genome-wide analysis of gene expression in adult anopheles

UC IrvineUC Irvine Previously Published Works

TitleGenome-wide analysis of gene expression in adult Anopheles gambiae

Permalinkhttpsescholarshiporgucitem4hb9w6jd

JournalInsect Biochemistry and Molecular Biology 15(1)

AuthorsMarinotti OsvaldoCalvo EricNguyen Quang Ket al

Publication Date2006

DOI101111j1365-2583200600610x

Supplemental Materialhttpsescholarshiporgucitem4hb9w6jdsupplemental Peer reviewed

eScholarshiporg Powered by the California Digital LibraryUniversity of California

Insect Molecular Biology (2006)

15

(1) 1ndash12

copy 2006 The Royal Entomological Society

1

Blackwell Publishing Ltd

Genome-wide analysis of gene expression in adult

Anopheles gambiae

O Marinotti E Calvodagger Q K NguyenDagger S Dissanayake J M C Ribeirodagger and A A Jamessect

Department of Molecular Biology and Biochemistry University of California Irvine CA USA

dagger

Laboratory of Malaria and Vector Research National Institutes of Health (NIHNIAID) Rockville MD USA

Dagger

Department of Biological Chemistry and

sect

Department of Microbiology and Molecular Genetics University of California Irvine CA USA

Abstract

With their genome sequenced

Anopheles gambiae

mosquitoes now serve as a powerful tool for basicresearch in comparative evolutionary and develop-mental biology The knowledge generated by thesestudies is expected to reveal molecular targets fornovel vector control and pathogen transmission block-ing strategies Comparisons of gene-expression pro-files between adult male and nonblood-fed female

Anopheles gambiae

mosquitoes revealed that roughly22 of the genes showed sex-dependent regulationBlood-fed females switch the majority of their metabo-lism to blood digestion and egg formation within 3 hafter the meal is ingested in detriment to other activi-ties such as flight and response to environmentstimuli Changes in gene expression are most evidentduring the first second and third days after a bloodmeal when as many as 50 of all genes showedsignificant variation in transcript accumulation Afterlaying the first cluster of eggs (between 72 and 96 hafter the blood meal) mosquitoes return to a nongono-trophic stage similar but not identical to that of 3-day-old nonblood-fed females Ageing andor the nutritionalstate of mosquitoes at 15 days after a blood meal isreflected by the down-regulation of simsimsimsim

5 of all genes Afull description of the large number of genes regulated

at each analysed time point and each biochemicalpathway or biological processes in which they areinvolved is not possible within the scope of this contri-bution Therefore we present descriptions of groupsof genes displaying major differences in transcriptaccumulation during the adult mosquito life Howevera publicly available searchable database (httpwwwangagepucibiouciedu) has been made available sothat detailed analyses of specific groups of genesbased on their descriptions functions or levels of geneexpression variation can be performed by interestedinvestigators according to their needs

Keywords

Anopheles gambiae

gonotrophic cycleageing sex-biased gene expression microarray

Introduction

The fruit fly

Drosophila melanogaster

is a proven modelfor studies of physiology biochemistry behaviour andother aspects of insect biology However the knowledgeobtained is not always directly applicable to other speciesdue mainly to the enormous diversification of insects andtheir adaptations to different niches Hematophagy withits multiple determining factors and consequences (host-seeking blood ingestion digestion and the unavoidablechallenge of dealing with a heavy load of iron moleculesingested at each meal and its direct implication in mosquitoreproduction and pathogen transmission) is just oneexample of a mosquito evolutionary adaptation that cannotbe addressed by studying

Drosophila

or other modelorganisms Based on these differences insect vectors ofdiseases comprising species of mosquitoes tsetse fliessand flies black flies fleas kissing bugs and bedbugs thattransmit viruses and parasites to humans have been theobjective of extensive investigation with the main purposeof finding novel and efficient ways to disrupt or diminishpathogen transmission The concluded venture to sequencethe genome of the human malaria vector

Anophelesgambiae

(Holt

et al

2002) and the ongoing projects tosequence the genomes of two other mosquito species

Aedes aegypti

(Severson

et al

2004) and

Culex quinque-fasciatus

represent major advances in this direction They

doi 101111j1365-2583200600610

Received 5 May 2005 accepted after revision 3 August 2005 Correspond-ence Dr Anthony A James Departments of Microbiology amp MolecularGenetics and Molecular Biology amp Biochemistry 3205 McGaugh Hall Uni-versity of California Irvine CA 92697ndash3900 USA Tel +1 949 824 5930fax +1 949 824 2814 e-mail aajamesuciedu

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Insect Molecular Biology

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are expected to present opportunities to improve vectorcontrol by revealing new gene targets for insecticides andinsect repellents and to advance novel strategies to controlpathogen transmission (Toure

et al

2004) However a keyquestion to be answered by vector biologists is how toexplore this sequence information in pursuit of these goalsThe tools of functional genomics represent a direct way toobtain answers to this question Determination of spatialand temporal expression patterns and the linking of genesto their biological functions are basic and fundamentalsteps to progress toward using sequence data for control-ling disease transmission In addition with their genomessequenced mosquitoes can now serve as powerful toolsfor basic research comparative evolutionary and develop-mental biological studies that should lead to the under-standing of the processes that shape insect genomes andtheir phenotypic expressions We have looked at globalchanges in gene expression in adult female

An gambiae

before and after a blood meal and at differences betweenmales and females and young and old mosquitoes It isimpossible to describe here the large number of genesregulated at each analysed time point and each biochemicalpathway or biological processes in which they are involvedThus descriptions are based on groups of genes display-ing major differences in transcript accumulation during theadult mosquito life However by using a publicly availablesearchable database (httpwwwangagepucibiouciedu)detailed analyses of specific groups of genes based on theirdescriptions functions or levels of gene expression variationcan be performed by any investigator according to need

Results and discussion

Identification of

An gambiae

-regulated transcripts and assignment of biological functions

The Gene Chip PlasmodiumAnopheles genome array(Affymetrix) which includes probe sets to 14 900

Angambiae

genes was used to evaluate sex-biased geneexpression in adult mosquitoes changes in transcript accu-mulation following blood meals in females and changesassociated with ageing The

An gambiae

genome isestimated to have approximately 15 000 genes (Holt

et al

2002) therefore this microarray platform represents acomprehensive tool for genome-wide enquiries of variationsin gene expression in this species

We developed a searchable database to facilitate theanalyses and add biological meaning to the large amountof microarray-derived data Consensus DNA sequences usedfor the microchip development (httpwwwaffymetrixcomsupportfile_downloadaffxonloadforward=analysisdownloadsdataPlasmodium_Anopheles_consensuszip)were used as input for B

LAST

searches performedlocally from executables obtained at the NCBI FTP site(Altschul

et al

1997) The electronic version of the

database complete with hyperlinks to web-based informa-tion and to B

LAST

results is available for download athttpwwwangagepucibiouciedu Online searchesalso are available through this site The annotation ofthe

An gambiae

genome and transcriptomes and con-sequently of the array elements is an ongoing processmostly due to low or lack of significant similarity of some

An gambiae

-derived cDNA sequences to known genesand transcripts of other organisms Approximately 35of all probe sets correspond to transcripts that have noidentified biological function and are without B

LAST

matches in gene ontology and protein families (PFAM)databases Furthermore in contrast to what is available for

D melanogaster

where a high proportion of sequencedgenes are linked to a verified biochemical function ormutant phenotype the putative functions assigned to

An gambiae

gene products are based almost exclusivelyon comparative sequence similarity and conserveddomains Although comparative genomic analysis improvesthe

An gambiae

gene annotation functional genomicsproteomics and high-throughput gene expression studiesare required to contribute and validate these predictedfunctions

