We have determined the gene expression patterns of 3,068 abdomen-derived cDNAs from adult femaleAn. gambiaemosquitoes representing 1906 unique transcripts were determined in the first two days following ingestion of a blood meal by microarray analysis. 413 unique transcripts were shown to be up-or down-regulated at least twofold in blood fed mosquitoes relative to sugar-fed mosquitoes at one or more of the following times: 5 min and 30 min following initiation of blood feeding and 0, 1, 3, 5, 12, 24, and 48 hr post-blood meal. These transcripts were clustered into three sets with different temporal patterns of expression that may reflect the major hormonal changes occurring within the mosquito during a gonotrophic cycle. These differentially expressed gene products were annotated putatively using sequence similarity searches and categorized by biological process to identify the major events occurring post blood meal ingestion in the female mosquito.
Multiple hormones interact to alter tissue states and to activate genes involved in the female mosquito's digestion of a blood meal, in oocyte development and in vitellogenesis. The three sets of differentially transcribed genes discerned in this study, the Early, Middle and Late genes may reflect differential hormonal responsiveness. After acquisition of a blood meal, the transcript levels of the Early genes which were abundant during blood feeding showed general declines until 24 hours PBM, after which a subset of transcript levels began to rise again. Expression of Early genes may be linked to the relatively high titers of JH present at the beginning of the first gonotrophic cycle and may then be repressed as a result of declining JH titers or of increasing 20-E. The expression of Middle gene transcripts followed an expression pattern that reflects the titers of 20-E: levels sharply increased by 12 hours PBM, remained stable or increased only slightly until 24 hours PBM, and then declined rapidly. OEH secreted by median neurosecretory cells stimulates the ovaries to secrete 20-E during vitellogenesis and the activity of this hormone begins to rise by 3–5 hours, peaks between 12 and 24 hours, and then declines to baseline levels by 48 hours post blood ingestion [4–6]. In contrast to the Middle genes, most Late gene transcripts exhibited baseline, steady state levels until 12 hours PBM after which they were induced at least twofold and continued to exhibit increased transcript levels at 48 hours PBM. This increase in transcript levels mirrored the increase in JH titer observed by 48 hours post blood meal ingestion . These results suggest that Middle genes products may be ecdysone-responsive whereas Late genes products may be JH-responsive.
Based on their finding that theDrosophila minidiscsgene product showed a primary response to JH, Dubrovsky et al.  have suggested that JH may transcriptionally regulate genes encoding maternally inherited products. Our Late gene, AS 806, shares sequence similarity withminidiscs. Additionally, the majority of mosquito gene products showing sequence similarity to maternally activeDrosophilagenes are categorized as Late genes. Whether transcription of Early genes is directly influenced by levels of JH or 20-E, cannot be determined easily because neither hormone is present at high levels during the first few hours following blood meal ingestion. Regardless of expression pattern, the gene products in each set reflect a diverse array of processes occurring in the female mosquito within 48 hours following initiation of blood feeding. The major processes initiated in response to blood feeding including digestion, peritrophic matrix formation, oogenesis and vitellogenesis, are discussed below with emphasis on the likely roles of particular gene products.
Digestion of the two different food sources, nectar sugars and blood, requires changes in the types of enzymes present within the digestive tract of the mosquito. The numbers of genes associated with sugar and protein metabolism within each set of genes may reflect the switch from sugar to protein metabolism. For instance, there are increases in transcript abundance of genes involved in carbohydrate metabolism and oxidative phosphorylation in the Early genes. However, both Early and Middle gene categories are enriched in genes involved in protein digestion. This result stems from the involvement of certain gene products in blood meal digestion that initiate a signaling cascade resulting in the up-regulation of other related proteolytic enzymes. The Middle genes contain the majority of gene products involved in amino acid metabolism, a process that follows protein digestion, whereas by 48 hours PBM, the time at which the majority of Late genes are induced, there is a generalized decrease in digestive enzyme transcripts.
