mosquito species (Figures S3 A
and B). Statistics on NBF and PBM library contig numbers passing
each stage of Blast2GO are shown in Table 2.
Comparison of the level two GO functions of the two libraries
(see Figure 1) shows that contigs with transporter, catalytic and
Table 1. Fat body Contig Statistics.
PBM170.8 3.79 379.742288 40
Fat Body Transcriptomes of Aedes aegypti
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nutrient reservoir activities are much more prevalent in the PBM
than in NBF library. In contrast, contigs with binding and
structural molecule activity functions, including all ribosomal
proteins, are heavily reduced in the PBM compared to NBF
library. Based on these results, we suggest that fat body gene
expression shifts from the production of transcripts associated with
the protein translation machinery to the expression of transcripts
involved with nutrient accumulation and utilization during
Novel transcript discovery
The fat body contig libraries generated in this study include a
collection of potential transcripts not previously identified in the
Ae. aegypti genome sequence in Vectorbase . We identified a
total of 123 of these, which are listed in Table S1. Examples of
some of these potential transcripts are shown in Table 3. The
majority of these contigs mapped to the reference genome where
there was no overlap with existing predicted genes. However,
there were exceptions. For instance, contig 6072 (Table 3) which,
based on blast results, encodes a putative sugar transporter,
overlaps with the gene encoding 4-hydroxyphenyl-pyruvate
dioxygenase (AAEL014600). While presently unclear, overlapping
contigs might be explained by a variety of mechanisms (e.g.
alternative splicing, pre-splice RNA, overlapping genes).
Transcript expression in the fat body
Alignment of our NBF and PBM transcriptome sequencing
reads to the Vectorbase Ae. aegypti reference transcripts produced a
total of 116,912 and 115,051 alignments, respectively. These
alignments covered 6,019 (NBF) and 7,625 (PBM) reference
transcripts. The top 4 transcripts aligned with on a percentage
basis from each sample are shown in Table S2 and all alignments
are shown in Table S3. Table S4 shows all transcripts found to
exhibit a statistically significant change in expression and the fold
difference in expression between the unfed and bloodfed state in
our libraries. The union of these two sets produced a set of 9,984
transcripts, representing all transcripts, including multiple iso-
forms, found to be expressed in either the NBF or PBM fat body
Of these 9,984 distinct transcripts, 4,974 exhibited at least a 2-
fold change in expression level. This is substantially lower than the
number of differentially expressed genes previously reported
(8,288 based on several repeats, but no statistical filter applied to
changes in transcript levels) in NBF vs 24 hr PBM whole
mosquitoes examined via microarray analysis . A major
contributor to the differences between these two studies may be
the difference in tissues: previously, the whole mosquito was
examined whereas only fat body/abdomen tissue was examined in
this study. It is highly likely that blood feeding affects the entire
organism at a transcriptional level, in different ways than the fat
body . Statistical analysis of our results [37,38] revealed that 236
(2.4% of all) transcripts our reads aligned with had significant
differential expression between samples.
Table 2. Results of contig processing with Blast2GO.
NBF (#contigs) PBM (#contigs)
Annotated, GO terms assigned,
GO terms assigned, Blastx results325 412
Blastx results748 797
No Blastx results84588710
True Contigs11588 11129
Figure 1. Gene Ontology of the Aedes aegypti fat body. Pre-and Post-Bloodmeal Level 2 GO functions for de novo assembled contigs. The x-axis
represents the total number of contigs with the given level 2 GO term.
Fat Body Transcriptomes of Aedes aegypti
PLoS ONE | www.plosone.org3 July 2011 | Volume 6 | Issue 7 | e22573
When all isoforms of each transcript are counted as one, we
identified 2,354 NBF and 2,746 PBM transcripts from the total of
6,019 and 7,625 distinct transcript isoforms. These numbers
represent estimates of the number of genes expressed in the fat
body under each physiological condition. They are comparable to
previous Drosophila melanogaster fat body gene expression estimates
(2261+ genes) [39,40].
Transcripts highly expressed in NBF fat body tissue
The condition of the mosquito fat body before the female takes
a blood meal has been described as a ‘‘previtellogenic state-of-
arrest’’ during which yolk proteins are not expressed . Fifteen
of our highly expressed transcripts found in our state-of-arrest
NBF fat body library (Table 4) encode ribosomal proteins (22%
of read alignments with reference transcriptome) and one
transcript encodes the eukaryotic translation elongation factor.
The last four of our highly expressed NBF transcripts potentially
encode serine hydroxymethyltransferase, NADH dehydrogenase
subunit 2, and two proteins of unknown function. All transcripts
shown in Table 4, with the exception of NADH dehydrogenase
subunit 2, were found to have statistically significant differential
Table 3. Twenty potential new genes identified using contigs.