Sex-biased gene expression

We first compared in a global perspective the expressionof genes in nonblood-fed (NBF) adult female mosquitoes(third day after emergence and fed only on raisins) withadult males (third day after emergence and fed on raisins)For this work genes differentially expressed were definedby statistical analysis as those showing variations with

P

-values lower than 0001A total of 3275 transcripts was identified as having

gender-specific or preferential expression (Fig 1 and Sup-plementary Material Table S1) Sex-biased gene expres-sion also is significant in

Drosophila

where 30ndash50 of thegenes were found to be differentially expressed (Jin

et al

2001 Arbeitman

et al

2002 Ranz

et al

2003) A similaranalysis performed recently comparing virgin males andfemales at 36 h posteclosion (Hahn amp Lanzaro 2005) found2901 genes differentially expressed (

P

lt 005) Our exper-iments used older mosquitoes that were allowed to mateand this could account for the difference in number of sex-biased genes found in each study Of a list of 167 highlysignificant sex-biased genes provided by Hahn amp Lanzaro(2005) we confirmed all but one Ag3R35570 which wasmore expressed in females (12-fold) in our experiment andless expressed in females (17-fold) in that Hahn amp Lanzaro(2005)

Approximately 47 of 3275 genes that exhibit sex-differential expression accumulate transcripts at higherlevels in males while the balance are found preferentiallyor specifically in females Five hundred of the transcriptsidentified as expressed preferentially or exclusively in

Gene expression in

Anopheles gambiae 3



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males correspond to transcripts that were not present in aset of 80 000

An gambiae

adult female-derived expressedsequence tags (ESTs) (Ribeiro 2003) providing additionalsupport for them being male-specific The majority of genesidentified as expressed preferentially in males but withsome representation in the female EST database hadlow numbers of sequenced ESTs For example 225ESTs were represented only once and 1187 had lessthan five sequenced cDNAs Genes represented in themale-enhanced transcript list (Fig 1 and SupplementaryMaterial Table S1) include several putative testis and spermcomponents such as those similar to the

D melanogaster

male-specific RNA 84DB a spermiogenesis-associatedtestis specific serinethreonine protein kinases expressedin

Homo sapiens

and

D melanogaster

an infertility-relatedsperm protein a protein with sequence similarity to sperm-specific thioredoxin 2 of

H sapiens

and other sperm- andspermatogenesis-associated transcripts

Several sensory reception-related transcripts such as anarrestin opsin and a gustatory receptor also were identified

as having male-enhanced expression (Fig 1) Genesencoding olfaction-related proteins expressed preferen-tially in males are probably involved in locating nectarsources for feeding or females for mating (Biessmann

et al

2005) Thirty-four transcripts with gene ontology annotationrelated to translation and transcription regulation alsowere enhanced in males and these may be involved incontrolling the gender-related differential gene expressionpatterns

Female-enhanced transcripts found prior to blood feed-ing (Fig 1 and Supplementary Material Table S1) corre-spond to a series of salivary gland components digestiveenzymes and ovary-expressed genes A total of 48 salivarygland-expressed transcripts showed female preferentialexpression Among these several were known previouslyto be expressed preferentially in females such as membersof the

D7-related

group an apyrase and a salivary peroxi-dase (Arca

et al

1999) This gender- and tissue-specificexpression has been interpreted to indicate that the prod-ucts of these genes represent adaptations of mosquitoes toblood feeding Accordingly genes equally expressed in thesalivary glands of both sexes such as

Maltase like I

and

AMY I

of

Aedes aegypti

and their homologues in anophe-line mosquitoes are probably involved in sugar or nectaringestion and digestion (Francischetti

et al

2002 Calvo

et al

2004)Midgut-expressed enzymes involved with blood diges-

tion generally are synthesized after a meal is ingestedHowever some of them are present in the midgut beforethe blood meal and participate in the initial steps of diges-tion presumably providing signals for the midgut cells toproduce the large bulk of digestive enzymes needed duringthe process Several proteins including digestive trypsinand peritrophin are known to be stored prior to a bloodmeal in secretory vesicles of midgut epithelial cells (Deven-port

et al

2004) Accordingly we find several transcriptsencoding serine proteases trypsin and chymotrypsinsaminopeptidases and carboxypeptidases in NBF femalesTranscripts corresponding to components of the peritrophicmatrix (a chitinprotein-containing acellular sheath that sur-rounds the blood meal and separates the food bolus fromthe midgut epithelium) are also found in the NBF mosquitoes

The behaviour of mature adult males is dictated mostlyby the requirements of mating They feed solely on sugarsources (nectar and fruit sources) and their digestivesystem is not adapted to respond to a blood meal Theirreproductive system continuously produces sperm and doesnot undergo substantial changes after adult emergenceand their flight-activity periodicities and responses to environ-mental stimuli do not change until the effects of ageing arenoticeable (Clements 1999) While the physiology of malesdoes not change much during adult life females go throughextensive changes after taking a blood meal (Holt

et al

2002 Ribeiro 2003 Dana

et al

2005)

Figure 1 Sex-biased gene expression in adult Anopheles gambiae (Top) Percentile representation of sex-biased gene expression of 14 900 An gambiae transcripts A total of 3194 probe sets representing 3002 transcripts are expressed at different levels (P-values lt 0001) in males and females (Supplementary Material Table S1) (Bottom) Selected genes displaying stronger accumulation in males (M 1ndash3) or females (F 1ndash3) The numbers 1 2 and 3 represent triplicate samples for each sex Transcript ID and annotation were obtained from the An gambiae microarray database (httpwwwangagepucibiouciedu) Expression data for each probe set were normalized and organized according to preferential accumulation in males or females Tree View software was used and red and green represent higher and lower relative expression respectively

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Global changes in transcript accumulation during a gonotrophic cycle

We examined variations of gene expression at several timepoints post blood meal (PBM) during a complete gono-trophic cycle and also determined changes in ageingmosquitoes 18 days after adult emergence (Figs 2 3 and 4)Gene expression at each developmental stage was com-pared with experimental time points immediately prior toand after it (Supplementary Material Tables S2ndash13) and alltime points were compared to NBF females (Supplemen-

tary Material Tables S2 S3 and S14 S15ndash23) which weconsidered as the standard nonvitellogenic stage As wellas comparisons of transcript accumulation based on

P

-values (lt 0001) (Figs 2 3 and Supplementary MaterialFig S1) genes expressed differentially at each stage of thegonotrophic cycle also were identified by

ANOVA

tests (Fig 4and Supplementary Material Tables S24ndash62) The follow-ing descriptions are based on groups of genes displayingmajor differences in transcript accumulation during theadult female mosquito life The searchable databasedeveloped here (httpwwwangagepucibiouciedu) allowsdetailed analyses of specific groups of genes based ontheir descriptions functions or levels of gene expressionvariation to be performed by any investigator in the researchcommunity

Figure 2 Numbers of Anopheles gambiae transcripts up- or down-regulated significantly (P-values lt 0001) in females with and without a blood meal The numbers of varying transcripts were calculated by comparison of each sample with its immediate subsequent experimental sample [nonblood-fed (NBF)] (A) or by comparing each time point with three-day-old NBF females (B) Numbers of regulated transcripts were extracted from Supplementary Material Tables S1ndash25

Figure 3 Global patterns of gene expression during oogenesis and ageing in Anopheles gambiae adult females Each row represents data for one probe set and each column corresponds to a developmental time point referred on the top as hours (h) or days (d) post blood meal (average of three determinations and the data for each experiment and standard deviations are available as Supplemental Material Tables S2 S3 S14 and S15ndash23) Expression values are relative to the reference 3-day post emergence nonblood-fed female Red indicates higher and green indicates lower levels of transcript accumulation Data were displayed using Tree View software Details of the expression patterns of selected genes mentioned in the text are presented in Supplementary Material Fig S1