Considering that blood contains large quantities of protein, the mosquito requires a variety of proteolytic enzymes to digest the recently acquired meal. In the present study, 11 genes were identified whose products are most likely required for protein digestion. These include 5 previously characterized digestive enzyme genes, two trypsins, a chymotrypsin, a serine protease and a carboxypeptidase. The majority of these digestive enzyme genes were transcribed at levels greater than twofold induction after 6 hours PBM. TheAn. gambiaeTrypsins 1 and 2 are both induced by a blood meal and exhibit similar expression profiles although Trypsin 1 is expressed at higher levels. Muller and coworkers, using an RT-PCR strategy, showed that Trypsin 2 mRNA is present at 8, 12, 24 and 28 hours post blood meal [18,52]. In our study, the Trypsin 2 gene (AS 569) also exhibited increased transcript abundance at 12 hours PBM with maximal expression occurring at 24 hours PBM, but these levels decreased by 48 hours. In contrast to Trypsins 1 and 2, Trypsins 3, 4, and 7 are constitutively expressed in non-bloodfed females. By 4 hours following a blood meal, levels of Trypsin 4 become undetectable by Northern and RT-PCR analysis and do not reappear until 20 hours PBM  and unlike Trypsins 1 and 2, Trypsin 4 reaches maximal expression by 48 hours, near the end of the gonotrophic cycle. Our study identified two clones corresponding to Trypsin 4 (AS 568) but the two cDNAs exhibited different expression patterns. One (Accession no. CD747033) reached maximal transcript abundance at 48 hours PBM, the expected expression pattern. However, the other cDNA (Accession no. CD747029) was expressed at high levels prior to 6 hours PBM and also reached at least twofold increased levels by 48 hours PBM. These cDNAs may not have clustered together for technical reasons intrinsic to Seqman II, or they may correspond to alternatively spliced variants of the same gene. The sequence alignment showed 97% nucleotide sequence identity between the two ESTs over the region common to both. However, the CD747033 EST is only 253 bp in length and it is possible that the full-length cDNA represents an alternatively spliced transcript. In addition to Trypsins 2 and 4, we identified a trypsin-like serine protease (AS 648) which shared the greatest amino acid similarity with Trypsin 4. However, this serine protease exhibited highest nucleotide identity to a different region of chromosome 3R than that containing all previously identified digestive trypsins. This gene product was induced greater than twofold within 6 hours PBM and was repressed during the height of digestion. Unlike Trypsins 3–7, it was not expressed at higher levels at 48 hours PBM. It is possible that this trypsin is not involved in digestion but in another proteolytic process that is down-regulated following a blood meal.
Barillas-Muryet al.  demonstrated that the early trypsin activity is essential to the transcription and subsequent expression of the late trypsins inAe. aegypti.An. gambiaeTrypsins 3–7 may indirectly activate transcription and increase the expression of Trypsins 1 and 2, the major endoproteolytic enzymes required for blood meal digestion . In bothAe. aegyptiandAn. gambiae, these early expressed digestive enzymes are presumed to act as signal transducers causing transcriptional up-regulation of the late expressed ones [14,53]. Thus the digestive process is regulated by an elaborate biphasic expression pattern of serine proteases. Additional evidence suggests that not only tryptic peptides but cleaved amino acids may serve as systemic signals regulating subsequent processes. In contrast to the trypsin by-products, cleaved amino acids may also function as negative regulators of food intake. AS 1158 shared weak sequence similarity with theDrosophila pumplessprotein, a larval fat body-expressed enzyme involved in glycine catabolism. In the fruit fly, larvae expressing thepumplessmutation are unable to pump food from the pharynx to the esophagus . These mutant animals do not feed, neither do they upregulate genes normally involved in responses to starvation. Because feeding amino acids to wild type larvae phenocopied effects of thepumplessmutation, Zinkeet al.  proposed that amino acids released from the fat body normally act as signals for cessation of feeding.