ContigMlenMis Gaps Blastx resultsAccession e-valdomainsEST
48 38231 1.97129100 hypothetical protein Tetrahymena thermophilaXP_977125.1 3.2 LC 1E-
hypothetical protein AaeL_AAEL000019XP_001647901.1 7.1
114 511 121.329 42530 sodium/potassium/calcium exchanger 5 precursor,
putative (Pediculus humanus corporis)
XP_002429936.1 7.1 LC, TM0
similar to potassium-dependent sodium-calcium
exchanger (Acyrthosiphon pisum)
8086548 1.77858401 hypothetical protein CpipJ_CPIJ018486
XP_001868660.1 2.E-06 SP, LC, TM0
vitelline membrane protein 32E (Drosophila erecta) ABO71727.10.034
8365767 1.17749500 chaperonin (Culex quinquefasciatus) .gb|EDS29066.1|XP_001869348.1 0.29 BT, CC, LC3E-07
1514 5283 1.49543941 ribosomal protein S27 (Aedes albopictus)AAV90719.1 8E-32 RS27e, BT0
16024637 1.36335202 no sig 1E-18
16515694 1.55 52116conserved hypothetical protein (Culex quinquefasciatus) XP_001848093.1 2E-38BT3.E-04
AGAP010003-PA (Anopheles gambiae str. PEST) XP_319147.4 2E-29
24685293 1.38947432 heat shock protein 70 B2 (Culex quinquefasciatus) XP_001864723.1 2E-32 TM, LC, EGF(BT) 0
heat shock protein 70 (Chironomus riparius) ADL27420.1 9E-32
2644 6653 1.56362213 Os02g0274400 (Oryza sativa Japonica Group) NP_001172895.1 5.4LC, BT1.E-04
3974 2804 1.13323221 NADH dehydrogenase subunit 6 (Aedes aegypti) YP_001649172.1 1.E-06TM, BT3E-09
40103503 1.10228110 Lian-Aa1 retrotransposon protein (Aedes aegypti) AAB65093.15E-14 BT1E-
4015 3323 1.363 31001 cytochrome c oxidase subunit II (Aedes aegypti)YP_001649164.1 5E-43 COX21E-
5669 4482 1.34936322 response regulator receiver domain-containing protein
YP_502925.10.49 LC, BT 2.E-05
hypothetical protein Phum_PHUM231450
(Pediculus humanus corporis)
6072 2272 1.119 18701 sugar transporter (Culex quinquefasciatus) XP_001846219.1 9.3 BT5E-98
6085 88531.371 70112 Senescence-associated protein (Brugia malayi) XP_001900327.1 1E-34BT0
Uncharacterized protein ART2 (Camponotus floridanus) EFN65036.1 6E-33
6816 4202 1.12 32912hypothetical protein CpipJ_CPIJ015859
XP_001865960.1 1E-11 SP, LC, TM, BT1E-
83774842 1.6839211 heat shock 70 Ba (Aedes aegypti) ACJ64195.1 6E-12LC, BT0
9356 7692 1.495 6261540S ribosomal protein S27 (Culex quinquefasciatus)XP_001847201.1 2E-44 LC, SP, TM, BT0
119352271 1.1519610 cytosolic large ribosomal subunit L27A
ACJ74464.1 6E-12L15, BT 1E-
14965 49721.115 39833 putative salivary odorant binding protein 1
AAR18408.1 1E-07 BT0
Contig name, length of contig, number of reads making up the contig, Ae. aegypti reference genome supercontig our contig matched against, length of the match,
number of base mismatches, number of base gaps, results from NCBI Blastx search, eval of Blastx result, pfam/SMART domains identified with eval greater than 1e-5, e-
val of best EST hit at vectorbase. LC - low complexity, TM – transmembrane, BT - below threshold, RS27e - ribosomal S27e, SP - signal peptide, CC - coiled coil, IR -
internal repeat, EGF - epidermal growth factor, COX2- Cytochrome C oxidase subunit II, L15 – Ribosomal protein L15.
Fat Body Transcriptomes of Aedes aegypti
PLoS ONE | www.plosone.org4 July 2011 | Volume 6 | Issue 7 | e22573
Cytosolic serine hydroxymethyltransferase is found in eukary-
otes and prokaryotes. It is a central enzyme of the one-carbon
metabolic pathway that catalyzes the production of the major one-
carbon donors for the biosynthesis of thymidylate, purines,
methionine and choline . NADH dehydrogenase, also called
Complex I, is a protein localized in the inner mitochondrial
membrane and the first protein of the oxidative phosphorylation
process in mitochondria. The eukaryotic translation elongation
factor 2 is part of the ribosomal protein synthesis machinery.
The strong presence of ribosomal protein transcripts in the NBF
library was expected since these messages have been shown to
accumulate in the fat body during the first days after eclosion of
adult Ae. aegypti . Translation of ribosomal proteins, transcrip-
tion of rRNAs, and ribosome assembly start directly after a blood
meal resulting in ribosome accumulation in the fat body. After
completion of vitellogenesis most of these ribosomes degrade and
the number of ribosomes returns to pre-blood meal levels.
