Vesicular ATPase-overexpressing Cells Determine the Distributionof Malaria Parasite Oocysts on the Midguts of Mosquitoes*

(Received for publication, December 4, 1998, and in revised form, January 29, 1999)

Stephane O. Cociancich‡§, Soon S. Park‡, David A. Fidock¶, and Mohammed Shahabuddin‡i

From the ‡Medical Entomology Section and the ¶Malaria Genetics Section, Laboratory of Parasitic Diseases, NIAID,National Institutes of Health, Bethesda, Maryland 20892-0425

In Plasmodium-infected mosquitoes, oocysts are pref-erentially located at the posterior half of the posteriormidgut. Because mosquitoes rest vertically after feed-ing, the effect of gravity on the ingested blood has beenproposed as the cause of such a biased distribution. Inthis paper, we examined the oocyst distribution on themidguts of mosquitoes that were continuously rotatedto nullify the effect of gravity and found that the typicalpattern of oocyst distribution did not change. Invasionof the midgut epithelium by ookinetes was similarlyfound to be biased toward the posterior part of the pos-terior midgut. We examined whether the distribution ofoocysts depends on the distribution of vesicular ATPase(V-ATPase)-overexpressing cells that Plasmodium ooki-netes preferentially use to cross the midgut epithelium.An antiserum raised against recombinant Aedes aegyptiV-ATPase B subunit indicated that the majority of V-ATPase-overexpressing cells in Ae. aegypti and Anophe-les gambiae are localized at the posterior part of theposterior midgut. We propose that the typical distri-bution of oocysts on the mosquito midgut is attribut-able to the presence and the spatial distribution ofthe V-ATPase-overexpressing cells in the midgutepithelium.

Designing rational strategies to block transmission of ma-laria can benefit from understanding how the Plasmodiumparasite has adapted to the mosquito midgut cellular environ-ment (1). Until now, the cellular and biochemical basis ofPlasmodium development in the mosquito has been poorlyunderstood. During most of their development in a vector mos-quito, malaria parasites remain associated with the midgut.The posterior midgut is the primary site of storage of ingestedblood, of the absorption of salts and fluids, and of the digestionof blood components. Within a day after ingestion, the para-sites develop in the posterior midgut as motile ookinetes thatescape from the blood bolus and invade the epithelium. Inva-sion of the mosquito midgut is obligatory for further develop-ment and transmission. After escaping from the invaded cells,

the ookinetes remain associated with the midgut under thebasal lamina for about 1 week and are then transformed intooocysts, which leads to the development of sporozoites that areinfectious to the vertebrate host.

In Plasmodium-infected mosquitoes, the oocysts are primar-ily distributed at the posterior portion of the posterior midgut.The anterior portion has relatively fewer oocysts, especiallywhen the infection rate is low (,100 oocysts/gut). Previously,this biased distribution was explained by the fact that mosqui-toes rest on a wall for a day or two after feeding, their abdomenbeing in a vertical position. It has therefore been proposed that,because of gravity, the ingested blood cells and parasites settleat the bottom portion of the midgut and that ookinetes only geta chance to invade this part of the midgut (2–4).

One argument against the above explanation comes from thefinding that diuresis rapidly compacts the blood meal longbefore the ookinetes invade the midgut epithelium (5), therebyreducing the possibility for the ookinetes to settle down. Here,we examine whether gravity is indeed involved in determiningthe distribution of oocysts on the mosquito midgut.

The mosquito midgut is a complex tissue composed of avariety of cell types (6). We have recently found that a specifictype of cell (Ross cells) is preferentially invaded by Plasmo-dium gallinaceum ookinetes (7). Ross cells are less basophilicthan other cells in the epithelium, they are devoid of microvillion their surface, and their cytoplasm appears less osmiophilicby electron microscopy. They also overexpress a vesicularATPase (V-ATPase).1 Because of the parasite’s preferentialinvasion of Ross cells, the development of the parasite wasthought to possibly depend on the presence, number, and loca-tion of these cells in the midgut.