In addition to the trypsins, three chymotrypsin genes have been characterized inAn. gambiae[19,20]. The expression of two of these digestive enzymes, AnChym 1 and 2, has been localized to the mosquito midgut by analysis of Northern blots . RT-PCR showed that both chymotrypsin genes are expressed at 12 hours PBM and are abundant until 48 hours PBM, unlike transcripts of Trypsins 1 and 2 which have decreased dramatically by this time . The cDNA representing chymotrypsin 2, AS 99, was categorized as a Late gene since maximal transcript levels were achieved after 24 hours PBM. In contrast, the other characterized chymotrypsin, AgChyL, exhibits changes in transcript abundance which is more similar to those of Trypsins 3–7. mRNA is present in non-blood fed females, detectable until 8 hours post blood meal after which mRNA can no longer be measured until 48 hours PBM . The cDNA corresponding to this chymotrypsin-like serine protease, AS 994, was induced more than twofold prior to the peak of digestion and clustered with the Early Genes. In addition to the aforementioned chymotrypsins, our study identified a previously uncharacterized chymotrypsin (AS 2243) also located on chromosome 2L but in a different region from both AnChym1 and AnChym2. Exhibiting an expression pattern different from both AnChym2 and AgChyL, this gene product was characterized as a Middle Gene with maximal transcript levels achieved between 12 and 24 hours PBM and a return to baseline by 48 hours PBM, similar to the expression patterns of Trypsins 1 and 2.
Edwardset al.  investigated expression levels ofAn. gambiaecarboxypeptidase A following blood meal ingestion. Northern blot analysis indicated that levels of Carboxypeptidase A mRNA rose rapidly to a ten-fold increase within 3–4 hours following a blood meal, then dropped to the pre-induction state by 24 hours PBM. We identified a carboxypeptidase gene located on chromosome 2L (AS 1742) that is transcribed in a manner similar to that of carboxypeptidase A. However another cDNA representing a carboxypeptidase (AS 44) exhibited a radically different expression pattern. Transcripts were present at low levels 1–5 hours PBM but increased more than twofold between 12 and 24 hours PBM, a pattern that resembled the enzymatic activity inAn. stephensiobserved by Jahanet al. , namely a rapid increase until 12 hours PBM, with a peak at 24 hours, followed by a steady decline over the next day.
In contrast to theAn. gambiaecarboxypeptidase A, the levels of aminopeptidase peaked around 30 hours inAn. stephensi. Additionally, Lemoset al.  recorded peak aminopeptidase activity at 24 hours PBM inAn. gambiae. We identified two aminopeptidases with at least two-fold increased expression during the 48 hours PBM. The first aminopeptidase, AS 340, reached peak transcript levels at 24 hours PBM, showing a similar expression pattern to the enzyme activity levels reported by Billingsley and Hecker  and Lemoset al. . In contrast, the other aminopeptidase, AS 430, showed amino acid similarity to theAe. aegyptiaminopeptidase N. This aminopeptidase was classified as a Late gene, due to increased transcript levels at 24 hours PBM but maximal levels were not achieved until 48 hours PBM. Jahanet al.  documented two different kinetic profiles of aminopeptidase enzymes inAn. stephensidepending on whether the enzyme was soluble or membrane-associated. The soluble aminopeptidase exhibited a kinetic profile similar to AS 340 and to that presented by Billingsley and Hecker  with peak activity at 24 hours PBM.
Geering  had suggested that phospholipase activity plays a role in blood digestion inAe. aegyptialthough no conclusive evidence was demonstrated. However, Geering and Freyvogel  demonstrated that lipolytic activity increased 15 hours after blood feeding. Of the three gene products that are characterized as being involved in Fatty Acid/Lipid Metabolism and Transport, two, ASs 1177 and 997, encoding an acetate-CoA ligase and a fatty acid binding molecule, respectively, are expressed at more than twofold greater abundance at 24 hours PBM. These gene products may be involved in fatty acid degradation of blood meal components and the transport of these lipids to the oocytes.