Our data support the finding of Niu and Fallon  that
ribosomal protein (rp) L34 is down-regulated after a blood meal.
In fact, the number of all transcripts encoding ribosomal proteins
are reduced in the PBM library (see Table 5). Interestingly, two
AAEL001849). These two proteins have an identity of 68% when
aligned and make up for 7% of all aligned transcripts in the NBF
Transcripts highly expressed in PBM fat body tissue
After a blood meal the fat body is activated and starts
vitellogenic gene expression . Several signals, including the
rise of hemolymph amino acids (coming from the midgut), the
steroid hormone ecdysone (secreted by the ovaries) and insulin-like
peptides (from the central nervous system), have been identified as
regulators of yolk protein expression [14,15,16]. At 24 h PBM the
process of vitellogenesis peaks and the fat body produces large
amounts of yolk proteins that are secreted into the hemolymph.
The 20 most highly expressed transcripts found in the 24 h PBM
library are presented in Table 5. All transcripts in Table 5, with
the exception of NADH dehydrogenase subunit 2 were found to
have differential expression.
Vitellogenin is the principal yolk protein
precursor in almost all oviparous vertebrates and invertebrates
[44,45]. It is a nutrient-rich glycolipoprotein that gives rise to
vitellin, the major yolk protein in eggs. Vitellogenins A, B and C
(AAEL010434, AAEL006138, AAEL006126) were found to have
differential expression between NBF and PBM samples.
proteases, usually located in the lysosomes, which are found in a
wide variety of organisms ranging from humans, rats and cattle to
papaya (as papain ) and insects. These proteases have a wide
variety of described functions: fetal myotube development, normal
and tumor angiogenesis, digestion, and vitellogenesis [47,48,
49,50,51]. In Ae. aegypti, vitellogenic cathepsin b (VCB) is
secreted into the hemolymph as 44 kDa subunits. It is taken up
by oocytes and processed during several stages of vitellogenesis.
Finally, it becomes a 33 kDa form during embryogenesis. This is
considered the active form and degrades vitellogenin .
We found three cathepsin transcripts (AAEL007590, AAEL00-
7585, AAEL007599) to be heavily up-regulated in the PBM fat
Table 4. Top 20 most highly expressed transcripts in fat body of non-blood fed mosquitoes.
Ensembl TranscriptIDName NBF PBMChange R-val% Pre% Post
AAEL009341 60S ribosomal protein L34 2189938 0.44 107.53.6 1.5
AAEL013221 60S ribosomal protein L10a 2065673 0.33 188.8.131.52
AAEL00184960S ribosomal protein L342036 859 0.43102.93.4 1.4
AAEL004149 unknown membrane protein 8683980.47 37.21.4 0.6
AAEL003877 ubiquitin/ribosomal protein L40796444 0.5720.8 1.3 0.7
AAEL01147160S ribosomal protein L17 737220 0.3 62.21.2 0.4
AAEL002510 serine hydroxymethyltransferase739 1940.2771.8 1.20.3
AAEL00003240S ribosomal protein S6699 2320.3451.6 1.2 0.4
AAEL013158 AAEL005901 40S ribosomal protein S3a696331 0.4827.5 1.10.5
AAEL017516 60S ribosomal protein L23a693276 0.438.8 1.1 0.4
AAEL00082360S ribosomal protein L35A 566 2350.4229.5 0.9 0.4
AAEL006698 60S ribosomal protein L31 560 238 0.43 27.9 0.9 0.4
AAEL003530 AAEL005027acidic ribosomal protein P1 543 153 0.2949.0 0.90.3
AAEL017413NADH dehydrogenase subunit 2 5115601.11 0.7 0.8 0.9
AAEL00417540S ribosomal protein S17 504 1920.3930.4 0.80.3
AAEL00810340S ribosomal protein S8 4982150.44 24.1 0.80.4
AAEL004500 eukaryotic translation elongation factor 2478 216 0.46 21.10.8 0.4
AAEL004851Unknown protein 474187 0.4 27.00.8 0.3
AAEL012686 40S ribosomal protein S12470 1720.37 184.108.40.206
AAEL00818860S ribosomal protein L6 467181 0.3927.40.8 0.3
Reference transcripts by number of reads aligned in the pre-blood meal sample. NBF, number of reads aligning to the shown transcript ID in pre-blood meal sample.
PBM, number of reads aligning in the post-blood meal sample. Change is the number of PBM reads divided by the number of reads in the NBF sample, with
normalization. Normalization was accomplished by multiplying the number of reads PBM by the ratio of the total number of NBF reads divided by the total number of
PBM reads. R-val is the computed R-value, R.9 is significant. %Pre is the percentage of unique alignments in the NBF sample the NBF alignments for this transcript
represents. %Post is the same for the PBM sample.
