Transcripts of the malaria vector Anopheles gambiae that are differentially regulated in the midgut upon exposure to invasive stages of Plasmodium falciparum

Authors

  • Sarah Bonnet,

    1. Ecologie des Systèmes Vectoriels, Institut Pasteur,
      25 rue du Dr Roux, 75724 Paris Cedex 15, France.
    2. IRD-OCEAC, PO Box 288, Yaoundé, Cameroon.
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    • The first two authors contributed equally to this work.

  • Ghislaine Prévot,

    1. Ecologie des Systèmes Vectoriels, Institut Pasteur,
      25 rue du Dr Roux, 75724 Paris Cedex 15, France.
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    • Present address: Institut Pasteur de la Guyane, 23 Avenue Pasteur, BP 6010, 97306 Cayenne Cedex, Guyane Française.

    • The first two authors contributed equally to this work.

  • Jean-Claude Jacques,

    1. Ecologie des Systèmes Vectoriels, Institut Pasteur,
      25 rue du Dr Roux, 75724 Paris Cedex 15, France.
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  • Christian Boudin,

    1. IRD-OCEAC, PO Box 288, Yaoundé, Cameroon.
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  • Catherine Bourgouin

    Corresponding author
    1. Ecologie des Systèmes Vectoriels, Institut Pasteur,
      25 rue du Dr Roux, 75724 Paris Cedex 15, France.
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*For correspondence. E-mail cabourg@pasteur.fr; Tel. (+33) 01 45 68 82 24; Fax (+33) 01 40 61 30 89.

Abstract

Understanding the interactions between the most deadly malaria parasite, Plasmodium falciparum, and its main vector, Anopheles gambiae, would be of great help in developing new malaria control strategies. The malaria parasite undergoes several developmental transitions in the mosquito midgut and suffers population losses to which mosquito factors presumably contribute. To identify such factors, we analysed An. gambiae midgut transcripts whose expression is regulated upon ingestion of invasive or non-invasive forms of P. falciparum using a differential display approach. Sixteen cDNA were studied in detail; 12 represent novel genes of An. gambiae including a gene encoding profilin. Four transcripts were specifically regulated by P. falciparum gametocytes (invasive forms), whereas the others were regulated by either non-invasive or both non-invasive and invasive forms of the parasite. This differential regulation of some genes may reflect the adaptation of P. falciparum to its natural vector. These genes may be involved in the development of P. falciparum in An. gambiae or in the defence reaction of the mosquito midgut towards the parasite.

Introduction

Plasmodium falciparum malaria remains one of the most important diseases in the world, especially in developing countries. This is mainly because of the spreading resistance of the parasite to several antimalarial drugs and to the selection of insecticide-resistant populations of its vector, the Anopheles mosquito. In subSaharan Africa, Anopheles gambiae is the most efficient vector of P. falciparum (Coluzzi, 1984). When ingested by a female mosquito during blood feeding on a gametocyte carrier, Plasmodium gametocytes pass through a series of developmental stages within the mosquito midgut: differentiation/maturation of the gametes and zygote and ookinete development. The motile ookinetes then cross the peritrophic matrix and the midgut epithelium reaching the basal membrane, where they differentiate further into oocysts. Sporozoites develop within oocysts and are released into the haemocoel upon oocyst rupture. Sporozoites then invade the mosquito salivary glands and can be injected to a new host when the mosquito feeds.

Although the sporogonic development of the parasite takes place mainly inside the mosquito midgut, our understanding of the molecular midgut–parasite interactions is still limited. The identification of mosquito genes involved in the sporogonic development would facilitate studies of the molecular interactions between the parasite and its vector. It could also lead to the development of new methods for interrupting malaria transmission (chemical products, antibodies or production of transgenic mosquitoes unable to transmit the parasite). Few mosquito factors involved in PlasmodiumAnopheles interactions have been identified (Shahabuddin et al., 1993; Billker et al., 1998; Luckhart et al., 1998). They were identified using various combinations of mosquito species and Plasmodium parasites, but not An. gambiaeP. falciparum. Although the previous findings are probably valid for the natural system involving Anopheles mosquitoes and P. falciparum, three lines of evidence suggest that the development of the human parasite in Anopheles, the only mosquito genus permitting its complete development, involves unique interactions. First, P. falciparum uses an intercellular route for crossing the mosquito midgut epithelium in Anopheles stephensi (Meis et al., 1989), whereas other species, such as Plasmodium berghei and Plasmodium gallinaceum, use an intracellular route in An. stephensi and Aedes aegypti respectively (Garnham et al., 1969; Meis et al., 1989; Torii et al., 1992; Han et al., 2000; Zieler et al., 2000). Secondly, an An. gambiae line refractory to the development of Plasmodium cynomolgi is also refractory to the development of several other species and strains of Plasmodium, but of limited refractoriness to African isolates of P. falciparum (Collins et al., 1986). Note that An. gambiae is found exclusively in Africa. Thirdly, Feldmann and Ponnudurai (1989) selected an An. stephensi line that limits the maturation of P. falciparum oocysts only (Feldmann et al., 1998).