Gene expression in

Anopheles gambiae 5



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Extensive variation in gene expression was observed inblood-fed (BF) females (Figs 2ndash4) A total of 10 631 probesets corresponding to 9724 transcripts were found to vary(

P

-values lt 0001) in accumulation during at least one ofthe analysed experimental time points Of these 5423varied at least two-fold and 2625 varied more than three-

fold (Supplementary Material Fig S2) While the majority oftranscripts varied less than three-fold 126 transcripts wereaccumulated at levels at least 100-fold different from thosedetected in NBF females

Thirty per cent (469714 900) of all analysed transcriptswere regulated immediately after the females fed on blood

Figure 4 A Welch ANOVA-based statistical analysis of gene expression during mosquito development with multiple testing correction Benjamini and Hochberg False Discovery Rate with a 0001 P-value cutoff Probe sets corresponding to mosquito genes regulated upon blood feeding and ageing were identified and grouped for similarity patterns using the K-Means clustering method generating 39 sets (hyperlinked lists of transcripts included in each set are in Supplementary Material Tables S24ndash63)

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(3 h PBM) with 2254 up-regulated and 2443 down-regulatedas compared with NBF females of the same age The totalnumber of genes regulated in BF females as comparedwith NBF females continued to increase during the three-day period that followed the meal reaching a maximumof 7494 at the third day (72 h PBM) which corresponds to

sim

50 of all genes (Fig 2B) The number of genes regu-lated decreased thereafter coincident with the end of thetwo major processes occurring in mosquitoes blood diges-tion and egg formation Changes in transcript accumula-tion particularly noticeable during the initial steps (NBF3 h PBM and 3ndash24 h PBM Fig 2B) of the gonotrophiccycle were not as large after egg laying (which occursbetween 72 and 96 h PBM) both in number of transcriptsand fold-variation These data represent the pattern of geneexpression characteristic of mosquitoes that do not take asecond blood meal during the gonotrophic cycle

Anophe-les gambiae

females may feed on blood several timesduring each gonotrophic cycle both in laboratory and fieldconditions and each meal triggers again the expressionand regulation processes of several genes involved indigestion and egg formation (Briegel amp Horler 1993 Koella

et al

1998 Nirmala

et al

2005) Therefore the patternsof gene expression described here may not reflect whathappens in nature

Immediate responses to a blood meal

Detailed analyses of variation in gene expression at 3 hPBM revealed a large number of up-regulated digestiveenzymes including those involved in proteolysis (trypsinschymotrypsins aminopeptidase carboxypeptidase) and thoserelated to lipid (lipases) and carbohydrate (

α

-glucosidasesand

α

-amylases) digestion These data are consistentwith a prompt reaction of the midgut cells to a blood mealresulting in the secretion of enzymes responsible for thedigestive process Other transcripts encoding moleculesinvolved with the digestive process such as peritrophinsand intestinal mucins also are up-regulated at that timepoint

Several of the salivary gland-expressed genes areregulated at higher or lower levels 3 h PBM During probingthe vertebrate host skin in search of a blood source mos-quitoes secrete saliva containing a variety of moleculeswith antihaemostatic activity Following a blood meal thesecreted salivary components are reduced significantly(Marinotti

et al

1990 Golenda

et al

1995) All of thesalivary components are synthesized

de novo

however therates of salivary gland replenishment varies for differentcomponents (Marinotti

et al

1990) Possibly some of thesalivary components need to be restored quickly to preblood-fed levels to allow mosquitoes to efficiently obtain anext meal while others could recover at a slower rate withoutaffecting the ability of the females to obtain further blood ornectar meals necessary for proper egg development

Hormone-related transcripts change in abundance at 3 hPBM The changes in expression of these genes areconsistent with the known hormone accumulation patternsobserved during mosquito adult life and particularly duringproduction of the first cluster of eggs (Redfern 1982 Lu ampHagedorn 1986 Hagedorn 2005) Ecdysone receptortranscripts are up-regulated in concert with the increasingconcentration of 20-hydroxyecdysone that follows a bloodmeal No experimental identification of juvenile hormoneesterases from mosquitoes has yet been performed how-ever transcripts encoding proteins similar to juvenilehormone esterases of other insects also are up-regulatedand their products putatively lead to a decrease in concentra-tion of circulating juvenile hormone (Kamita

et al

2003)Down-regulation of transcripts related to vision gustatory

and olfactory reception and flight activity is already initiatedat 3 h PBM This is consistent with what was described inmosquitoes at 24 h PBM (Ribeiro 2003) (Fig 3) Opsinsarrestins and odourant-binding proteins involved with envi-ronment perception and muscle structural componentsflightin and troponin as well as pyrroline-5-carboxylatereductase and proline oxidase involved in the utilization ofthe amino acid proline as an energy substrate during flightare expressed at lower levels in BF mosquitoes This is con-sistent with the inhibition of flight activity in BF mosquitoes(Jones amp Gubbins 1978) Some ovary-expressed genessuch as

oskar

and several immune-related transcripts arealso down-regulated during these initial steps of the gono-trophic cycle We interpret this to indicate that most of theenergetic and biosynthetic effort in mosquitoes is directedtowards the initiation of an efficient digestive process andthis comes at the expense of other activities such as flightand ovary development However ovary development isquickly resumed as noted at 24 h PBM

Blood meal digestion and yolk protein synthesis

Previous analyses of blood meal-induced changes in geneexpression in

An gambiae

were based on sequencingcDNA libraries constructed with RNA extracted from eitherNBF females or BF females at 24 h PBM and these iden-tified 168 (Holt

et al

2002) or 435 (Ribeiro 2003) genetranscripts to be significantly more or less transcribed inBF mosquitoes The list of regulated transcripts at 24 hPBM determined in our study includes those previouslydescribed but also is much larger reflecting more sensitivedetection limits of the techniques applied in our analysesHowever in spite of the different techniques the expressionprofiles of 80 of regulated transcripts identified by thoseEST analyses are consistent with what is observed inmicroarray experiments (Marinotti

et al

2005) A total of6584 transcripts vary in concentration between the timemosquitoes ingest a blood meal and 24 h later (Fig 2)While 3961 (

sim

60) are up-regulated 2623 (

sim

40) aredown-regulated at this physiological stage Significant

Gene expression in

Anopheles gambiae 7



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differences in BF females 24 h PBM reflect a continuationof the variations seen at 3 h PBM with a qualitative andquantitative decrease in transcripts associated with loco-motion and response to environment stimuli and presentnew trends such as the activation of secretory activity bythe midgut cells (which results in the formation of theperitrophic matrix and accumulation of digestive enzymesin the lumen of the midgut) activation of fat body cells(responsible for the accumulation of lipid reserves and syn-thesis of vitellogenic proteins) and progress of oogenesisThe set of genes up- or down-regulated at 24 h PBMis therefore not identical to those regulated earlier at3 h PBM Several of the digestive enzymes expressedabundantly earlier during digestion are now repressedand replaced by different enzymes This phenomenon isconsistent with the two-phase blood digestion processdescribed previously wherein early serine proteasesexpressed prior to or immediately subsequent to a bloodmeal and related to the initiation of the digestion generatesignal molecules that in turn modulate mosquito-wide phys-iological changes leading to the completion of digestionand egg production (Barillas-Mury

et al

1995 Muller

et al

1995) Vitellogenin vitellogenic cathepsin B andother transcripts either not seen as up-regulated or regul-ated at low levels at the beginning of the gonotrophic cycleare up-regulated remarkably at 24 h PBM The accumulationof large quantities of these transcripts and their translationresult in massive yolk protein synthesis and deposition in

fast growing oocytes These proteins are deposited in gran-ules and serve as amino acid reserves for future embryonicdevelopment (Ahmed

et al

2001 Raikhel

et al 2002)