The erythrocyte membrane contains a number of glycoproteins. It is therefore possible that enzymes normally associated with carbohydrate metabolism of nectar meals could also be involved in blood digestion. Almost all of the Simple/Complex Carbohydrate Metabolism and Transport genes identified in our study as being at least twofold upregulated were categorized as Early genes. Within the first 6 hours PBM, their transcripts are present at higher levels than in sugar-fed females and thereafter, they decrease steadily until 12 and 24 hours PBM. This expression profile does not exclude these genes from having a role in RBC glycoprotein metabolism. Several glycosidases are present within the midgut ofAn. stephensi, either associated with the lumen or with epithelial lysosomes . The enzymatic activity of α-glucosidase, the major midgut glycosidase inAn. stephensi, increased from 6 hours PBM to maximal levels by 24 hours and decreased to basal levels by 36 hours PBM in the anterior midgut. The transcript abundance of the α-glucosidase, AS1786, characterized in this study followed a different pattern than the enzymatic activity ofAn. stephensiα-glucosidase. It showed greatest amino acid sequence similarity toDrosophila melanogastergene product CG8690 α-glucosidase and was categorized as an Early Gene with minimal transcript levels occurring at and after 24 hours PBM.
Peritrophic matrix formation
Shen and Jacobs-Lorena  characterized theAn. gambiaeperitrophic matrix protein Peritrophin 1 (Ag-Aper1) by analysis of Northern blots, and demonstrated that transcripts were present 6 hours PBM, increased by 12 hours, and remained elevated between 24–48 hours PBM. The present study identified several genes encoding proteins with a chitin-binding domain (InterPro ID IPR002557: Chitin binding Peritrophin-A) similar to the one found in Peritrophin 1. The Early gene AS 928 contains the Peritrophin-A chitin binding domain and maps to chromosome 3Lin silico, corresponding to agCP10685. Another Early gene, AS 13, shows high identity to Peritrophin 1. A Middle gene, AS 516, is expressed by 6 hours PBM but does not reach maximal levels until 24 hours PBM. This gene product mapsin silicoto chromosome 2L, exhibits a similar transcript profile, and also shares 98% amino acid identity with Peritrophin 1. It is not clear why two sets of ESTs, both identified as Peritrophin 1, should exhibit different transcription patterns unless they are derived from differentially regulated genes. Another Middle gene, AS 1164, contains the Peritrophin-A chitin binding domain in addition to a prenyl-group binding site (InterPro ID IPR001230: CAAX box). This Middle gene may not be involved in peritrophic matrix formation but in some other process coinciding with protein digestion. Two other cDNAs, Accessions CD746211 and CD746202, both in AS 13, could also be localized to chromosome 2L. However, their ESTs exhibited greatest nucleotide identity to the gene predicted as ENSANGG00000020776 located 4 kb 3' to Ag-Aper1. These two cDNAs clustered as Early and Late genes respectively, and may represent alternatively spliced gene products.
As early as an hour after the adult female has taken a blood meal, secretory vesicles previously present in the apical brush border of midgut epithelial cells are no longer detectable [Staubli et al., 1966 as cited in ]. These apical granules presumably contain precursors of the peritrophic matrix. The Middle and Late peritrophin gene products may be packaged into vesicles in preparation for a subsequent blood meal. In contrast, the Early peritrophin genes may be transcribed in response to blood meal acquisition and their products used immediately in the formation of the peritrophic matrix.
Ovarian cycle and oogenesis
The extensive literature on genes involved inDrosophilaovarian development and early embryogenesis opens windows into interpreting ourAn. gambiaemicroarray results and understanding mosquito egg development. The majority ofAn. gambiaegenes upregulated at least twofold following a blood meal appear to function in egg production. The only gene possibly involved in oogenesis during the early phases of the ovarian cycle is the Early gene AS 670. It shares sequence similarity withpeter pan, aDrosophilagene product required during oogenesis. Oocytes inpeter panmutants often have an incorrect number of associated nurse cells, suggesting that thepeter panprotein influences the separation of cells within the germaria . The identification of other genes involved in the early stages of mosquito oogenesis may be facilitated by the construction of cDNA libraries from the abdomens or ovaries of recently blood fed female mosquitoes.