We investigated the effects of P. falciparum on mosquito gene expression in a blood meal ingested by An. gambiae. As the production of in vitro infective gametocytes of P. falciparum is very delicate, infection of An. gambiae was carried out in an endemic area (Cameroon) by experimental feeding on blood of naturally infected gametocyte carriers. We focused on the identification of genes differentially expressed in the midgut of mosquitoes fed either on parasite-free blood or on blood of P. falciparum gametocyte or asexual stage carriers. We used the differential display approach (Liang and Pardee, 1992) to identify such genes: five transcripts were identified. Four correspond to genes selectively triggered by the presence of P. falciparum gametocytes in the blood meal and one to a gene overexpressed in the presence of both gametocytes and asexual stages of P. falciparum. We also describe the expression of seven other mRNAs in the midgut of An. gambiae after a blood meal containing P. falciparum. This is the first analysis of the interaction between the human malaria parasite and the An. gambiae midgut at the molecular level.

Results

Selection of An. gambiae midgut mRNAs expressed differently after a parasite-free and a P. falciparum-containing blood meal

We tried to identify two sets of genes: (i) those expressed in the midgut after a parasite-free blood meal; and (ii) those regulated by the presence of P. falciparum in the blood meal. Accordingly, two differential display experiments were performed. From the first display, we selected 88 amplicons corresponding to RNA expressed during at least two consecutive times after a parasite-free blood meal but not in unfed An. gambiae. Thirty-four amplicons were efficiently reamplified by one or the other reamplification methods (see Experimental procedures). Nine amplicons were analysed in detail in addition to two others generated during the course of the reverse transcription–polymerase chain reaction (RT–PCR) analysis. The second differential display compared the expression pattern of midgut RNA isolated from mosquitoes fed on parasite-free blood, blood containing gametocytes and blood containing only the asexual stages of P. falciparum. RNA was isolated 14 h after the blood meal, at which time P. falciparum develops into motile ookinetes. An example of the expression pattern observed is given in Fig. 1. Over 100 amplicons corresponding to RNAs regulated by the presence of either gametocytes or asexual stages in the blood meal were detected. However, only 20 amplicons gave reproducible expression profiles. Five were successfully reamplified and sequenced. Three amplicons corresponded to RNA induced by the presence of both gametocytes and asexual stages (14Yde, 58Yde and 70Yde). Expression of amplicon 70Yde was, however, stronger in the presence of gametocytes. The fourth amplicon (67Yde) was upregulated in mosquitoes fed on gametocyte-containing blood. The fifth amplicon (55Yde) corresponded to an RNA expressed only in parasite-free midguts.

Figure 1.

Example of expression pattern obtained by differential display on mRNA from midgut of an An. gambiae female fed on P. falciparum-containing blood. Each series (1,2,3) and (1′,2′,3′) corresponds to different pools of RNA isolated 14 h after feeding from mosquitoes fed on: 1 and 1′, parasite-free blood meal; 2 and 2′, blood containing P. falciparum gametocytes; 3 and 3′, blood containing P. falciparum asexual forms. PCRs were duplicated in each line. Arrow indicates one band of reproducible pattern in each condition and selected for further analysis. DDRT–PCR amplification was performed with upstream primer U15 (5′-GATCAATCGC) and downstream primer PMA (5′-(T)11NA).

Sequence analysis of selected amplicons

The sequences of all 16 selected amplicons were determined and compared with sequences in public databases. Seven showed significant similarity to known sequences. No matches were found for nine (Table 1). Of interest, 20P2b corresponds to the Antryp1 gene, and 34P2 is very similar to the Drosophila profilin. This latter amplicon contains a conserved stretch of amino acids corresponding to a profilin motif SWQDYVD. Amplicons 17P1, 34P2 and 58Yde contain one EGF-like repeat, and amplicons 23P2, 24P2, 55Yde and 67Yde contain ferredoxin, the iron–sulphur-binding region signature. The other amplicons do not contain motifs other than potential N-glycosylation, N-myristoylation and phosphorylation sites. For each cDNA fragment corresponding to mRNA, genomic DNA was amplified using the primers used for the RT–PCR: all correspond to exons with no intron (not shown).