We have cloned the Aedes aegypti V-ATPase B subunit cDNAand used the recombinant protein to prepare an antibody toinvestigate the distribution of V-ATPase-overexpressing cellsin the midgut of Ae. aegypti and Anopheles gambiae. The oocystdistribution was studied in mosquitoes kept in a device de-signed to nullify the effect of gravity, and the location of P.gallinaceum ookinete invasion of the midgut epithelium wasassessed at the peak invasion period. We provide evidence thatRoss cells, rather than gravity, determine the typical distribu-tion pattern of the oocysts in infected mosquitoes.

EXPERIMENTAL PROCEDURES

Mosquitoes and Parasites—Ae. aegypti black-eye strain and An. gam-biae G3 were raised under standard conditions (26 °C, 80% humidity,photoperiodism (12 h dark-12 h light)) and fed on diluted Karo syrup(CPC International Inc., Englewood Cliffs, NJ). P. gallinaceum (8Astrain) was maintained by serial subpassages in 4–5-week-old whiteLeghorn chickens.

Detection of the Distribution of Oocysts on Mosquito Midguts—Adult

* This work was supported, in part, by Grant 950476 (to Louis H.Miller) from the United Nations Developmental Program/World Bank/World Health Organization Special Program for Research and Trainingin Tropical Diseases. The costs of publication of this article were de-frayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submittedto the GenBankTM/EBI Data Bank with accession number(s) AF092934.

§ Supported by the Vector Biology Network of the John D. and Cathe-rine T. MacArthur Foundation.

i To whom correspondence should be addressed: Laboratory of Para-sitic Diseases, NIAID, National Institutes of Health, Bldg. 4, Rm. B2-39, 9000 Rockville Pike, Bethesda, MD 20892-0425. Tel.: 301-496-9389;Fax: 301-402-8536; E-mail: mohammed_shahabuddin@nih.gov.

1 The abbreviations used are: V-ATPase, vesicular ATPase; PCR,polymerase chain reaction; PBS, phosphate-buffered saline; PIPES,1,4-piperazinediethanesulfonic acid.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 18, Issue of April 30, pp. 12650–12655, 1999Printed in U.S.A.

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mosquitoes were fed on an infected chicken for 20 min. Fed mosquitoeswere transferred into two cylindrical containers (9 cm high, 9 cm indiameter). One container was kept on an immobile rack, thus allowingmosquitoes to rest on the wall. The other container was rotated for 48 hat a speed of 10 rpm. The rotation prohibited any specific orientation ofthe abdomens of the blood-fed mosquitoes. The mosquitoes were thenkept in an insectary and dissected for oocyst counting at 8 days post-blood meal. Oocysts were counted separately in the anterior and pos-terior halves of the posterior midgut with a microscope equipped withan eyepiece micrometer. The statistical comparison (nonparametric)and the significance test of the oocyst numbers between two groups ofinfected mosquitoes were determined using Wilcoxon rank sum analy-sis. All of the mosquitoes in the test and control groups that received ablood meal were included in analysis regardless of whether they hadoocysts.

Preparation of the Mosquito V-ATPase-specific Probe—Two degener-ate primers designed from the predicted amino acid sequence of theV-ATPase B subunit from yeast (8) were as follows: sense primer,59-GG(ACGT)TT(TC)CC(ACGT)GGITA(TC)ATGTA(TC)A-39 (degenera-cy 5 1/128); and antisense primer, 59-GC(TC)TC(TC)TCICCIAC(ACG-T)AC(ACGT)GC(TC)TTC-39 (degeneracy 5 1/128). Thirty-five cycles ofPCR were performed with these primers using 1 ng of Ae. aegyptigenomic DNA as target. The conditions for the PCR were 30 s at 94 °C,30 s at 45 °C, and 1 min at 72 °C, performed in an automatic thermalcycler (GeneAmp PCR System 9600, Perkin-Elmer). The amplifiedproduct was subcloned into the pCR2.1 cloning vector (Invitrogen,Carlsbad, CA) and its nucleotide sequence determined.