Several differentially expressed gene products found in the present study may be involved in the formation of ring canals, structures necessary for the delivery of maternal factors to oocytes. In particular, bulk transfer of cytoplasmic content from nurse cells to oocytes depends on actin structures . A Middle gene product, AS 679, shares sequence similarity to theDrosophilagene CG13388 encoding the protein kinase anchor protein 200,Akap200.Akap200protein localizes to ring canals during oogenesis, regulates protein kinase C activity, and controls their morphology . Late gene product AS 1317 shows sequence similarity to theDrosophila pendulingene product, encoded by CG4799. This gene product is also required for assembly of fully functional ring canals.pendulinencodes an importin-α2, a protein necessary for the localization of thekelchgene product, CG7210.kelchencodes an actin organizer without which the ring canals become occluded and nurse cell-oocyte cytoplasmic transport is inhibited [59,60]. Though we found an apparentpendulingene, we did not findkelch. A Late gene product, AS 578, is the homolog ofCdc42, which encodes a small monomeric RHO GTPase involved in signal transduction. Rohatgiet al.  suggested thatCdc42protein most likely links signal transduction to the actin cytoskeleton inXenopus. InDrosophilaovaries, mutations inCdc42caused nurse cells to deflate and coalesce, and inhibited transfer of nurse cell cytoplasm to oocytes in late stage egg chambers .
Drosophilanurse cells transcribe thebicoidanterior determinant gene and the resulting mRNA is transported to the anterior region of developing oocytes via polarized microtubules .bicoiddoes not appear to be an anterior determinant in other insects, but other genes important for its localization are conserved. The Early gene AS 2047 shares similarity with theDrosophila cornichongene, CG5855. InDrosophila,cornichonis required for formation of a functional microtubular cytoskeletal scaffold used to transportbicoidmRNA and the posterior grouposkargene product to their proper location within the embryo . The Late gene product, AS 1044 exhibits greatest sequence similarity toD. virilis exuperantia, a gene whose product is also required for properbicoidmRNA localizationD. melanogaster[65,66]. The Late gene AS 2222 is putatively identified as theAn. gambiaehomolog ofDrosophila Notch. InDrosophila,Notchsignaling regulates a large number of ovarian events beginning with cyst development in the germarium and extending through oogenesis . The mechanisms by whichNotchsignaling activates transcription of its target genes are reviewed by Barolo and Posakony . Since we identifiedNotchas a Late gene, its activities may be more restricted inAn. gambiae, and/or reflect fundamental differences in ovarian biology. AS 1391, a Late gene product, shares sequence similarity withDrosophila Rab-protein 11. ThisDrosophilasmall monomeric RAB GTPase is also involved in the polarization of the microtubules for the organization of the posterior pole and foroskarlocalization there .
To regulate the progress of oogenesis and embryogenesis, stored maternal mRNAs are translationally repressed during early oocyte development. The Middle gene product AS 2031 shares sequence similarity with theDrosophilagene productBicaudal C, a RNA binding protein that may play a role in translational silencing of maternal mRNAs in addition to its role in eggshell patterning . Mutations inBicaudal Cresult in premature translation ofoskarmRNA before it has reached the posterior region of the oocyte . The Middle gene product AS 1490 is the putative homolog for theDrosophilagene productvasa(CG3506), an ATP dependent helicase involved in pole plasm assembly that may also be involved in translational modification of maternal mRNAs . The Late gene AS 453 shares sequence similarity with theDrosophila cupprotein (CG11181).cupprotein interacts withnanos, the posterior determinant, and a translational regulator of the gap genehunchbackmRNA during oogenesis, although the exact function of thecupprotein still remains unknown . A DEAD box protein encoded byvasaalso influences oocyte differentiation and the development of theDrosophilaembryo body plan via translation ofoskar,nanos, and gurkenduring oogenesis [74–77]. In amphibians, several mRNA binding proteins have been identified that are only present in oocytes and not post cleavage embryos [78,79]. OneAn. gambiaeMiddle gene product, AS 2449, shared sequence similarity with theXenopus laevispoly(A)-specific ribonuclease and also has mRNA binding motifs, thus it may also repress translation of mRNAs in embryos.