Spatial distribution of gene expression in gonotrophic females

The accumulation of specific transcripts in isolated fatbodies midguts and ovaries of females at 24 h PBM wasalso determined (Fig 5) This time point was selected forthe extensive changes in gene expression occurring invitellogenic mosquitoes and the selected tissues are in-volved in intensive protein synthesis associated with blooddigestion and egg formation as discussed above We ident-ified 2714 genes expressed in all three tissues and corre-sponding to housekeeping genes and generated lists ofgenes expressed exclusively in fat bodies (481) ovaries (3721)or midguts (190) (Supplementary Material Tables S63ndash65)Vitellogenin vitellogenic cathepsin B hexamerin and lipo-phorin are proteins known to be expressed in the fat bodiesof mosquitoes (Zakharkin et al 1997 Sun et al 2000Raikhel 2004) and this is confirmed by our analysisSimilarly several serine proteases aminopeptidasesperithophin the microvillar protein G12 and other genespreviously known to be expressed in midgut cells (Mulleret al 1993 Vizioli et al 2001 Xu et al 2005) were con-firmed by our results The ovaries have the largest numberof tissue specific transcripts These components include avast assortment of RNA species that support early embry-onic development For example the dorsalventral andanteriorposterior axes of the future embryo are laid downduring oogenesis (Goltsev et al 2004) The somatic follic-ular epithelium surrounding each egg chamber is also animportant source of structural proteins for the eggshellAnalysis of other dissected tissues should provide anaccount of all genes expressed in the gonotrophic femalesand provide further support to the tissue specificity indicatedby the partial analysis presented here

Completion of the gonotrophic cycle

After an impressive change in the pattern of geneexpression during the first and second days PBM quanti-tative variations between the second and third days areless noticeable (Fig 2B) However important qualitativechanges occur (Figs 3 and 4) At 48 h PBM those genesdetermined as strongly up-regulated at 24 h PBM includ-ing vitellogenin vitellogenic cathepsin B and severaldigestive enzymes are down-regulated and return to lowerlevels similar to those seen in NBF females However othergenes are up-regulated at this stage Several odourantbinding protein transcripts are up-regulated between 24and 48 h PBM and they may represent females regainingfull capacity to respond to host odours andor their abilityto search for cues that will lead them to oviposition sitesA third group of genes regulated at 48 h PBM includes

Figure 5 Three-way comparison of gene expression in fat bodies ovaries and midguts of adult female Anopheles gambiae at 24 h post blood meal (PBM) Genes were considered as expressed in a tissue when its respective transcript was assigned as present by GCOS analysis in all three samples derived from that tissue Up- down- or no regulation where derived from the comparisons of transcript accumulation between nonblood-fed and 24 h PBM females performed with whole mosquitoes (httpwwwangagepucibiouciedu)

8 O Marinotti et al

copy 2006 The Royal Entomological Society Insect Molecular Biology 15 1ndash12

those with sequence similarity to chorion genes of otherinsect species and have an expression pattern consist-ent with their function as components of the mosquitoeggshell

A new increase in the number of regulated transcripts isobserved between 72 and 96 h PBM corresponding tooviposition and the return of the female to a resting orprevitellogenic stage Several of the transcripts seen as up-regulated at these latter time points represent residual tran-scripts of genes with strong expression earlier during thedevelopment For example vitellogenin is up-regulatedmore than 100-fold at 24 h after feeding but only two-foldat 72 h PBM Other genes have their transcripts accumu-lated to the maximum level at 72 h PBM This groupincludes those with sequence similarity to the bicaudalexuperantia bicoid hunchback nanos and oskar genesof D melanogaster These fruit fly maternally-expressedmRNAs are deposited in the oocytes and are involved withthe determination of antero-posterior axis and segmenta-tion of the developing embryos (St Johnston amp Nusslein-Volhard 1992) Their orthologous genes in An gambiaeexhibit similar patterns of expression and are consideredto be involved in the same biological processes (Goltsevet al 2004 Calvo et al 2005) The diminished amountof these transcripts at 96 h PBM is consistent with theirproposed functions reflecting the oviposition of the firstcluster of eggs and a cessation of follicular development

Ageing in adult mosquitoes

We compared patterns of transcript accumulation in mos-quitoes at 15 days PBM with mosquitoes of the same age(18 days) that had never been fed on blood with 3-day-oldNBF mosquitoes and with females at 96 h PBM Thereis little variation in gene expression between 96 h PBMfemales and 15-day PBM females and among these twosamples and 18-day-old females that never took a bloodmeal These comparisons show only three up-regulatedtranscripts between 96 h PBM and 15 days PBM samplescorresponding to a putative infection-responsive shortpeptide a putative chitinase expressed in response tobacteria and pathogen infection and another similar to theD melanogaster gene CG16995 that has been annotatedin the gene ontology database as a molecule related to thedefence response to biotic stimuli Therefore these genescould represent the response of mosquitoes to environ-mental contamination (fungi and bacteria) instead oftrue ageing (senescence) The down-regulated genescorrespond to a variety of components including mitochon-drial proteins salivary gland proteins fat body-expressedmolecules and digestive enzymes However the down-regulation is in most cases relatively minor with a morethan 10-fold variation in transcript accumulation occurringfor only 13 transcripts (Supplementary Material Tables S12and S13)

Considerable variation was observed in comparisonsof NBF females (third day after emergence) and 18-day-old adult NBF or BF females (Supplementary MaterialTables S22 and S23) In this comparison it is still evidentthat only 10 genes are up-regulated in older mosquitoeswhile a group of 768 genes have their expression levelsdecreased In addition to ageing these variations mayreflect the nutritional status of the mosquitoes that have notbeen fed on a second blood meal

In D melanogaster senescence is characterized as adynamic process with approximately 2 of transcriptschanging in their representation with age (Zou et al 2000)However when reproduction-associated female-specificchanges in gene expression also are considered this per-centage increases to nearly 23 of the genome (Pletcheret al 2002) Changes in gene expression were analysedduring the lives of mice and rhesus monkeys indicatingrespectively that roughly 2 and 6 of the genes areregulated with age According to our analyses 5 of themosquito genome has its expression modulated duringthe ageing process Our comparison of nonvitellogenicfemale mosquitoes at 3 and 18 days after emergence wasdesigned to eliminate most of the reproduction-relatedgene expression effects Therefore our result (5 of themosquito genome varying in expression in an age-dependentmanner) agrees with the data reported previously for otherorganisms

An important consideration for investigations of geneexpression regulation during the ageing process is thatdiets imposing calorific restriction are accompanied by aslowing of the age-related changes in transcript levels inD melanogaster (Pletcher et al 2002) A detailed compar-ison of the An gambiae genes regulated during ageingandor restricted nutritional status with those identified inD melanogaster is yet to be performed

Constitutive and abundant expression in adult mosquitoes

An interesting subset of An gambiae genes is that re-presented by the most abundantly and constitutivelyexpressed transcripts These genes were identified by thesum of all signal values determined throughout mosquitodevelopment creating a list from which the 200 genesshowing the strongest signals and constitutive expressionpatterns in mosquitoes were analysed further (Fig 6 andSupplementary Material Table S66) Forty-eight per centof these genes correspond to transcripts related to proteinsynthesis and modification with the majority encodingribosomal proteins Other transcripts related to protein syn-thesis including translation initiation factors and elongationfactors as well as proteins of the transcription machinery(RNA polymerase II) and RNA editing (splicing factor) arealso found Genes encoding mitochondrial proteins includingADPATP translocase ATP synthase and cytochrome coxidase are among those strongly expressed and correspond