A cDNA library made with the mRNA of unfed and blood-fed midgutsof Ae. aegypti and the Lambda Zap Express cloning vector (Stratagene,La Jolla, CA) was a kind gift of Dr. Anthony James (University ofCalifornia at Irvine). The unamplified library consisted of 12.9 3 106 lphages, of which 95.3% were recombinant as determined by blue/whitescreening. This library was amplified before screening (final titer 52.3 3 109 plaque-forming units/ml in Escherichia coli XL1-Blue cells).The library was screened with a digoxygenin-labeled PCR-amplifiedfragment of the Ae. aegypti V-ATPase B subunit gene (see above). Thepreparation, hybridization, and detection of the probe were performedusing the DIG/Genius kit (Boehringer Mannheim). Nine positive cloneswere purified and excised in vivo to produce pBK-CMV recombinantphagemids (Stratagene). Once the identity of the clones was confirmedby initial sequencing, the complete nucleotide sequence of one clone wasperformed by primer walking using a modified dideoxynucleotide-basedsequencing method (ABI model 377; Applied Biosystems, Foster City,CA).

For cloning, the ORF of the Ae. aegypti V-ATPase B subunit cDNAwas amplified by PCR with a 59 primer containing the start codon ATGand an antisense 39 primer containing the stop codon TAA. NdeI andBamHI sites were added for directional cloning. The conditions for thePCR were the same as above, except that the extension time was 3 min.The 1.5-kilobase ORF was cloned into the pET11a bacterial expressionvector (Stratagene) and introduced into E. coli BL21(DE3) cells (Strat-agene). Expression of the recombinant protein was induced by adding 1mM isopropyl-b-D-thiogalactopyranoside into the growth medium andwas assessed by SDS-polyacrylamide gel electrophoresis on cells har-vested 3 h post-induction.

To produce antiserum, the Coomassie Blue-stained recombinant V-ATPase B subunit band from a preparatory SDS-polyacrylamide gelwas excised, homogenized with Freund’s complete adjuvant in PBS, andused to immunize a New Zealand White rabbit (Spring Valley Labora-tories, Sykesville, MD). Subsequent boosts, performed at 14-day inter-vals, were done with incomplete adjuvant. After the third boost, theserum was tested by Western blot analysis. To deplete E. coli-specificantibodies, the antiserum was preabsorbed twice with heat-denaturedand sonicated extracts of E. coli BL21(DE3).

Western Blot Analysis with Mosquito Proteins—Midguts from sugar-fed, blood-fed, and infected blood-fed mosquitoes were dissected in PBSand heated at 80 °C for 30 min in Laemmli sample buffer. Chickenblood (negative control) was treated the same way. After SDS-polyacryl-amide gel electrophoresis, proteins were transferred to nitrocellulose(Nytran, Schleicher & Schuell). Membranes were blocked overnightwith 5% milk in TBS (20 mM Tris, 500 mM NaCl, pH 7.5) and probedwith a 1:100 dilution of the preabsorbed polyclonal antiserum. Blotswere developed using an alkaline phosphatase-conjugated goat anti-rabbit antibody (dilution 1:500, Kirkegaard & Perry Laboratories,Gaithersburg, MD) and the Western Blue kit (Promega, Madison, WI).

Immunofluorescence Assay—To detect V-ATPase-overexpressingcells, sugar-fed mosquitoes were dissected 4–6 days after emergence.Gut tissues were immediately fixed in 4% paraformaldehyde, 2 mM

MgSO4, 1 mM EGTA, and 0.1 M PIPES buffer (7). Fixed tissues weredehydrated by incubating 5 min each with 20, 40, 60, and 80% methanolin PBS, pH 7.2, and then for 15 min with 100% methanol. Tissues wererehydrated by sequentially washing them with decreasing concentra-tions of methanol in PBS. Rehydrated and permeabilized tissues wereblocked overnight at 4 °C with PBTG (1% bovine serum albumin, 0.1%Triton X-100, and 2% goat serum in PBS) and incubated overnight at4 °C with preabsorbed V-ATPase antiserum diluted in PBTG. Afterthree washes (1 h each) with PBTG, the tissues were incubated for 4 hwith a fluorescein isothiocyanate-conjugated goat anti-rabbit antibody(Pierce) diluted in PBTG and 0.005% bisbenzimide H33258 fluoro-chrome (nuclear stain, Calbiochem).