Fat Body Transcriptomes of Aedes aegypti
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body and a fourth (AAEL009637) that appears to have a lesser
degree of up-regulation (Figure 2A & B). The changes in
expression levels for all four were found to be statistically
significant. Our phylogenetic analysis shows AAEL007599 and
AAEL007585 to be the most closely related while AAEL009637
diverges the most from this small group. Analysis using SMART
software  shows that all four of these VCBs have an identical
protein domain organization with a signal peptide followed by a
single cysteine protease domain (Figure 2 C).
Vitellogenic carboxypeptidase (VCP).
to be one of the most up-regulated genes in the fat body after a
blood meal. Serine carboxypeptidases are found in many different
species, from invertebrates to humans, and in many cell types in
those organisms.The described
carboxypeptidase family is the removal of one or more amino
acids from the carboxy terminal end of an amino acid chain
[53,54]. In Ae. aegypti, vitellogenic carboxypeptidase (VCP) has
been found to be transcribed and translated in the fat body and
exported to the developing oocytes after a blood meal, with the
peak of transcription at 24 hours post blood meal and very little
transcription by 48 hours post blood meal . Once in the
oocytes, VCP surrounds the vitellin yolk in the same manner as
cathepsin B [53,56]. VCP is modified from a 53 kDa form to a
48 kDa form at the onset of embryogenesis and is rendered into
small amino acid sequences by the time the embryo reaches the
first instar. The function of VCP has not been described to our
knowledge. Similar carboxypeptidases have been shown to have a
We observed VCP
functionfor the serine
role activating or modifying other enzymes and molecules, but it is
not thought to act upon vitellogenin .
Vitelline membrane proteins
important part of the vitellogenic process, forming the inner
layer of the Ae. aegypti eggshells. We were surprised to find them
highly expressed in the vitellogenic fat body, but we were able to
confirm these results via RT-PCR repeatedly (data not shown).
VMPs have been described as being exclusively secreted by the
follicular epithelium , but there is some evidence that they are
also produced in the fat body. We also found ESTs encoding
VMPs in a collection of Ae. aegypti fat body specific ESTs within the
Unigene and dbEST database [59,60]. We found transcripts for
AAEL006670, AAEL013027 and AAEL014561 to be highly
upregulated post blood-meal.
VMPs are an
Fat body membrane transporters
Since the fat body is a key player in mosquito metabolism,
efficient transport across its cell membrane is extremely important
for homeostasis. The mosquito fat body must import nutrients
derived from a blood meal for the rapid and energetically costly
process of vitellogenesis. A wide variety of transporter families
capable of transporting a plethora of substrates have been
identified by our sequencing. We mapped reads to 54 out of 58
of the families represented by the list of transporters we obtained.
Figure 3 shows the total number of transporter reads aligned by
Table 5. Top 20 most highly expressed cDNAs PBM, reference transcripts by number of reads aligned in the PBM sample.
Ensembl TranscriptIDName, references NBF PBMChange R-val % Pre% Post
VCB-A [15,56,78]7 4120 5981234.3 0.016.6
AAEL006138vitellogenin-B  14 3306240 971.60.025.3
AAEL010434 Vitellogenin-A 133194 250939.8 0.025.1
AAEL007599VCB-B [15,56,78]1 2507 2548 760.00.002 4.0
AAEL006126vitellogenin-C 6 2141363 635.90.01 3.5
AAEL007590 VCB-C [15,56,78]3 1936 656580.7 0.0053.1
AAEL006563 VCP-A 4 1721 437513.0 0.0072.8
AAEL009341 ribosomal protein L342189 9380.44 220.127.116.11
AAEL001849 60S ribosomal protein L342036 8590.43 102.9 3.41.4
AAEL006542 VCP-B 3 806 273237.8* 0.0051.3
AAEL014561 vitelline membrane protein homolog 0 699 n/a 212.9*0 1.1
AAEL013221 ribosomal protein L10a 20656730.33 18.104.22.168
AAEL017413 NADH dehydrogenase subunit 2511 5601.110.7 0.80.9
AAEL003877 Ubiquitin precursor796 4440.5720.8 1.3 0.7
AAEL013027 vitelline membrane protein 0 403n/a122.7*0 0.7
AAEL004149 unknown membrane protein868 398 0.4737.2 1.40.6
AAEL009637 VCB-D [15,56,78] 73383 5.3351.3 0.10.6
AAEL006670 vitelline membrane protein0 371 n/a113.0*0 0.6
AAEL003345argininosuccinate lyase263 3651.41 4.00.4 0.6
AAEL013158 ribosomal protein S3a696 331 0.48 27.5* 1.20.5
NBF, number of reads aligning to the shown transcript ID in NBF sample. PBM, number of reads aligning in the post-blood meal sample. Change is number of post-BM
reads divided by number of reads in the NBF sample, with normalization. Normalization was accomplished by multiplying the number of reads PBM by the ratio of the
total number of NBF reads divided by the total number of PBM reads. R-val is the computed R-value, R.9 is significant. %Pre is the percentage of unique alignments in
the NBF sample the number of alignments in this sample represents. %Post is the same for the PBM sample.