Gene expression in Anopheles gambiae 9


to the second most abundant group at 25 of the 200selected genes It is interesting to note that despite variationsof these mitochondrial products during the gonotrophiccycle overall they remain among the most abundant prod-ucts in mosquitoes Actin myosin and α-tubulin transcriptsare also found in this group and this is consistent with theirubiquitous expression and participation in the cytoskeletalorganization of cells A cecropin expressed abundantly inmosquitoes may reflect the continuous response of themosquitoes to environmental pathogens The ferritin geneis also expressed abundantly throughout adult life andthere is no evidence of its regulation upon blood feedingIn contrast ferritin is up-regulated after blood feeding inAe aegypti (Geiser et al 2003) therefore either An gambiaeresponds differently to the load of iron ingested with theblood or the induction of ferritin gene expression occursshortly after the meal and the peak of expression was notdetected within the sampling intervals used here Arrestinsand opsins are important and abundant components ofthe visual system of insects (Graf et al 1996) and areexpressed continually at high levels Polyubiquitins also areexpressed abundantly in mosquitoes These genes encodeprecursor molecules that are cleaved in the cytoplasm togenerate ubiquitin monomers that are involved in numer-ous cellular functions including chromatin structuremodification DNA repair cell cycle modulation proteindegradation and stress response The polyubiquitin geneof An gambiae is expressed constitutively in larvae pupaeand adults and shares high identity at the DNA sequencelevel to polyubiquitins of various organisms rangingfrom 759 in Saccharomyces cerevisiae to 855 in

D melanogaster (Beard et al 1996) Because of its strongand continuous expression the D melanogaster polyubiq-uitin gene promoter has been used in insect transgenesisexperiments Genetically transformed insects includingAn albimanus carrying a chimeric polyubiquitinpromoter-DsRed1 transgene express the marker proteinthroughout development (Handler amp Harrell 1999 Pereraet al 2002 Allen et al 2004) Genes encoding membersof a second group of ubiquitin molecules that fuse toribosomal proteins are also among the strongly expressedgenes in An gambiae These ubiquitin fusion genes areinvolved in ribosome biogenesis (Finley et al 1989)Interestingly 14 genes abundantly expressed in An gam-biae are related to products with unknown functions Theyhave no significant match in BLAST searches to proteinswith known functions (nonredundant database) or conserveddomains [gene ontology (GO) and PFAM databases] It willbe important to determine the biological functions of thesemolecules

Final comments

In conclusion this study exploited a commercial DNAmicroarray platform to identify differentially expressedgenes during the adult life of An gambiae The embryoniclarval and pupal stages are yet to be studied and the deter-mination of gene expression profiles in whole insects at allthese stages plus the determination of expression patternsin specific organs and cells will provide a comprehensiveknowledge of transcription in An gambiae Further studiesare needed to define the relationships among these genesand their products that are involved in mosquito develop-ment and reproduction Although much of this analyticalwork is descriptive the output is rich in identifying manynew gene products that may be associated with fundamen-tal aspects of An gambiae biology Acknowledging thatmicroarray derived results must be taken with caution thedatabase that we are providing which includes gene anno-tations and expression patterns for 14 900 An gambiaegenes is a valuable tool for researchers working in all fieldsof malaria vector biology

Experimental procedures

Mosquitoes

The Pink-eye strain of An gambiae (Githeko et al 1992) wasmaintained at 25 degC 75ndash85 relative humidity and a 186 hlightdark cycle Larvae were fed on finely powdered fish food(Tetramind Tetra Werke Germany) mixed 1 1 with yeast powderAdults (males and females) were kept in cages with access adlibitum to raisins and water For blood feeding adult femalemosquitoes were fed on mice anaesthetized with a mixture ofketamine and xylazine Males and females were kept together inthe cage until the blood meal Blood-fed females were transferredto a separate cage and kept without males for the remaining

Figure 6 Functional distribution of 200 abundantly expressed transcripts of Anopheles gambiae mosquitoes The sequences were classified according to their functions related to cs cytoskeleton (including muscle related proteins) dg digestion em extracellular matrix im intermediate metabolism imm immunity nr nuclear regulation pmps protein synthesis and modification st signal transduction tm transcriptional machinery tr transporters uk unknown Legends indicate classification total number of transcripts and percentages they represent in each category respectively Details in Supplementary Material Table S64

10 O Marinotti et al


duration of the experiment The transferred females were kept withaccess to raisins and water and were offered a cup with water asoviposition site at all times Egg laying was observed between 72and 96 h PBM Three samples composed of five mosquitoeseach were collected for each experimental time point and the RNAwas immediately extracted

Target preparationprocessing for GeneChip analysis

Isolated total RNA samples were processed as recommendedby Affymetrix Inc (Affymetrix GeneChip Expression AnalysisTechnical Manual Affymetric Inc Santa Clara CA USA) In brieftotal RNA was initially isolated using TRIzol Reagent (Gibco BRLLife Technologies Rockville MD USA) and purified further bypassing it through an RNeasy spin column (Qiagen ChatsworthCA USA) Eluted total RNAs were quantified and a portion ofthe recovered material adjusted to a final concentration of 1 microgmicrolThree independent samples were evaluated to each time point andeach experimental sample was compared to a common reference[nonblood-fed females 3 days after emergence (NBF)] All startingtotal RNA samples were quality-assessed prior to beginning targetpreparationprocessing steps by resolving a small amount of eachsample (typically 25ndash250 ngwell) on to a RNA Laboratory-On-A-Chip (Caliper Technologies Mountain View CA USA) that wasevaluated on an Agilent Bioanalyser 2100 (Agilent TechnologiesPalo Alto CA USA) Single-stranded then double-stranded cDNAwas synthesized from the poly (A)+ mRNA present in the isolatedtotal RNA (10 microg total RNA starting material each sample reaction)using the SuperScript Double-Stranded cDNA Synthesis Kit(Invitrogen Carlsbad CA USA) and poly (T)-nucleotide primersthat contained a sequence recognized by T7 RNA polymeraseA portion of the resulting ds-cDNA was used as a template to gen-erate biotin-tagged cRNA from an in vitro transcription reactionusing the BioArray High-Yield RNA Transcript Labeling Kit (T7)(Enzo Diagnostics Inc Farmingdale NY USA) 15 microg of theresulting biotin-tagged cRNA were fragmented to strands of 35ndash200 bases in length following prescribed protocols (AffymetrixGeneChip Expression Analysis Technical Manual) Subsequently10 microg of this fragmented target cRNA was hybridized at 45 degCwith rotation for 16 h (Affymetrix GeneChip Hybridization Oven640) to probe sets present on an Affymetrix GeneChipreg Plasmo-diumAnopheles Genome Array The GeneChip arrays werewashed and then stained (SAPE streptavidin-phycoerythrin) onan Affymetrix Fluidics Station 450 followed by scanning on aGeneChip Scanner 3000 The results were quantified and ana-lysed using GCOS software (version 111 Affymetrix Inc) usingdefault values (scaling target signal intensity = 500 normalizationall probe sets parameters all set at default values)

Statistical analysis and data display

The signal values generated by GCOS 111 software enabled usto perform a Bayesian t-test using a web-based statistical analysispackage Cyber-T at httpvisitoricsuciedugenexcybert (Longet al 2001) and generate a list of probe sets with a P-value of lessthan 0001 To monitor the expression of genes over the differentexperimental time points an expression profile and a K Meansclustering were carried out using the software package Gene-Spring (Silicon Genetics Redwood City CA USA) according tothe supplierrsquos recommendations Cluster and Tree View softwares(Eisen et al 1998 httpranalblgovEisenSoftwarehtm) wereused for data display and editing

Acknowledgements

The authors thank Dr Denis Heck from the UCI DNA ArrayCore Facility for help performing the microarray experimentsand discussions and Lynn Olson for help in preparing themanuscript This investigation was supported by the UNDWorld BankWHO Special Programme for Research andTraining in Tropical Diseases to OM and NIH (AI 29746) toAAJ

References

Ahmed AM Maingon R Romans P and Hurd H (2001)Effects of malaria infection on vitellogenesis in Anophelesgambiae during two gonotrophic cycles Insect Mol Biol 10347ndash356