Maternal nurse cells not only provide the biosynthetic machinery and mRNA needed for oocyte axis determination, but also many transcripts and proteins required for zygotic development through the cellular blastoderm stage. The Middle gene product AS 1032 shares sequence similarity withnop5, encoding a maternally derived product of theDrosophilaCG10206 gene, a component of the small nucleolar ribonucleoprotein (snoRNP) complex involved in rRNA processing . The Late gene product AS 806, referred to above in the context of its possible regulation by JH, shares sequence similarity with theDrosophila minidiscsgene product, an amino acid transporter. InDrosophilaovarian nurse cells, JH induces the expression ofminidisksand its transcripts are most likely transferred to the oocyte during nurse cell cytoplasmic streaming .
Similar to the oocyte, the eggshell undergoes dorsal-ventral patterning. Crucial to this process is the correct placement of the oocyte relative to the maternal somatic follicle cells. InDrosophila, the localization of the oocyte depends on cadherin-associated adhesion . The Late gene product AS 1890 is the homolog of α-catenin, the CG17947 gene product. A cytoskeletal anchor protein, α-catenin is required for positioning of the oocyte relative to the posterior follicle cells during germ cell rearrangement inDrosophila. The RAS 1 signaling cascade is an important means of cell communication during embryo and eggshell patterning [41,42]. The Middle gene product AS 657 is weakly similar to theDrosophila Starprotein, a RAS 1 enhancer involved in the EGF receptor signaling pathway, either upstream or in parallel to EGFR, during formation of the embryonic ventral midline.Starencodes a single pass transmembrane protein that may be involved in the processing ofgurkenprotein. The DNA damage checkpoint14-3-3epsilonprotein also participates in RAS 1 signaling, normally functioning downstream or in parallel to RAF, but upstream of transcription factors. The Middle gene AS 106 shares sequence similarity withDrosophila 14-3-3epsilon. The14-3-3epsilonprotein is also capable of binding to a large number of other proteins in a phosphorylation-dependent manner. One of its functions may be to alter the cell cycle by binding Cyclin B and appears to have homologs in most if not all eukaryotes [82,83]. TheAn. gambiae Cyclin Bhomolog, AS 1357, grouped as a Late gene.
Mutation screens inDrosophilahave led to the identification of a number of other gene products that may be involved in RAS 1 signaling.TppII (tripeptidyl-peptidase II), andsmt3(SUMO) were discovered in a search for lethal mutations that could enhance a weak RAS 1 eggshell phenotype . The Middle gene product AS 1268 shares sequence similarity withDrosophila tripeptidyl-peptidase II, the CG3991 gene, encoding a serine protease that degrades neuropeptide signals . The Late gene product AS 922 is the homolog ofDrosophila smt3, CG4494, whose product is ubiquitin-like protein that may tag proteins for nuclear localization or retention in the cytoplasm [86,87].smt3protein may modulate activity of transcription factors in the follicle cells downstream of EGFR activation. However, we feel thatsmt3is likely to be a minor player in RAS 1 signaling in the events following blood ingestion in the mosquito, because its mRNA reaches maximal expression after 12–24 hours PBM, unlike the other gene products we identified as potentially influencing RAS 1 signaling.
smt3protein may also play a role inTollsignaling. This signal transduction pathway is known to be necessary for dorsal/ventral patterning of theDrosophilaembryo.smt3protein binds the NFκB homologdorsalprotein and targets this Rel transcription factor for migration to the nucleus [88,89]. Bhaskaret al.  demonstrated thatsmt3conjugation to thedorsalprotein enhanced its transcriptional activity.smt3protein may play other roles in the cell by altering the interactions of septins, cytoskeletal proteins involved in cytokinesis [90,91]. InDrosophila, septins have been found in the cytoplasm of nurse cells and at the baso-lateral surfaces of follicle cells . These results suggest even more pleiotrophc roles for thesmt3gene product in oogenesis. We also found that the Late gene AS 2034, a homolog ofDrosophila Aos1, the CG12276 gene, thesmt3(SUMO) activating enzyme, was also expressed at least twofold more abundantly during the height ofsmt3expression. This result reinforces the importance ofsmt3in the events occurring between 24–48 hours PBM.