To examine the early invasion of the midgut epithelium by ookinetes,P. gallinaceum-infected blood-fed mosquitoes were treated the sameway, except that the blood-fed midguts were dissected in PBS 42 hpost-feeding and cut longitudinally to remove the blood meal. An addi-tional incubation with a rhodamine-labeled anti-Pgs28 monoclonal an-tibody (dilution 1:500) was performed to detect the parasites that hadinvaded the epithelium. The tissues were then washed five times withPBTG (1 h each), mounted, and examined with a Leitz Ortholux 2fluorescent microscope using appropriate filter blocks for rhodamineand fluorescein isothiocyanate. In a defined area of the mosquito tis-sues, the total number of cells, the number of V-ATPase-overexpressingcells, and the number of ookinetes that invaded the epithelium weredetermined, respectively, by nuclear staining, V-ATPase staining, andPgs28 staining.

RESULTS

Distribution of P. gallinaceum Oocysts on the Mosquito Mid-gut—In a preliminary experiment, 12 infected blood-fed mos-quitoes were individually kept head down in small tubes. After48 h, which is enough for the food bolus to concentrate in themidgut epithelium and for the majority of ookinetes to invadethe midgut epithelium, the mosquitoes were transferred into acage until dissected and examined for the distribution of oo-cysts. The distribution of oocysts in these mosquitoes remainedbiased toward the posterior portion of the posterior midgut(data not shown), similar to the oocyst distribution in thecontrol mosquitoes. We repeated the experiment with 60 fullyengorged mosquitoes that were rotated for 48 h to avoid orien-tation in a particular direction. A control group of mosquitoesfrom the same cohort, fed on the same infected chicken, was

FIG. 1. Distribution of P. gallinaceum oocysts on midguts ofundisturbed or rotated Ae. aegypti. Each line links the number ofoocysts on the anterior part (Ant.) of the posterior midgut to the numberof oocysts on the posterior part (Post.) of the posterior midgut of a singlemosquito. Rotated mosquitoes were kept for 48 h post-blood meal in arotating device.

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kept in a cage and allowed to rest vertically after feeding. Eightdays later, oocyst distributions were determined for the midg-uts of mosquitoes from both groups. No significant difference(p . 0.1) was observed in the mean number of oocysts permidgut between the control (74.2 6 15.8, n 5 13) and test(102.9 6 8.6, n 5 60) groups. This result suggests that therotation does not significantly influence the total number ofoocysts per midgut; however, the difference between the meannumbers of oocysts present on the anterior part (32.6 6 2.8)and on the posterior part (70.3 6 6.3) of the posterior midgutwas highly significant (p , 0.001) in the rotated mosquitoes.This finding indicates that gravity does not determine theoocyst distribution. Indeed, all of the mosquitoes in the controlgroup had a majority of the oocysts distributed toward theposterior part of the posterior midgut as expected (Fig. 1; seealso Fig. 5A). Of the 60 mosquitoes that were rotated, only twohad slightly more oocysts on the anterior part of the posteriormidgut. In four other mosquitoes, the oocysts were equallydistributed between the anterior and the posterior parts of theposterior midgut. All of the other mosquitoes (90%) had oocystsprimarily in the posterior half of the posterior midgut. The lackof difference in the distribution of oocysts between the controland test mosquitoes suggests that gravity does not have adetectable effect on oocyst distribution in the posterior midgutof the mosquito.