Allen ML Handler AM Berkebile DR and Skoda SR (2004)PiggyBac transformation of the New World screwworm Coch-liomyia hominivorax produces multiple distinct mutant strainsMed Vet Entomol 18 1ndash9

Altschul SF Madden TL Schaffer AA Zhang J ZhangZ Miller W and Lipman DJ (1997) Gapped BLAST andPSI-BLAST a new generation of protein database searchprograms Nucleic Acids Res 25 3389ndash3402

Arbeitman MN Furlong EEM Imam F Johnson E NullBH Baker BS Krasnow MA Scott MP Davis RW andWhite KP (2002) Gene expression during the life cycle ofDrosophila melanogaster Science 297 2270ndash2275

Arca B Lombardo F de Lara Capurro M della Torre ADimopoulos G James AA and Coluzzi M (1999) TrappingcDNAs encoding secreted proteins from the salivary glands ofthe malaria vector Anopheles gambiae Proc Natl Acad SciUSA 96 1516ndash1521

Barillas-Mury CV Noriega FG and Wells MA (1995) Earlytrypsin activity is part of the signal transduction system thatactivates transcription of the late trypsin gene in the midgut of themosquito Aedes aegypti Insect Biochem Mol Biol 25 241ndash246

Beard CB Cornel AJ and Collins FH (1996) The polyubiquitingene of the mosquito Anopheles gambiae structure andexpression Insect Mol Biol 5 109ndash117

Biessmann H Nguyen QK Le D and Walter MF (2005) Geneexpression in sensory tissues of the malaria mosquitoAnopheles gambiae Insect Mol Biol 14 575ndash589

Briegel H and Horler E (1993) Multiple blood meals as a repro-ductive strategy in Anopheles (Diptera Culicidae) J Med Ento-mol 30 975ndash985

Calvo E Andersen J de Francischetti IML Capurro M deBi-anchi AG James AA Ribeiro JM and Marinotti O (2004)The transcriptome of adult female Anopheles darlingi salivaryglands Insect Mol Biol 13 73ndash88

Calvo E Walter M Adelman ZN Jimenez A Onal SMarinotti O and James AA (2005) Nanos (nos) genes of thevector mosquitoes Anopheles gambiae Anopheles stephensiand Aedes aegypti Insect Biochem Mol Biol 35 789ndash798

Clements AN (1999) Sensory reception and behaviour In TheBiology of Mosquitoes Vol 2 p 740 CABI Publishing NewYork

Dana AN Hong YS Kern MK Hillenmeyer ME Harker BWLobo NF Hogan JR Romans P and Collins FH (2005)



Gene expression patterns associated with blood-feeding in themalaria mosquito Anopheles gambiae BMC Genomics 6 5

Devenport M Fujioka H and Jacobs-Lorena M (2004) Storageand secretion of the peritrophic matrix protein Ag-Aper1 andtrypsin in the midgut of Anopheles gambiae Insect Mol Biol 13349ndash358

Eisen MB Spellman PT Brown PO and Botstein D (1998)Cluster analysis and display of genome-wide expressionpatterns Proc Natl Acad Sci USA 95 14 863ndash14 868

Finley D Bartel B and Varshavsky A (1989) The tails of ubiquitinprecursors are ribosomal proteins whose fusion to ubiquitinfacilitates ribosome biogenesis Nature 338 394ndash401

Francischetti IM Valenzuela JG Pham VM Garfield MKand Ribeiro JM (2002) Toward a catalog for the transcriptsand proteins (sialome) from the salivary gland of the malariavector Anopheles gambiae J Exp Biol 205 2429ndash2451

Geiser DL Chavez CA Flores-Munguia R Winzerling JJand Pham DQ (2003) Aedes aegypti ferritin A cytotoxic pro-tector against iron and oxidative challenge Eur J Biochem270 3667ndash3674

Githeko AK Brandling-Bennett AD Beier M Atieli F OwagaM and Collins FH (1992) The reservoir of Plasmodium falci-parum malaria in a holoendemic area of Western Kenia TransR Soc Med Hyg 86 355ndash358

Golenda CF Klein T Coleman R Burge R Ward RA andSeeley DC (1995) Depletion of total salivary gland protein inblood-fed Anopheles mosquitoes J Med Entomol 32 300ndash305

Goltsev Y Hsiong W Lanzaro G and Levine M (2004) Differ-ent combinations of gap repressors for common stripes inAnopheles and Drosophila embryos Dev Biol 275 435ndash446

Graf R Godknechi A Nakano M Li X Ackermann U andHelbling P (1996) Cloning and expression analysis of Aedesaegypti opsin adaptation of an in situ hybridization protocol formosquitoes Insect Mol Biol 5 173ndash180

Hagedorn HH (2005) Mosquito endocrinology In Biology ofDisease Vectors 2nd edn (WC Marquardt ed) pp 317ndash328Elsevier Academic Press London

Hahn MW and Lanzaro GC (2005) Female-biased geneexpression in the malaria mosquito Anopheles gambiae CurrBiol 15 192ndash193

Handler AM and Harrell RA (1999) 2nd Germline transforma-tion of Drosophila melanogaster with the piggyBac transposonvector Insect Mol Biol 8 449ndash457

Holt RA Subramanian GM Halpern A Sutton GGCharlab R et al (2002) The genome sequence of the malariamosquito Anopheles gambiae Science 298 129ndash149

Jin W Riley RM Wolfinger RD White KP Passador-Gurgel Gand Gibson G (2001) The contributions of sex genotype andage to transcriptional variance in Drosophila melanogasterNat Genet 29 389ndash395

Jones MDR and Gubbins SJ (1978) Changes in the circadianflight activity of the mosquito Anopheles gambiae in relation toinsemination feeding and oviposition Physiol Entomol 3 213ndash220

Kamita SG Hinton AC Wheelock CE Wogulis MDWilson DK Wolf NM Stok JE Hock B and HammockBD (2003) Juvenile hormone (JH) esterase why are you soJH specific Insect Biochem Mol Biol 33 1261ndash1273

Koella JC Sorensen FL and Anderson RA (1998) Themalaria parasite Plasmodium falciparum increases the fre-

quency of multiple feeding of its mosquito vector Anophelesgambiae Proc R Soc Lond B Biol Sci 265 763ndash768

Long AD Mangalam HJ Chan BY Tolleri L Hatfield GWand Baldi P (2001) Improved statistical inference from DNAmicroarray data using analysis of variance and a Bayesianstatistical framework Analysis of global gene expression inEscherichia coli K12 J Biol Chem 276 19937ndash19944

Lu YH and Hagedorn HH (1986) Egg development in themosquito Anopheles albimanus Int J Reprod Dev 9 79ndash94

Marinotti O James AA and Ribeiro JMC (1990) Diet andsalivation in female Aedes aegypti mosquitoes J Insect Physiol36 545ndash548

Marinotti O Nguyen QK Calvo E James AA and RibeiroJMC (2005) Microarray analysis of genes showing variableexpression following a bloodmeal in Anopheles gambiaeInsect Mol Biol 14 365ndash373

Muller HM Catteruccia F Vizioli J della Torre A and CrisantiA (1995) Constitutive and blood meal-induced trypsin genes inAnopheles gambiae Exp Parasitol 81 371ndash385

Muller HM Vizioli I della Torre A and Crisanti A (1993)Temporal and spatial expression of serine protease genes inAnopheles gambiae Parassitologia 35(Suppl ) 73ndash76

Nirmala X Marinotti O and James AA (2005) The accumula-tion of specific mRNAs following multiple blood meals inAnopheles gambiae Insect Mol Biol 14 95ndash103

Perera OP Harrell IIRA and Handler AM (2002) Germ-linetransformation of the South American malaria vector Anophelesalbimanus with a piggyBacEGFP transposon vector is routineand highly-efficient Insect Mol Biol 11 291ndash297