Table 1.  Search results for 16 sequences selected by differential display of Anopheles gambiae female midgut RNA.
CodeAmplicons size (bp)HomologyP-value
8P1a267D. melanogaster unknown proteinb3.5 e-10
17P1a359None
20P2-90a102Ae. aegypti ribosomal RNA genec1.5 e-16
20P2-200a200Ae. aegypti ribosomal RNA genec3.9 e-45
20P2-450a472An. punctulatus ribosomal RNA genec2.5 e-129
20P2ba862An. gambiae trypsin1 gened0
23P2a269None
24P2a172None
29P1a298None
34P2a387Drosophila profilin genee5.7 e-20
36P2a180None
14Ydef421None
55Ydef170None
58Ydef145None
67Ydef228An. gambiae unknown geneg7 e-44
70Ydef97None

Expression pattern of selected amplicons after a parasite-free blood meal

Studying the expression patterns of the selected amplicons in different organs of male and female mosquitoes may give clues as far as their function is concerned. The expression pattern of all selected amplicons was analysed using RT–PCR in midguts and carcasses of fed and unfed female and male mosquitoes. Patterns of expression were diverse (Table 2, Fig. 2). In female midguts, the expression of three RNAs (trypsin 20P2b, 8P1 and, to a lesser extent, 55Yde) was upregulated by the blood meal, whereas the expression of others was downregulated (at least at one time point) or unaffected. In female carcasses, the expression of 24P2 and 36P2 increased 24 h after the blood meal when the expression of all other RNAs was downregulated. All RNAs were found in both sexes. However, four RNAs were more abundant in unfed female midguts than in male midguts (36P2, 58Yde, 67Yde and 70Yde). The abundance of all the RNAs, except RNA 24P2, was higher in unfed female carcasses than in male carcasses. Only the trypsin RNA (20P2b) was tissue restricted (in both male and female mosquitoes). Nevertheless, the expression levels of RNA 17P1 and 29P1 were very low in female midguts. 24P2 RNA was absent from unfed female carcasses.

Figure 2.

Graphic representation of the expression levels of selected mRNAs in An. gambiae: sex and tissue dependence and regulation by a parasite-free blood meal. Expression levels were assessed using RT–PCR in the midgut and carcass of males (M), unfed females (F) and of blood-fed females 24 h and 48 h after feeding. PCR products were quantified (see Experimental procedures). Number of PCR cycles used was 40 cycles for 23P2, 29P1, 34P2 and 30 cycles for all other cDNAs. The amount of polymerase was double for cDNA 29P1. Results were normalized with values obtained for actin mRNA after 22 PCR cycles.

Table 2.  Expression patterns of 13 cDNA clones identified by differential display from mRNA of An. gambiae female midgut.
 SexTissueBlood meal effectEffect of parasite presence
CodeMidgutCarcassFemaleMaleMidgutCarcassGametocytesAsexual forms
  • Sex- and tissue-dependent mRNA in male (M) and unfed female (F) mosquito midgut (MG) and carcass (whole An. gambiae minus midgut) (C). The effect of parasite-free blood meal was analysed at 24 h and 48 h after feeding. The effect of parasite in the blood meal was analysed at 18 h, 24 h and 30 h after feeding.

  • a

    . cDNA selected as induced by a human blood meal at least two consecutive times from 14 h to 28 h after feeding.

  • b

    . cDNA selected as regulated by the presence of Plasmodium falciparum in a human blood meal at 14 h after feeding.

8P1aM > FF > MC > MGMG = CInductionRepressionNoneInduction
17P1aM > FF > MC > MGMG = CNoneRepressionRepressionRepression
20P2baF = MMGMGInductionNoneRepressionNone
23P2aM > FF > MC > MGMG > CNoneRepressionNoneInduction
24P2 aF = MM > FMG > CMG > CRepressionInductionInductionInduction
29P1aM > FF > MC > MGC > MGNoneRepressionUnsuccessfulUnsuccessful
34P2aM > FF > MC = MGMG > CRepressionRepressionInductionInduction
36P2aF > MF > MMG > CMG > CRepressionInductionNoneInduction
14YdebF = MF > MC > MGC = MGRepressionRepressionInductionInduction
55YdebM > FF > MC > MGMG > CLow inductionRepressionRepressionNone
58YdebF > MF > MC > MGC > MGRepressionRepressionInductionInduction
67YdebF > MF > MC > MGMG > CRepressionRepressionInductionInduction
70YdebF > MF > MC > MGC = MGRepressionRepressionInductionInduction