In addition to genes regulating the polarity of the embryo and eggshell, genes involved in cellular growth and differentiation were differentially expressed during the 48 hours PBM ingestion. AS 337 and 495 shared sequence similarity with theAe. aegyptiornithine decarboxylase antizyme, a protein that modulates polyamine synthesis. The homologousDrosophila ornithine decarboxylase antizymegene, formerly known asgut feeling, has been shown to be important in developing oocytes. It is one target ofSex lethalwhich encodes an RNA binding protein that regulates mRNA splicing and the mitotic events in early germ cells via regulating Cyclin B . The Late gene product AS 2073 shares sequence similarity with theDrosophila poloCG12306 gene product, a protein kinase required for cytokinesis and another regulator of Cyclin B . The Early gene AS 1972 shows identity with theDrosophila black pearlCG5268 gene product. This protein contains DnaJ domains implying that it is necessary for cellular growth . Northern blot analysis ofblack pearlRNA from various developmental stages showed two transcripts with greatest expression inDrosophilaembryos 0–6 hours old , the stages in which DNA replication recurs most rapidly. The Late gene AS 2268 shares sequence similarity with theDrosophila Imaginal disc growth factor4(Idgf4), a mitogen with a non-functional chitinase domain. Transcripts ofIdgf4are detected inDrosophilanurse cells, oocytes, and in the yolk cytoplasm of early embryos . The Middle gene product AS 2273 shares sequence similarity withDrosophila β Integrin.An. gambiaeβ Integrin may interact with the Middle gene AS 985 product to promote somatic cell adhesion and cell migration during oogenesis and embryogenesis. This is due to the similarity of the AS 985 gene toDrosophila Receptor of activated protein kinase C, RACK1. RACK1 can bind a number of different signaling and cell adhesion molecules including the activated form of protein kinase C (PKC), Src family kinases, and β Integrins [97–99]. Coxet al.  demonstrated that, in a mammalian system, RACK1 organizes focal adhesions and directional cell migration via its Src-binding site. Mahairakiet al.  found that theAn. gambiaeβ Integrin gene was expressed at highest levels 48 hours PBM, whereas we found that the β integrin homolog reached at least twofold increased expression by 24 hours PBM.
A number of genes have been implicated in the development of the egg shell structures. Our screen does not appear to have identified any homologs of the several endochorionic structural proteins characterized inAe. aegypti[102,103]. This was unexpected because Northern blot analysis had indicated that transcription of the vitelline membrane proteins 15a-1, 15a-2, 15a-3 was induced rapidly between 10 and 24 hours PBM, reached maximal levels between 30 and 40 hours PBM, and decreased to baseline levels between 50 and 60 hours PBM [102,103]. Our study also did not identify aDopa decarboxylase (Ddc)gene, Ddc is an enzyme involved in the tyrosine metabolic pathway necessary for eventual chorion melanization inAe. aegypti, and other melanization events. The gene is up-regulated in response to blood meal with transcripts initially detectable by 12 hours PBM, and maximal levels achieved between 24 and 48 hours PBM . However, we did identify a gene encoding another enzyme involved in tyrosine metabolism. AS 1340, a Middle gene product, shared sequence similarity with theAe.aegyptiDopachrome conversion enzyme . This enzyme is required for processing of dopachrome to melanin. It is interesting that its mRNA is constitutively expressed inAe. aegyptifemales, but becomes upregulated when they are infected withDirofilaria. Since insect melanins can be produced via any of three intermediates, Dopa, Dopamine, or Dopachrome, it may be thatAn. gambiaediffers fromAe. aegyptiin the substrate metabolized to produce chorionic melanin.