Pletcher SD Macdonald SJ Marguerie R Certa U StearnsSC Goldstein DB and Partridge L (2002) Genome-widetranscript profiles in aging and calorically- restricted Drosophilamelanogaster Curr Biol 12 712ndash723

Raikhel AS (2005) Vitellogenesis of disease vectors from phys-iology to genes In Biology of Disease Vectors 2nd edn (WCMarquardt ed) pp 329ndash346 Elsevier Academic Press London

Raikhel AS Kokoza VA Zhu J Martin D Wang SF Li CSun G Ahmed A Dittmer N and Attardo G (2002) Molec-ular biology of mosquito vitellogenesis from basic studies togenetic engineering of antipathogen immunity Insect BiochemMol Biol 32 1275ndash1286

Ranz JM Castillo-Davis CI Meiklejohn CD and Hartl DL(2003) Sex-dependent gene expression and evolution of theDrosophila transcriptome Science 300 1742ndash1745

Redfern CPF (1982) 20-hydroxy-ecdysone and ovarian develop-ment in Anopheles stephensi J Insect Physiol 28 97ndash109

Ribeiro JM (2003) A catalogue of Anopheles gambiae transcriptssignificantly more or less expressed following a blood mealInsect Biochem Mol Biol 33 865ndash882

Severson DW Knudson DL Soares MB and Loftus BJ(2004) Aedes aegypti genomics Insect Biochem Mol Biol 34715ndash721

St Johnston D and Nusslein-Volhard C (1992) The origin ofpattern and polarity in the Drosophila embryo Cell 68 201ndash219

Sun J Hiraoka T Dittmer NT Cho KH and Raikhel AS(2000) Lipophorin as a yolk protein precursor in the mosquitoAedes aegypti Insect Biochem Mol Biol 30 1161ndash1171

Toure YT Oduola AM and Morel CM (2004) The Anophelesgambiae genome next steps for malaria vector control TrendsParasitol 20 142ndash149



Vizioli J Catteruccia F della Torre A Reckmann I and Muller HM(2001) Blood digestion in the malaria mosquito Anophelesgambiae molecular cloning and biochemical characterizationof two inducible chymotrypsins Eur J Biochem 268 4027ndash4035

Xu X Dong Y Abraham EG Kocan A Srinivasan P GhoshAK Sinden RE Ribeiro JM Jacobs-Lorena M KafatosFC and Dimopoulos G (2005) Transcriptome analysis ofAnopheles stephensindashPlasmodium berghei interactions MolBiochem Parasitol 142 76ndash87

Zakharkin SO Gordadze AV Korochkina SE MathiopoulosKD Della Torre A and Benes H (1997) Molecular cloningand expression of a hexamerin cDNA from the malariamosquito Anopheles gambiae Eur J Biochem 246 719ndash726

Zou S Meadows S Sharp L and Jan YN (2000) Genome-widestudy of aging and oxidative stress response in Drosophilamelanogaster Proc Natl Acad Sci USA 97 13726ndash13731

Supplementary Material

The following material is available for this article online

Tables S1minusminusminusminus23 Statistical analysis of sex- oogenesis- and ageing-related variations of transcript accumulation in Anopheles gambiae mosquitoes Expression values (columns D-I) were used as input to a Bayesian t-test at Cyber-T (httpvisitoricsuciedugenexcybert) The Cyber-T output including P-values (column AD) and fold variations (column AF) are presented for each probe set Known or putative functions of genes represented by each probe set can be retrieved using the hyperlinks of the tables to access httpwwwangagepucibiouciedu

Tables S24minusminusminusminus62 A Welch ANOVA-based statistical analysis of gene expression during mosquito development with multiple testing correction Benjamini and Hochberg False Discovery Rate with a 0001 P-value cutoff was performed Genes regulated during mosquito development were identified and grouped according to similar patterns of expression using the K-Means clustering method and Genespring software A graphical representation of the clustering is displayed in Fig 4 and the hyperlinked lists of probe sets included in each cluster included here

Tables S63minusminusminusminus65 Hyperlinked Excel spreadsheets containing the lists of genes expressed exclusively in the fat bodies (63) ovaries (64) or midguts (65) of mosquitoes at 24 h post blood meal (PBM) Genes were considered

as having tissue specific expression when their transcripts were assigned as present (GCOS analysis) in all three samples of that tissue and absent from the samples of the other two tissues analysed Transcripts present in more than one tissue but with more than 90 of the total signal in one of them were also assigned as tissue specific

Table S66 Abundantly and constitutively expressed Anopheles gambiae transcripts were identified by the sum of all signal values determined by microarray experiments throughout mosquito development The 200 transcripts showing the strongest sum of signals and constitutive expression were analysed further Functional assignments were derived from the Annotated Gene Chip PlasmodiumAnopheles genome array database The identified transcripts were classified according to their functions related to cs cytoskeleton (including muscle related proteins) dg digestion em extracellular matrix im intermediate metabolism imm immunity nr nuclear regulation pmps protein synthesis and modification st signal transduction tm transcriptional machinery tr transporters uk unknown Numbers of blood-fed and nonblood-fed expressed sequence tags were derived from Ribeiro (2003)

Figure S1 Global patterns of gene expression during oogenesis and ageing in Anopheles gambiae adult females Selected genes mentioned in the text that undergo regulation in expression following a blood meal (A) serine proteases (B) other digestive enzymes including salivary components (C) oogenesis-related transcripts (D) environmental perception-related transcripts and (E) muscle hormone and transporter transcripts The arrow indicates sequential time points identical those in Fig 3 Transcript IDs and additional annotation was obtained from the microarray database at httpwwwangagepucibiouciedu

Figure S2 Distribution of transcripts showing statistically significant differences in accumulation (P-value lt 0001) following a blood meal when compared with nonblood-fed females Absolute values of fold variation were considered and the transcripts grouped according to their levels of fold variation A total of 9724 transcripts varied in accumulation at least one of the times examined Of these 5423 varied two-fold or more and 2625 at least three-fold Only 126 of the regulated transcripts varied more than 100-fold after a blood meal

This material is available as part of the online article from httpwwwblackwell-synergycom

Insect Molecular Biology (2006)

15

(1) 1ndash12


1

Blackwell Publishing Ltd

Genome-wide analysis of gene expression in adult

Anopheles gambiae

O Marinotti E Calvodagger Q K NguyenDagger S Dissanayake J M C Ribeirodagger and A A Jamessect

Department of Molecular Biology and Biochemistry University of California Irvine CA USA

dagger

Laboratory of Malaria and Vector Research National Institutes of Health (NIHNIAID) Rockville MD USA

Dagger

Department of Biological Chemistry and

sect

Department of Microbiology and Molecular Genetics University of California Irvine CA USA

Abstract

With their genome sequenced

Anopheles gambiae

mosquitoes now serve as a powerful tool for basicresearch in comparative evolutionary and develop-mental biology The knowledge generated by thesestudies is expected to reveal molecular targets fornovel vector control and pathogen transmission block-ing strategies Comparisons of gene-expression pro-files between adult male and nonblood-fed female

Anopheles gambiae

mosquitoes revealed that roughly22 of the genes showed sex-dependent regulationBlood-fed females switch the majority of their metabo-lism to blood digestion and egg formation within 3 hafter the meal is ingested in detriment to other activi-ties such as flight and response to environmentstimuli Changes in gene expression are most evidentduring the first second and third days after a bloodmeal when as many as 50 of all genes showedsignificant variation in transcript accumulation Afterlaying the first cluster of eggs (between 72 and 96 hafter the blood meal) mosquitoes return to a nongono-trophic stage similar but not identical to that of 3-day-old nonblood-fed females Ageing andor the nutritionalstate of mosquitoes at 15 days after a blood meal isreflected by the down-regulation of simsimsimsim