In Vivo Invasion of P. gallinaceum Ookinetes in Ae. aegyptiMidguts—To determine the stage at which the spatial distri-bution of oocysts is defined, we examined the early invasion ofookinetes in the mosquito midgut. The number of parasitesthat invaded the midgut epithelium was estimated at 42 h afterinfection, when the majority of invasion is complete but nooocysts are yet formed (9). The invaded parasites were detectedon the surface of intact midguts using rhodamine-labeledmonoclonal antibodies raised against the ookinete surface pro-tein Pgs28 (10). The majority of parasites were found in theposterior half of the posterior midgut, whereas only a fewinvasion events were detected in the anterior half (see Fig. 5, Band C), suggesting that the oocyst distribution is determined ator prior to the time of invasion. Because in Ae. aegypti theparasites preferentially invade a particular type of cell known

as Ross cells (7), we examined whether the distribution of thesecells in the mosquito midgut could determine the location of theoocysts. One of the characteristics of Ross cells is their overex-pression of a V-ATPase. We therefore chose to use the Ae.aegypti V-ATPase as a marker to examine the distribution ofthese specialized cells in the mosquito midgut.

Spatial Distribution of V-ATPase-overexpressing Cells in thePosterior Midgut of Mosquitoes—The sequence of the B subunitof V-ATPase enzymes is well conserved among organisms asevolutionarily separated as bacteria and humans. We preparedtwo degenerate primers from the amino acid sequence of theyeast V-ATPase B subunit, ranging from amino acid 303 to 310for the 59 primer and amino acids 417 to 424 for the 39 primer(8). A PCR product of the expected size (365 base pairs) wasamplified from genomic DNA prepared from newly emergedlarvae of Ae. aegypti. Data base comparisons using the se-quenced PCR product demonstrated 100% identity at theamino acid level with V-ATPase B subunit sequences from thedipterans Culex quinquefasciatus (11) and Drosophila melano-gaster (12) and the lepidopterans Manduca sexta (13) and He-licoverpa virescens (14). This identity suggests that the ampli-fied DNA fragment derives from the Ae. aegypti V-ATPase Bsubunit gene.

A specific digoxygenin-labeled probe made from this PCR-amplified gene fragment was used to screen an Ae. aegyptimidgut cDNA library prepared from a pool of sugar-fed anduninfected blood-fed adult mosquito midgut mRNA. Nine pos-itive clones were identified and found by sequence analysis tocontain the PCR probe sequence. The complete sequence of oneof these cloned cDNAs was determined by primer walking. ThiscDNA is 2538 base pairs long and contains a 1491-base pairORF encoding a 496-amino acid protein (Fig. 2). The calculatedmass of the protein is 55,221 Da and is within the predictedsize of V-ATPase B subunits from other organisms (range54–58 Da). A Prosite analysis identified the signature se-quence for the V-ATPase B subunit (PPVNVLPSLS; aminoacids 376–385). The predicted amino acid sequence is 98.4%identical with the C. quinquefasciatus V-ATPase B subunit(11), 97.8% identical with the D. melanogaster protein (12), and97.0% identical with the M. sexta protein (14). This sequence

FIG. 2. Nucleotide and deducedamino acid sequences from Ae. ae-gypti V-ATPase B subunit cDNA. The1491-base pair ORF starts with the in-itiation codon at position 235 and endswith a stop codon at position 1723. Thesignature sequence for V-ATPase A and Bsubunits is underlined (amino acids376–385).

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also shares high similarities (50–95%) with its counterpartsfrom organisms as different as bacteria, fungi, plants, para-sites, and vertebrates (Fig. 3).

The ORF from the cloned Ae. aegypti V-ATPase B subunitcDNA was amplified by PCR and inserted downstream of theT7 promoter of the pET11a vector between the NdeI andBamHI sites. This recombinant plasmid (pSC1) was then in-troduced into E. coli BL21(DE3). After induction with isopro-pyl-b-D-thiogalactopyranoside, the transformed E. coli pro-duced high levels of the recombinant protein (Fig. 4, lane 2). Noexpression was detected in uninduced transformed bacteria(Fig. 4, lane 1) or in induced nontransformed bacteria (data notshown). The estimated mass of the recombinant protein issimilar to the predicted size of the protein encoded by the ORFof the cloned cDNA.