*Also significant by DESeq results (p,0.05).
Fat Body Transcriptomes of Aedes aegypti
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Figure 2. The VCB family of Aedes aegypti. A. Neighbor joining tree showing evolutionary relationships of Ae. aegypti VCBs. B. Number of VCB
reads identified in the NBF and PBM libraries. All cathepsins identified in Ae. aegypti which had reads align using the methods used for sequence
alignment and data analysis, are represented. C. Domain structure of Ae. aegypti VCB proteins. Cy - Cystatin-like domain, Pept_C1 - Papain family
cysteine protease domain, I29 - Cathepsin propeptide inhibitor domain, SO - Somatomedin B -like domain. Signal peptides are labeled red.
Fat Body Transcriptomes of Aedes aegypti
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The total number of transporter reads decreases PBM by
approximately one third. This is approximately the number of
reads taken up by yolk protein reads in the PBM library, which
may reduce the number of reads against other transcripts.
Three of the six aquaporins encoded in the Ae. aegypti genome
were identified, AAEL003512 (0 reads NBF/2 reads PBM),
AAEL005001 (20 NBF/2 PBM) and AAEL005008 (34 NBF/8
PBM). While not significant by our statistical analyses, these
numbers may show a trend which supports our previous findings
that expression of these aquaporins decreases post-blood meal
. We also see a trend which suggests an increase in the
presence of oligo- and polypeptide transporter transcripts
Immunity-related fat body transcripts
As mentioned previously, the fat body is an important player in
mosquito immunity. Synthesis of a variety of anti-microbial
peptides to control and erradicate pathogens throughout the
mosquito as a whole occurs within this organ [11,62]. We were
able to identify 44 transcripts across our NBF and PBM samples
which are thought to encode protein products related to immunity;
Table 6 contains selected examples. Included are transcripts
thought to encode defensins, cecropins, gram negative and
peptidoglycan recognition proteins and dicer, among many others.
Defensins are classically known as small (4 kDa) cationic
peptides up regulated in response to bacterial challenge in insects.
They are capable of forming ion channels into gram positive cells
. However, Ae. aegypti and A. gambiae defensins have been
shown to respond to, and have an effect on, infections by certain
stages of Plasmodium, the causative organism of malaria [63,64,65].
We identified two defensins in the fat body with our sequencing
(AAEL003849 and AAEL003841) and found them to be present in
both our PBM and NBF libraries. They were not found to have
statistically significant differential expression.
Cecropins are similar to defensins in size and function . In
Ae. aegypti they have been shown to work in conjuction with
defensins against bacterial and plasmodium infections . Addi-
tionally, cecropins may play a role in Ae. aegypti’s response to
infection with filarial worms, the causative agent of filiariasis, a
major health problem in many parts of the world and subject of a
World Health Organization erradication program [67,68]. In our
sequencing we identified reads for cecropin (AAEL000621) in our
NBF sample and in our PBM sample, but did not find statistically
significant differential expression. Increasing sequencing depth in
the future would increase both the number of immune related
genes identified in the fat body, and provide more data on their
differential expression within this organ, pre- and post-bloodmeal.
In order to verify differential expression of selected genes
identified via 454 sequencing in the analysis of our libraries, we
used quantitative real-time PCR directed at transcripts of interest.
Transcripts and results are shown in Table 7 where they are
compared with our 454-pyrosequencing data and data from a
microarray analysis of blood meal induced transcription changes
in whole mosquitoes . Although the qPCR data supports the
trends we observed in the analysis of our pyrosequencing results,
the levels of change tend to be dissimilar among methods for each
gene. We attribute differences in the level of change to the
differences in technologies used to obtain gene expression data.
To date, only a handful of projects describing the transcriptome
of Ae. aegypti or one of its organs have been reported on. Two
projects have utilized microarrays to describe gene expression in
whole Ae. aegypti mosquitoes  or mosquito midguts . To
identify early changes post-blood feeding an RNA-seq (illumina)
investigation of whole mosquitoes, before and five hours after
blood feeding, has also been performed . An earlier
transcriptome analysis performed on the Ae. aegypti vitellogenic
fat body involved a very small number of randomly selected cDNA
ESTs from 24 h PBM fat bodies . Our present study now
builds upon these investigations by describing in great depth the
Figure 3. Transporters identified in the Aedes aegypti fat body. Number of reads in the NBF and PBM samples by transporter type.