Expression patterns of selected amplicons after a P. falciparum-containing blood meal

The display comparing the effect of parasite-free or parasite-containing blood meal (PBM) at 14 h after feeding led to the selection of five cDNAs whose expression was regulated by the presence of P. falciparum. RT–PCR analysis was used to control their expression patterns, and the same overall expression profile was recovered (Table 2, Fig. 3A). The RNAs were also studied in midguts isolated 18 h, 24 h and 30 h after feeding the mosquitoes with parasite-containing blood or parasite-free blood samples (Fig. 3B). 14Yde was upregulated at 14 h and 24 h, but not at 18 h and 30 h PBM when mosquitoes fed on both gametocytes and asexual stages containing blood. For all other amplicons, the effect of gametocytes upon the transcript profile was not prolonged, although the effect of the asexual stages was continuous or pronounced.

Figure 3.

Midgut expression pattern of An. gambiae cDNAs selected as differentially regulated by P. falciparum parasite. The expression level was analysed:

A. 14 h after feeding with a parasite-free blood meal, a blood meal containing gametocytes (blood sample E) or blood containing only asexual forms (blood sample F). The number of PCR cycles was 35 cycles for all samples. Results were normalized with values obtained for actin mRNA after 35 PCR cycles.

B. 18 h, 24 h and 30 h after feeding with a parasite-free blood meal, a blood meal containing gametocytes (blood sample B) or blood containing only asexual forms (blood sample C). The number of PCR cycles was 25 cycles for all samples. Results were normalized with values obtained for actin mRNA after 25 PCR cycles. RNA 55Yde and 70Yde were assayed only once owing to lack of material.

The expression patterns of the RNAs selected during the display comparing unfed mosquitoes and mosquitoes fed on parasite-free blood meal were determined in the presence of parasites (Fig. 4). 8P1, 23P2 and 36P2 were all upregulated in the presence of asexual stages at 18 h PBM. Only the expression of RNA 24P2 and 34P2 was clearly upregulated by both gametocytes and asexual stages at 18 h and 30 h PBM and 18 h and 24 h PBM respectively. The expression of 17P1 was downregulated by the presence of gametocytes at 18 h and 30 h PBM and by the presence of asexual stages at 24 h PBM. However, the expression of Antryp2 (20P2b) was downregulated at 18 h PBM by gametocyte-containing blood meals. RNA 29P1 was not successfully amplified.

Figure 4.

Midgut expression pattern, in the presence of P. falciparum, of An. gambiae RNAs selected as differentially regulated by a parasite-free blood meal. The expression level was analysed at 18 h, 24 h and 30 h after feeding with a parasite-free blood meal, a blood meal containing gametocytes or blood containing only asexual forms (blood samples A and D for 34P2 and 20P2b and blood samples B and C for all other RNA respectively). The number of PCR cycles was 35 (8P1, 34P2 and 20P2b) or 40. A double amount of RNA was used for amplicons 17P1, 34P2 and 20P2b. Results were normalized with values obtained for actin mRNA after 35 PCR cycles for 34P2 and 20P2b and 25 cycles for all other RNA. RNA 36P2, 24P2 and 17P1 were assayed only once owing to lack of material.

Discussion

Identification of new An. gambiae genes differentially expressed in the mosquito midgut

We found that the reliability of the differential display method can be improved by duplicating the PCRs and using two sets of RNA and also by improving the reamplification method (Bonnet et al., 1998). In the first run, we used the stability of expression over two consecutive time points after blood feeding as a way of reducing the selection of false positives. Nevertheless, only two amplicons out of the nine studied showed an expression profile compatible with the selection criteria. When the reactions were performed in duplicate, as in the second experiment, all five amplicons selected had the expected expression profile.

One of the selected amplicons is similar to the Antryp1 gene (Müller et al., 1993). This is not surprising, as it encodes a protease involved in blood processing that is overexpressed after a blood meal (Billingsley and Hecker, 1991; Müller et al., 1993; Chege et al., 1996). We observed the regulation in female midguts. The expression level in the male midguts was similar to that observed in unfed female midguts, disagreeing with previous reports (Müller et al., 1993). Trypsin expression was downregulated by the presence of gametocytes in the blood meal at 18 h PBM, at which time parasites presumably reached the ookinete stage. Ookinetes of P. gallinaceum are sensitive to trypsin in vitro (Gass and Yeates, 1979): possibly, these mobile developmental stages are able to downregulate trypsin expression as a defence mechanism.