A polyclonal rabbit antiserum was raised to Ae. aegypti V-ATPase B subunit recombinant protein. The antiserum waspreabsorbed with heat-denatured and sonicated extracts of theparent E. coli BL21(DE3) strain. As determined by Westernblot, this preabsorbed serum recognized the recombinant pro-tein (Fig. 4, lane 3) but no other E. coli or chicken bloodantigens (Fig. 4, lanes 3 and 4). Therefore, the preabsorbedantiserum was used for all subsequent experiments.

To determine whether the native mosquito V-ATPase B sub-unit was recognized by the antiserum, we performed a Westernblot analysis with mosquito proteins prepared from sugar-fedadult Ae. aegypti midguts. One polypeptide of the same esti-mated size as the recombinant protein was detected in the

FIG. 4. Expression of recombinant Ae. aegypti V-ATPase B sub-unit cDNA in a bacterial expression vector and of the nativeprotein in Ae. aegypti. The PCR-amplified, full-length cDNA wascloned into the pET11a expression vector and expressed into E. coliBL21(DE3) as described under “Experimental Procedures.” Lane 1,uninduced transformed bacteria; lane 2, induced transformed bacteria.A strong band, with relative migration at 55 kDa, is present and highlyexpressed only in the induced bacteria (arrow). The band was used toprepare an antiserum that was preabsorbed with the parent bacterialstrain and used in Western blot. Lane 3, recombinant V-ATPase (Rec.V-ATPase; positive control); lane 4, chicken blood (negative control);lane 5, sugar-fed (Sug) Ae. aegypti midguts; lane 6, blood-fed (Bld) Ae.aegypti midguts; lane 7, infected (Inf) blood-fed Ae. aegypti midguts. Nonotable difference in the intensity of expression was detected in any ofthese last three samples. IPTG, isopropyl-b-D-thiogalactopyranoside.

FIG. 3. Sequence comparison of the Ae. aegypti V-ATPase B subunit with V-ATPase B subunits from evolutionary diverseorganisms: insects (D. melanogaster), vertebrates (Homo sapiens), plants (Arabidopsis thaliana), nematodes (Caenorhabditiselegans), fungi (Neurospora crassa), and parasites (P. falciparum). Alignments were performed using the Clustal method. Dots representamino acids that are identical to the mosquito sequence. Dashes represent gaps introduced to maximize the alignments.

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midgut tissue (Fig. 4, lane 5), suggesting that the antiserumprimarily recognizes the mosquito V-ATPase. Protein extractsprepared from midguts of sugar-fed mosquitoes and uninfectedor infected mosquitoes 24 h after the blood meal were comparedby Western blot analysis (Fig. 4, lanes 5–7). An equivalent oftwo midguts was used in each case. Within the sensitivityrange of the Western blot, no difference was noted in theintensity of the bands between these differently fed midguts.This indicates that blood feeding or infection with Plasmodiumdoes not affect V-ATPase expression in the mosquito midgut.

We then used immunofluorescence assay to determinewhether the antiserum detects V-ATPase-expressing cells insugar-fed Ae. aegypti midguts. As seen in Fig. 5, E and G, theantiserum recognizes a subset of cells in the mosquito midgut.This is similar to our previous finding with a C. quinquefascia-tus V-ATPase antiserum (7).

We have examined the distribution of V-ATPase-overex-pressing cells in the posterior midgut of Ae. aegypti. Mostnotable during this analysis was the nonrandom distribution ofthese cells in this region; ;90% were present at the posteriorportion of the posterior midgut versus ;10% at the anteriorpart of the posterior midgut (Fig. 5, D and E).