Transporters were identified as described in the text and the number of reads aligned were pulled from the aligned reads generated for our EST
expression in the fat body overview.
Fat Body Transcriptomes of Aedes aegypti
PLoS ONE | www.plosone.org8 July 2011 | Volume 6 | Issue 7 | e22573
transcriptome of fat body tissue before blood meal and at the
metabolic peak of vitellogenesis after a blood meal has been taken.
In female mosquitoes vitellogenesis involves specific cellular
signaling events and transport of large quantities of nutrients in the
fat body, an extremely important metabolic and reproductive-
associated organ. The transition from a state-of-arrest to
vitellogenic fat body is primed by blood feeding and culminates
in the deposition of yolk protein into developing eggs. 454
pyrosequencing has enabled us to take a detailed look at the fat
body transcriptome of the yellow fever mosquito Ae. aegypti, an
important disease vector and model organism. The two tran-
scriptomes we compared were considerably different from each
other in terms of mRNA expression and reflect the different
physiological stages of the fat body in NBF versus PBM mosquitoes
undergoing the process of vitellogenesis. This key tissue and its
physiological functions are a prime target for novel vector control
strategies and our results represent a first in-depth examination of
a mosquito fat body transcriptome. In the future, we will extend
this study by further analysis of the mosquito fat body
transcriptome under different physiological and environmental
conditions. One of our long term goals is to identify potential
targets for the development of novel insecticides. We suggest that
fat body proteins, especially transporters and ion channels, are
rational targets for that.
The research plan used for this work involving animals was
specifically approved by the Institutional Animal Care and Use
Committee (IACUC) at New Mexico State University under
approval ID #2008-034. All procedures and care are described in
Table 6. Immune genes expressed in the fat body.
AAELNBFPBM Vectorbase description
Expressed in both samples
AAEL00384926 Defensin ,Anti-Microbial Peptide Precursor
AAEL00977030 66 Ubiquitin-conjugating enzyme E2 I
AAEL000621 101 Cecropin, Anti-Microbial Peptide.
AAEL003841105 Defensin-A Precursor (AaDef)
AAEL01052475 Hypothetical protein (Putative Tumor Necrosis Factor)
AAEL01046631Mitogen activated protein kinase kinase kinase 4, mapkkk4, mekk4
AAEL01017193 Peptidoglycan Recognition Protein (Long)
AAEL004675105 Conserved hypothetical protein (Toll pathway related)
AAEL00076026 Clip-Domain Serine Protease, family B.
AAEL0066743095 Clip-Domain Serine Protease, family B.
unique to NBF
AAEL00762630 Gram-Negative Binding Protein (GNBP)
unique to PBM
AAEL00414202 Phagocyte signaling-impaired protein
NBF is reads NBF, PBM is reads PBM. Reads were normalized as described in Tables 4 and 5. None were found to have statistically significant differential expression by
the R-test we performed. Dicer (AAEL006794) was found to have differential expression (P=0.026, P,0.05 significant) with DESeq.
Table 7. Comparision of the number of reads in NBF and PBM samples for the transcripts selected for qPCR.
Name Vectorbase IDReads NBF/PBM
Vitellogenin AAEL01043413/3194246* 78951487
Cathepsin BAAEL0153127/3753536* 529 1347
trypsin AAEL00781834/20.05*0.02 0.005
Arginase AAEL00267551/1 0.02* 0.02 0.1
Serine-Type EndopepidaseAAEL008781 22/0- 0.080.1
Serine-Pyruvate AminotransferaseAAEL01048094/5 0.05* 0.4 0.9
DDCt:2‘-((CT sample-CT housekeeping gene1) - (CT calibrator - CT housekeeping gene2)).
CT sample: average CT of 3 qPCR repeats of PBM gene.
CT housekeeping gene1: average CT of 3 qPCR repeats of ribosomal protein S7 PBM.
CT housekeeping gene2: average CT of 3 qPCR repeats of ribosomal protein S7 NBF.
CT calibrator: average CT of 3 qPCR repeats of NBF gene.
*significant, R.9. Trypsin, Arginase and Serine-Pyruvate Aminotransferase also significant by DESeq, p,0.05.
Fat Body Transcriptomes of Aedes aegypti
PLoS ONE | www.plosone.org9 July 2011 | Volume 6 | Issue 7 | e22573
the New Mexico State University Animal Care Facility Standard
Operating Procedure and on file in the IACUC office there. All
persons involved in animal work successfully completed Animal
Welfare Training at New Mexico State University and were
specifically trained in protocols used in the research plan. All New
Mexico State University IACUC care and protocols follow the
NIH guidelines described in Guide for the Care and Use of
Laboratory Animals: Eighth Edition, ISBN-10: 0-309-15400-6.