67Yde corresponds to a gene of unknown function cloned independently by A. J. Cornel et al. (unpublished) and G. Dimopoulos et al. (unpublished). This cDNA has thus been isolated twice by differential display analysis of mosquito midgut RNA (G. Dimopoulos et al.; this study), suggesting that its corresponding RNA is abundant in mosquito midgut cells.

We also identified 12 new genes of An. gambiae. Reflecting the intrinsic features of the differential display reverse transcription (DDRT) technique, the majority of the cDNA fragments isolated correspond to 3′ untranslated regions of RNA and, therefore, similarity searches with known sequences are not very informative. Nevertheless, five sequences shared similarity with sequences present in the databases.

Amplicon 34P2 is similar to profilin sequences: the highest similarity is with the Drosophila sequences. This amplicon is probably the profilin of An. gambiae. Profilin is a ubiquitous cytoplasmic protein in eukaryotic cells. Profilins regulate cytoskeleton actin dynamics and are also linked to major signal transduction pathways. Profilins bind at least three different ligands: actin, polyphosphoinositides and poly-l-proline (Machesky and Pollard, 1993). Interestingly, mosquitoes produce a proline-rich decapeptide, the so-called mosquito oostatic factor (Borovsky et al., 1990), which is able to block the intracellular locomotion of Listeria in cell cultures by titrating intracellular profilin (Southwick and Purich, 1995). In mosquitoes, this factor is produced by the ovaries and inhibits egg development as well as biosynthesis of trypsin and chymotrypsin-like enzymes in the midgut (Borovsky et al., 1990). Possibly, An. gambiae profilin titrates the oostatic factor in response to blood feeding or is involved in microorganism invasion. Profilin expression is constitutive in some models but is tightly regulated during development in a tissue- and isoform-dependent manner in others. The Drosophila profilin is encoded by two transcripts, one being expressed throughout the organism in both males and females and the second only in the ovary (Cooley et al., 1992). Southern blot analysis revealed that An. gambiae harbours two closely related profilin genes (data not shown). It is possible that corresponding transcripts are regulated differently by blood meals. The transcript we detected was downregulated by a parasite-free blood meal but upregulated when the blood meal contained both gametocytes and asexual stages of P. falciparum. It may be implicated in the locomotion of the parasite within midgut cells, or its upregulation may more simply reflect the stimulation of the digestion process by the presence of parasites (either gametocytes or asexual stages) in the blood meal.

Three amplicons are similar to 18S rRNAs in various mosquitoe species (Brown and Knudson, 1997; Beebe et al., 2000) and probably correspond to the 18S rRNA subunit of An. gambiae. Each has a specific expression pattern upon blood feeding (not shown), suggesting that they might correspond to different copies of 18S rRNA. In carcasses, only one is upregulated by the blood meal, although one might expect all of them to be upregulated, at least in carcasses, as reported for Ae. aegypti (Hotchkin and Fallon, 1987). As the An. gambiae genome harbours a large number of rDNA copies, the fragments we isolated might correspond to copies regulated in an unusual manner.

Differential gene expression in female mosquitoes fed on parasite-free blood and in male mosquitoes

Ingestion of a blood meal by female mosquitoes triggers two events: activation of the digestion process in the midgut and initiation of the vitellogenesis process in the fat body (Clements, 1992). Accordingly, sets of genes are presumably upregulated in the midgut and the fat body. We observed two such genes expressed in the midgut (the trypsin gene and 8P1) and two in the carcasses containing the fat body (24P2 and 36P2). Ingestion of a blood meal may also downregulate sets of genes. This could be for resource reallocation if the corresponding gene products are not required or concern genes involved in the negative regulation of genes required for blood digestion or vitellogenesis. Most of the genes we identified were regulated in this manner in the midgut, carcasses or both. Any modification of this basic pattern of regulation in mosquitoes fed on parasite-containing blood could be informative as far as the host–parasite interaction is concerned (see below).

All the genes identified were expressed in both males and females. However, the expression levels and patterns differed in the two sexes for all the genes, except 58Yde and 36P2. The last two may have similar functions in both males and females. For the others, the expression is generally higher in the female carcasses. This might reflect the physiological status of the females, which were all analysed as nulliparous females.