V-ATPase-overexpressing Cells and P. gallinaceum Ookinete

Invasion in An. gambiae Midguts—An. gambiae G3 mosquitoesare vectors for human malaria but are considered to be refrac-tory to P. gallinaceum development (15). However, in a smallproportion of these mosquitoes, a few parasites develop asoocysts, again at the posterior half of the posterior midgut.2 Inour previous attempts with Culex V-ATPase antiserum, wefailed to detect V-ATPase-overexpressing cells in An. gambiaemidguts, precluding the study of whether cells similar to Rosscells were present in Anopheles mosquitoes. Here, we examinedwhether Ae. aegypti V-ATPase antiserum can detect V-ATPase-overexpressing cells in An. gambiae. By Western blot, we de-tected a single protein of similar size to the one found in Ae.aegypti (Fig. 6A). We then used the antiserum in an immuno-fluorescence assay to detect cells that express the enzyme.Similar to Ae. aegypti, the antiserum specifically recognizedV-ATPase-overexpressing cells in the An. gambiae midgut ep-ithelium (Fig. 6D). Because our V-ATPase antiserum was ableto detect the Anopheles enzyme and the cells that express thisenzyme, we examined whether P. gallinaceum also invadesV-ATPase-overexpressing cells in An. gambiae and whetherthe oocyst distribution is determined by the presence of thesecells.

An. gambiae were fed with P. gallinaceum-infected blood,

2 S. O. Cociancich and M. Shahabuddin, unpublished observations.

FIG. 5. P. gallinaceum ookinete invasion and oocyst develop-ment in the Ae. aegypti posterior midgut. A, oocyst distribution 8days post-blood meal. The arrowhead points to the middle of the Ae.aegypti posterior midgut. The closed arrow identifies the junction of theanterior and posterior midgut, and the open arrow shows the junction ofthe posterior midgut and the hindgut. B, ookinete invasion 42 h post-blood meal in the anterior half, and C, in the posterior half of theposterior midgut. V-ATPase-overexpressing cells at D, the anterior half,and E, the posterior half of the posterior midgut. F, the same field as inG but with staining for epithelial cell nuclei. G, magnification of theboxed area in E.

FIG. 6. Detection of V-ATPase and of cells that overexpressthis enzyme in An. gambiae posterior midguts. Sugar-fed (Sug),blood-fed (Bld), and infected blood-fed (Inf) An. gambiae midguts weretreated as described under “Experimental Procedures.” A, the Ae. ae-gypti antiserum recognizes a sharp band of 55 kDa. B, immunofluores-cence assay studies reveal a colocalization of V-ATPase-overexpressingcells with the invasion of a P. gallinaceum ookinete. The biased distri-bution of the V-ATPase-overexpressing cells toward the posterior end ofthe posterior midgut can be seen in C–F: C, V-ATPase staining of theanterior part of the posterior midgut; D, V-ATPase staining of theposterior part of the posterior midgut; E, total cells in the same area asC; F, total cells in the same area as D.

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and 36–40 h later, their midguts were dissected and probedwith V-ATPase B subunit and Pgs28 antisera. We found that,as in Ae. aegypti, P. gallinaceum ookinetes invade An. gambiaecells that express high levels of V-ATPase (Fig. 6B). When thedistribution of V-ATPase-overexpressing cells was examined,we found that the anterior half of the posterior midgut had veryfew of these cells (less than 10% of the total V-ATPase-overex-pressing cells) and that more than 90% of them were present atthe posterior half. These data indicate that although An. gam-biae G3 is refractory to P. gallinaceum, P. gallinaceum ooki-netes do invade functionally related cells in both Ae. aegyptiand An. gambiae.