Mosquito Rearing & Blood Feeding
Aedes aegypti Rockefeller strain eggs were obtained from MR4
(available as MRA-734) and used to start a laboratory colony
[72,73]. The colony had been maintained in the laboratory for
approximately one year at the time the work described in this
paper began. Eggs produced by the lab colony were hatched under
25inHg vacuum in 27uC water deoxygenized for 30 minutes. The
eggs were left under vacuum for 15 minutes, then eggs and
hatched larvae were transferred to pans containing water at 27uC
and placed in an incubator maintained at 27uC and 80% relative
humidity. Mosquitoes were fed daily a 1:1:1 mix of albumin,
ground cat food and yeast. Pupae were transferred to a cup
containing 27uC water and placed in a cage to hatch. Mosquitoes
were maintained with free access to 20% sucrose solution until
competent for blood feeding 72 h after emergence.
Mosquitoes were blood fed by placing a live chicken (Gallus
gallus domesticus) on top of their cage for approximately 30 minutes.
Total RNA quantities in the fat body tissue PBM
RNA was extracted and purified from fat body tissue (four
samples each, NBF and PBM, consisting of 5 fat bodies per
sample) and then had concentration measured as described in fat
body dissection and RNA isolation.
Fat body Dissection, RNA Isolation, and poly A+ RNA
200 mosquito abdomens were dissected each from two groups of
mosquitoes 72 hours post eclosion and 24 hours post-blood meal
in groups of 10. Malpighian tubules, midgut, ovaries and crop
were removed, then the abdomens were transferred to eppendorf
tubes with 0.5 ml of Trizol reagent (Invitrogen, Carlsbad, CA).
They were then homogenized with pellet pestle and handheld
homogenizer. Another 0.5 ml of Trizol was added to the cap
afterwards and mixed by inversion. RNA was isolated and
precipitated following the manufacturer’s protocol. RNA pellets
were dissolved in RNase-free water. Concentration was measured
on a Thermo Scientific nanodrop 1000 (Thermo Scientific). RNA
quality was checked using a Bioanalyzer 2100 (Agilent). mRNA
isolation was accomplished by separation using poly-A tails with
Oligotex solution (Qiagen, Valencia, CA) following the instruc-
tions of the manufacturer.
454 cDNA library Construction
The cDNA libraries were constructed using the Clontech SMART
cDNA Library Construction Kit (Invitrogen) following the manufac-
turers instructions with modifications . For the reverse transcrip-
GCC GAC ATG T(4)GT(9)CT(10)V N-39 was used (primer mix:
V=A, G or C; N=A, C, G or T). An amplification step was
performed using the Advantage 2 PCR kit (Invitrogen) with a
modified 39PCR primer: 59-ATT CTA GAG GCC GAG GCG
GCC GAC ATG T(4)GT CT(4)G TTC TGT(3)CT(4)V N-39. The
first strand was synthesized by combining 3 ul of RNA, (.200 ng),
1 ul modified 39 RT primer and 1 ul SmartIV oligo (Invitrogen).
for 2 min and then combined with 2 ul of 106first strand buffer, 1 ul
of DTT, 1 ml of dNTP mix, 1 ul of MMLV-reverse transcriptase,
then incubated at 42uC for 90 minutes. 20 ul of water was added and
firststrandcDNAstoredat220uC.Ina0.2 mlPCRtube,2 uloffirst
strand cDNA, 5 ul of 106buffer, 1 ul of dNTP-Mix (10 mM),1 ul of
modified 39 PCR primer (10 um), 1 ul of Advantage Polymerase and
39 ul of water were combined. In an Eppendorf MasterCycler, the
following program was run: 94uC22 min, 24 cycles: 94uC220 sec ,
65uC220 sec, 68uC26 min. 50 mg of cDNA was synthesized per
experimental sample and purified by phenol/chloroform extraction.
Sequencing and De Novo Assembly
The cDNA library samples described above were run on a 1/6
picotitre plate and Roche 454 Genome Sequencer FLX
instrument. Binary SFF files generated by the sequencing
hardware were converted into fasta, fasta.qual and xml qual files
by the sff_extract program . The extracted fasta files and
fasta.qual files were then used to perform a de novo assembly of the
reads using the assembler Mira . The EST and de novo options
were used to run the assembly software.
Contig files from our assembly were analyzed using Blast2GO.
This program uses a pipeline involving a BlastX step followed by
gene ontology and annotation steps. For most contigs the default
Blast options within Blast2GO were used with the exception of 7
contigs which required lower numbers of results returned due to
the size of the results and the maximum size Blast2GO can report.
Following Blastx, we performed a sanity check outside of
Blast2GO on our contigs by analyzing the species of the best
BlastX result for each contig. BlastX results then had gene
ontology and annotation steps applied, and were grouped by GO
term. Only contigs successfully passing all stages of the pipeline
were used in GO term analysis. Level 2 GO terms for each contig
were used for our analyses .