Several Middle and Late genes encoding antioxidants were upregulated at least twofold 12–48 hours PBM. The Middle gene, AS 2033, a glutathione S-transferase D3, and the three Late genes, ASs 1684, 35, and 2156, encoding glutathione S-transferase 1–6 class theta, and homologs ofDrosophilathioredoxin andAe. aegypti2-Cys thioredoxin peroxidase, may have roles in regulating reactive oxygen species that can be produced from the highly reactive quinones which are normally cross-linked into melanin immediately after they are formed.
Ovarian cycle and vitellogenesis
Paramount to the development of the embryo is the massive accumulation of vitellogenin by the oocyte. InAn. gambiaethere is a small, polymorphic tandem array of vitellogenin genes and a single dispersed vitellogenin gene, all located on Chromosome 2R in division 18B (P. Romans and M. Sharakhova, unpublished observations). Vitellogenin mRNA becomes detectable by Northern blot analysis by 8 hrs PBM, though it is detectable earlier by RT-PCR, increases dramatically by 12 hours, reaches maximal levels by 24 hours, and declines to undetectable levels by 48 hours PBM . Our microarray study identified three cDNAs, all Middle gene products and greater than twofold induced, as vitellogenin gene homologs. Two of the ESTs were not conjoined during EST assembly because they represented non-overlapping 5' and 3' ends of the Vg1 gene. The third EST included the more closely resembled the sequence of the dispersed vitellogenin gene (P. Romans and A. Dana, unpublished). As expected, all three vitellogenin clones exhibited expression profiles similar to the overall pattern previously described .
Following synthesis in the fat body, vitellogenins are released into the hemolymph. Eventually, they diffuse through channels between the cells of the follicular epithelium and are accumulated by the oocyte by receptor-mediated endocytosis in clathrin-coated pits . The increased number of gene products involved in receptor-mediated endocytosis before and after the height of vitellogenin gene transcription, 12–24 hours PBM in this study, may reflect a preparation for the increase in receptor-mediated endocytosis when the oocytes are accumulating vitellogenins and other yolk constituents during the trophic phase of the ovarian cycle. These genes included an Early gene, AS 1974, similar to theDrosophilaAdaptin subunit,AP-1σ, CG5864, and the Late gene, AS 2086, homolog of anotherDrosophilaclathrin-associated protein,AP-50, CG7057.
When vitellogenesis has ceased, the biosynthetic machinery in the fat body is degraded in lysosomes . InAe. aegypti, the lysosomal cathepsin D-like aspartic protease (AeLAP) exhibited a similar transcription profile to vitellogenin . Choet al.  also identified a Cathepsin B-like thiol protease, vitellogenic Cathepsin B or VCB, which is secreted from the fat body with a peak at 24 hours PBM and incorporated into oocytes. It appears to be involved in the degradation of vitellin in embryos. The Middle gene AS 996, a Cathepsin B, shares identity with thisAe. aegyptiprotein, exhibits the same expression profile, and may be its homolog. At approximately 30 hours PBM, 6 hours after peak production of vitellogenin, the activity of four other lysosomal enzymes, arylsulfatase A, acid phosphatase-1, β-galactosidase, and Cathepsin D, has dramatically increased to reach maximal levels by 36–42 hours PBM [108,109]. The two Late gene products, ASs 1254 and 2231, were identified putatively as the lysosomal enzymes, acid phosphatase-1 and Cathepsin F, respectively. These genes also may be involved in the termination phase of vitellogenesis. Cathepsin F is necessary for oocyte growth in a teleost fish and has been suggested to be associated with yolk protein processing . It will be a very interesting example of gene co-evolution, should processing of vitellogenins, proteins conserved between egg-laying vertebrates and non-Brachyceran insects, actually be accomplished by similarly conserved cathepsins.