5 of all genes Afull description of the large number of genes regulated

at each analysed time point and each biochemicalpathway or biological processes in which they areinvolved is not possible within the scope of this contri-bution Therefore we present descriptions of groupsof genes displaying major differences in transcriptaccumulation during the adult mosquito life Howevera publicly available searchable database (httpwwwangagepucibiouciedu) has been made available sothat detailed analyses of specific groups of genesbased on their descriptions functions or levels of geneexpression variation can be performed by interestedinvestigators according to their needs

Keywords

Anopheles gambiae

gonotrophic cycleageing sex-biased gene expression microarray

Introduction

The fruit fly

Drosophila melanogaster

is a proven modelfor studies of physiology biochemistry behaviour andother aspects of insect biology However the knowledgeobtained is not always directly applicable to other speciesdue mainly to the enormous diversification of insects andtheir adaptations to different niches Hematophagy withits multiple determining factors and consequences (host-seeking blood ingestion digestion and the unavoidablechallenge of dealing with a heavy load of iron moleculesingested at each meal and its direct implication in mosquitoreproduction and pathogen transmission) is just oneexample of a mosquito evolutionary adaptation that cannotbe addressed by studying

Drosophila

or other modelorganisms Based on these differences insect vectors ofdiseases comprising species of mosquitoes tsetse fliessand flies black flies fleas kissing bugs and bedbugs thattransmit viruses and parasites to humans have been theobjective of extensive investigation with the main purposeof finding novel and efficient ways to disrupt or diminishpathogen transmission The concluded venture to sequencethe genome of the human malaria vector

Anophelesgambiae

(Holt

et al

2002) and the ongoing projects tosequence the genomes of two other mosquito species

Aedes aegypti

(Severson

et al

2004) and

Culex quinque-fasciatus

represent major advances in this direction They

doi 101111j1365-2583200600610

Received 5 May 2005 accepted after revision 3 August 2005 Correspond-ence Dr Anthony A James Departments of Microbiology amp MolecularGenetics and Molecular Biology amp Biochemistry 3205 McGaugh Hall Uni-versity of California Irvine CA 92697ndash3900 USA Tel +1 949 824 5930fax +1 949 824 2814 e-mail aajamesuciedu

2

O Marinotti

et al



15

1ndash12


et al


An gambiae



Identification of

An gambiae



Angambiae


An gambiae


et al



LAST


et al



LAST


An gambiae


An gambiae


LAST


D melanogaster


An gambiae


An gambiae




P



Drosophila


et al

2001 Arbeitman

et al

2002 Ranz

et al


P



Gene expression in

Anopheles gambiae 3



15

1ndash12


An gambiae


D melanogaster


Homo sapiens

and

D melanogaster


H sapiens




et al



D7-related


et al


Maltase like I

and

AMY I

of

Aedes aegypti


et al

2002 Calvo

et al



et al



et al


et al

2005)


4

O Marinotti

et al



15

1ndash12




P


ANOVA




Gene expression in

Anopheles gambiae 5



15

1ndash12


P





6

O Marinotti

et al



15

1ndash12


sim


Anophe-les gambiae


et al

1998 Nirmala

et al




α

-glucosidasesand

α



et al

1990 Golenda

et al


de novo


et al



et al



oskar




An gambiae


et al


et al


sim


sim


Gene expression in

Anopheles gambiae 7



15

1ndash12


et al

1995 Muller

et al



et al

2001 Raikhel

et al 2002)






8 O Marinotti et al















Final comments



Mosquitoes










Acknowledgements


References





































































2

O Marinotti

et al



15

1ndash12


et al


An gambiae



Identification of

An gambiae



Angambiae


An gambiae


et al



LAST


et al



LAST


An gambiae


An gambiae


LAST


D melanogaster


An gambiae


An gambiae




P



Drosophila


et al

2001 Arbeitman

et al

2002 Ranz

et al


P



Gene expression in

Anopheles gambiae 3



15

1ndash12


An gambiae


D melanogaster


Homo sapiens

and

D melanogaster


H sapiens




et al



D7-related


et al


Maltase like I

and

AMY I

of

Aedes aegypti


et al

2002 Calvo

et al



et al



et al


et al

2005)


4

O Marinotti

et al



15

1ndash12




P


ANOVA




Gene expression in

Anopheles gambiae 5



15

1ndash12


P





6

O Marinotti

et al



15

1ndash12


sim


Anophe-les gambiae


et al

1998 Nirmala

et al




α

-glucosidasesand

α



et al

1990 Golenda

et al


de novo


et al



et al



oskar




An gambiae


et al


et al


sim


sim


Gene expression in

Anopheles gambiae 7



15

1ndash12


et al

1995 Muller

et al



et al

2001 Raikhel

et al 2002)






8 O Marinotti et al















Final comments



Mosquitoes










Acknowledgements


References





































































Gene expression in

Anopheles gambiae 3



15

1ndash12


An gambiae


D melanogaster


Homo sapiens

and

D melanogaster


H sapiens




et al



D7-related


et al


Maltase like I

and

AMY I

of

Aedes aegypti


et al

2002 Calvo

et al



et al



et al


et al

2005)


4

O Marinotti

et al



15

1ndash12




P


ANOVA




Gene expression in

Anopheles gambiae 5



15

1ndash12


P





6

O Marinotti

et al



15

1ndash12


sim


Anophe-les gambiae


et al

1998 Nirmala

et al




α

-glucosidasesand

α



et al

1990 Golenda

et al


de novo


et al



et al



oskar




An gambiae


et al


et al


sim


sim


Gene expression in

Anopheles gambiae 7



15

1ndash12


et al

1995 Muller

et al



et al

2001 Raikhel

et al 2002)






8 O Marinotti et al















Final comments



Mosquitoes










Acknowledgements


References





































































4

O Marinotti

et al



15

1ndash12




P


ANOVA




Gene expression in

Anopheles gambiae 5



15

1ndash12


P





6

O Marinotti

et al



15

1ndash12


sim


Anophe-les gambiae


et al

1998 Nirmala

et al




α

-glucosidasesand

α



et al

1990 Golenda

et al


de novo


et al



et al



oskar




An gambiae


et al


et al


sim


sim


Gene expression in

Anopheles gambiae 7



15

1ndash12


et al

1995 Muller

et al



et al

2001 Raikhel

et al 2002)






8 O Marinotti et al















Final comments



Mosquitoes










Acknowledgements


References





































































Gene expression in

Anopheles gambiae 5



15

1ndash12


P





6

O Marinotti

et al



15

1ndash12


sim


Anophe-les gambiae


et al

1998 Nirmala

et al




α

-glucosidasesand

α



et al

1990 Golenda

et al


de novo


et al



et al



oskar




An gambiae


et al


et al


sim


sim


Gene expression in

Anopheles gambiae 7



15

1ndash12


et al

1995 Muller

et al



et al

2001 Raikhel

et al 2002)






8 O Marinotti et al















Final comments



Mosquitoes










Acknowledgements


References





































































6

O Marinotti

et al



15

1ndash12


sim


Anophe-les gambiae


et al

1998 Nirmala

et al




α

-glucosidasesand

α



et al

1990 Golenda

et al


de novo


et al



et al



oskar




An gambiae


et al


et al


sim


sim


Gene expression in

Anopheles gambiae 7



15

1ndash12


et al

1995 Muller

et al



et al

2001 Raikhel

et al 2002)






8 O Marinotti et al















Final comments



Mosquitoes










Acknowledgements


References





































































Gene expression in

Anopheles gambiae 7



15

1ndash12


et al

1995 Muller

et al



et al

2001 Raikhel

et al 2002)






8 O Marinotti et al















Final comments



Mosquitoes










Acknowledgements


References





































































8 O Marinotti et al















Final comments



Mosquitoes










Acknowledgements


References









































































Final comments



Mosquitoes










Acknowledgements


References












































































Acknowledgements


References





































































genome-wide analysis of gene expression in adult anopheles

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