Novel Transcript Discovery
To identify potential new transcripts, we aligned our contigs
against the reference transcripts available from Vectorbase, using
the tool Blat . Those contigs which did not align with the
reference transcripts were then aligned with the reference genome
using Blat. From this set of contigs (those which did not align with
the reference transcripts but aligned with the reference genome)
we selected only alignments over 100 bp that matched the genome
for at least 75% of the contig. The alignments had to have greater
than 99% similarity over the match with the genome and less than
0.7% gaps with the reference genome along the length of the
match. Further analysis of these contigs consisted of BlastX at
NCBI to identify potential functions from similar protein products
if the contigs were to be translated, use of Blast and the genome
browser at Vectorbase to verify alignment with the genome and a
lack of known protein product, and identification of potential
protein domains using SMART  and the pfam databases .
Sequence Alignment and Data Analysis
Aligned sequences were generated by using Blat to align
generated ESTs to reference transcript sequences from Vector-
base. The aligned output sequences were analyzed for statistically
significant differential expression between samples using the R test
[37,38] and a negative binomial distribution was performed with
Fat Body Transcriptomes of Aedes aegypti
PLoS ONE | www.plosone.org 10July 2011 | Volume 6 | Issue 7 | e22573
From membranetransport.org  we retrieved a list composed of
orknown amino acid sequences.Tothislist weaddedanadditionalsix
genes for potential transporters, which we identified through searches
of transcript information for Ae. aegypti at Vectorbase (AAEL002527,
AAEL003136, AAEL006509, AAEL012596, AAEL006650 and
AAEL007809). We then searched for and identified matches in our
aligned transcript data for the genes on this list.
Immunity Related Transcript Identification
From our GO results, contigs with the term ‘‘immune system
process’’ were selected and their sequences Blasted against the Ae.
aegypti database at Vectorbase. Results obtained produced e-values
typically less than 1e-50, with the majority of results being less than
1e-100, except where noted. Resulting AAEL numbers were then
used to look up read numbers and differential expression statistics
from our sequence alignment data.
Quantitative Real-time PCR
Gene-specific primers were developed using Primer BLAST .
specific RNAs were isolated after dissection of samples from 30
individual mosquitoes including previtellogenic females 72 h after
eclosion and females 24 h post-blood meal. Transcripts were analyzed
and quantified with quantitative RT-PCR (qPCR) using iQ Supermix
the standard. Primers were as follows: Vitellogenin-A1: Forward
primer CTC GTT CCC GCT CTG GCA GC, reverse primer TGT
AGC CGC GACCAA TGT CGG,product length 282; Cathepsin b:
Forward primer AGG GTG CAC AGC ACG TAG AGA, Reverse
primer TGC CGG AGG TTT CGG GTT GC, Product length 308;
Trypsin: Forward primer GCC AAG CTG CAA CGC TGT CC,
Reverse primer GGC GCG CAA CAA CGT GTT CA, Product
length 449; Arginase: Forward primer GCA ACA TGC TGC GCG
GAA AAC A; Reverse primer GCC CAC ATC GCT GCA GTG
CT, Product length 449; Serine-Type Endopeptidase: Forward primer
AGG TGG CCC TTT TCG AGA CGG A , Reverse primer TGA
TTT TCT TCC ACC CGG ATG CAA, Product length 450; Serine-
Pyruvate Aminotransferase, Forward primer ACT ACTGAT GGG
TCC AGG CCC A, Reverse primer AAG CGA GGC AAC CGT
GTC CA, Product length 496; RPS7: Forward Primer TCA GTG
CGC TCA CTT ATT AGA TT. A total of eight RNA samples (4
NBF, 4 PBM) were prepared using the same method for the
measurement of total RNA in fat bodies. Samples were then converted
into cDNA using an Omniscript RT kit (Qiagen) following the
manufacturer’s supplied protocol.
Library from fat bodies of NBF mosquitoes; (B) Library from fat
bodies of mosquitoes 24 h PBM.
A and B Size distribution of EST library reads. (A)
PBM (B) samples.
A and B. GC content of contigs from NBF (A) and
species NBF(A) and PBM(B).
A and B Blast2GO Blastx results broken down by
Putative new genes.
transcript alignments (multiple isoforms counted as one transcript).
Percentage of the total number of unique read-
transcripts from Vectorbase. R-value.9 is considered significant,
meaning high likelihood of being differentially expressed between
the two conditions.
All reads aligned using blat to Ae. aegypti reference
in expression between blood fed and not-blood fed state.
Transcripts reads aligned with using blat, fold change
We thank Jessica Aguirre, Hao Feng, and Nabeeh Hassan for technical
Performed the experiments: DPP IAH LLD. Analyzed the data: DPP VN
AC PH BM LLD JEG IAH. Wrote the paper: DPP IAH. Edited the
manuscript: VN AC PH BM LLD JG.
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