DISCUSSION

In this paper, we provide evidence that the distribution ofPlasmodium oocysts on the posterior midgut of mosquitoes isrelated to the presence and distribution of V-ATPase-overex-pressing Ross cells. We also present evidence indicating thatgravity plays no major role in the phenomenon. Indeed, whenmosquitoes were kept under conditions in which gravity couldnot have any effect on the distribution of the ingested bloodcells and parasites in the posterior midgut, we found that thepattern of oocyst distribution remains biased toward the pos-terior end of the posterior midgut. In only 10% of mosquitoesdid we find oocysts distributed in equal or greater numbers onthe anterior half of the posterior midgut (Fig. 1). The reasonsfor this unusual distribution pattern are unclear but may in-volve individual differences in the vector or alternative distri-bution of Ross cells or other invaded cell types in the midgut.We have also shown that the initial invasion of the midgutepithelium by ookinetes primarily occurs at the posterior partof the posterior midgut. When the distribution of V-ATPase-overexpressing cells in Ae. aegypti and An. gambiae was exam-ined, we found that these cells were also primarily located atthe posterior part of the posterior midgut.

V-ATPases are members of the class of ATP-hydrolyzingproton pumps ubiquitous among living organisms (16). TheATPase family can be divided into three distinct groups (17): i)the P-ATPases, which are localized in plasma membranes;these are composed of a single subunit, and the degradation ofATP involves a phosphorylated intermediate; ii) the F0/F1-ATPases, which are multisubunit molecules found in eubacte-ria and in organellar membranes (mitochondria and chloro-plasts); and iii) the V-ATPases, which were first identified invesicular membranes but have recently been found in the cy-toplasmic membrane of epithelial cells. They resemble the F0/F1-ATPases both in their structure and in the way they cata-lyze ATP but are classified as a distinct family because they arenot sensitive to F0/F1-ATPase inhibitors. V-ATPases are com-posed of nine subunits organized in two domains: one trans-membrane domain and one cytoplasmic (catalytic) domain. Thecatalytic domain includes three copies of both the A (catalytic)and B (regulatory) subunits. Taking advantage of the highdegree of sequence conservation among organisms as differentas bacteria, fungi, plants, insects, and vertebrates, we havecloned and expressed the B subunit of the Ae. aegyptiV-ATPase. The production of an antiserum raised against therecombinant protein allowed us to study both Ae. aegypti and

An. gambiae cells that overexpress this enzyme.The role of V-ATPases in the mosquito midgut is not known.

In M. sexta larvae midguts, specialized cells resembling verte-brate goblet cells overexpress V-ATPase and appear to be in-volved in ion and water transportation from the midgut lumento the hemolymph (18). It has also been shown that malpighiantubules, which play an important role in fluid absorption, over-express a V-ATPase at their surface (19). It might therefore bepossible that the V-ATPase-overexpressing cells present in theposterior midgut of mosquitoes are involved in the diuresis thatoccurs immediately after blood feeding. However, it is notknown why the parasites prefer to use these cells to cross themidgut.

This study was undertaken in the context of how the humanmalaria parasite Plasmodium falciparum develops in its vectorand is transmitted. P. gallinaceum is better adapted to use inthe laboratory and hence is commonly used as a model formalaria transmission-blocking studies. Several genes havebeen cloned from P. gallinaceum and reveal greater homologywith P. falciparum than with any other Plasmodium spp. (20).Also, the blocking of P. gallinaceum oocyst development inAedes mosquitoes by the chitinase inhibitor allosamidin can bereproduced with P. falciparum in Anopheles mosquitoes (21).Therefore, the results presented here might apply to P. falcip-arum, allowing us to initiate work on V-ATPase-overexpressingcells in An. gambiae to determine their role in human malariatransmission.

Acknowledgments—We thank Dr. Anthony A. James for permissionto use the Ae. aegypti midgut cDNA library. We are indebted to Drs.Louis H. Miller and Jose M. C. Ribeiro for their support and criticalcomments throughout the work. We thank Dr. David C. Kaslow for thePgs28 antiserum. We acknowledge the technical assistance of AndreLaughinghouse and Kevin Lee in rearing mosquitoes and cultivatingparasites. We also thank Brenda Rae Marshall for preparing themanuscript.

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