Ingestion of gametocytes and asexual stages of P. falciparum affect the expression of An. gambiae genes in the midgut

When a mosquito feeds on a gametocyte carrier, it usually ingests both asexual stages and gametocytes. The method we developed can discriminate between the effect of gametocytes and that of asexual stages. The expression of 12 of the 16 RNAs identified was affected by the presence of P. falciparum in the blood meal. Some RNAs were upregulated by gametocytes alone, others by gametocytes and asexual stages and others by asexual stages alone. Some were downregulated by gametocytes or asexual stages.

Plasmodium asexual stages do not develop further within the mosquito midgut. Their degradation during the digestion process probably overstimulates the digestive response. They (or their remnants) might also be perceived as ‘foreign’, presumably leading to the activation of some type of immune response. Thus, genes upregulated after the ingestion of asexual stages of P. falciparum may reflect the overstimulation of the digestion process. Indeed, the upregulation of most of the genes was higher early after the ingestion of parasites. Alternatively, some of them may encode elements of the mosquito defence system, or their upregulation may be a secondary effect of the stimulation of the mosquito immune system. Under our experimental conditions, the expression of several immune-responsive genes, such as Nos and defensin, was analysed to confirm that mosquito midguts were immunocompetent (not shown; R. Tahar and C. Bourgouin, unpublished). We report several genes upregulated by asexual stages but not by gametocytes. If they encode immune-responsive molecules or are activated as an indirect effect of the immune system activation, this result suggests that P. falciparum sexual stages maturing from the gametocytes may evade or even repress the immune response of its natural host. However, we cannot exclude the possibility that the absence of activation may reflect the low number of parasites present in the gametocyte-containing blood meals.

Plasmodium gametocytes do develop within the mosquito midgut. During the first 24 h after their uptake, they undergo several developmental transitions, leading to the production of the ookinetes that cross both the peritrophic matrix and the midgut epithelium before maturing into oocysts. There is some evidence that mosquito factors contribute to the sporogonic development of the parasite (Shahabuddin, 1998). The clearest evidence is that immunization of a vertebrate host with mosquito midgut leads to a diminution of the establishment and further development of the parasite, as described for various parasite–vector systems (Ramasamy and Ramasamy, 1990; Lal et al., 1994; Srikrishnaraj et al., 1995; Ramasamy et al., 1997). Only two factors have been identified so far: GAF, contributing to the exflagellation of male gametocytes (Nijhout, 1979;Garcia et al., 1997; Billker et al., 1998); and a trypsin that facilitates the passage of the ookinetes through the peritrophic matrix (Shahabuddin et al., 1993; 1996). Other mosquito factors probably limit the sporogonic development of the parasite. Indeed, parasite numbers fall sharply in the mosquito midgut (Vaughan et al., 1994; Gouagna et al., 1998). Recently, mosquito ‘immune-related molecules’ have been shown to contribute to this effect. Mosquitoes react to the presence of parasites by triggering humoral and cellular immune effectors (Richman and Kafatos, 1996; Dimopoulos et al., 2001). Some of these responses are elicited by Plasmodium spp. and include the activation of genes encoding antimicrobial peptides (Dimopoulos et al., 1997; 1998; Richman et al., 1997), NOS (Luckhart et al., 1998; Han et al., 2000), a protease inhibitor (Oduol et al., 2000) and activation of the prophenol oxidase cascade (Collins et al., 1986; Paskewitz et al., 1989).

Ingestion of P. falciparum gametocytes induced 58Yde, 67Yde and 70Yde. These may encode products important for the sporogonic development of P. falciparum in An gambiae. These RNA were downregulated by a parasite-free blood meal, suggesting that the ingestion of gametocytes modifies the normal effect of a blood meal. However, they may also be genes involved in host defence. Their specific regulation by sexual stages is consistent with An. gambiae having a stage-specific reactivity towards P. falciparum, as described previously towards P. berghei (Richman et al. 1997). One RNA (55Yde) deserves particular attention, as its expression was selectively downregulated by gametocytes. 55Yde might encode a product detrimental to the development of P. falciparum. As the invasive stages of the parasite presumably activate different sets of genes in the mosquito, it is also possible that, to maintain the homeostasis status, other genes, whose products are less important in such a situation, are expressed at a reduced level

In conclusion, we report for the first time that the ingestion of gametocytes of P. falciparum selectively affects the expression of An. gambiae midgut genes in a developmental stage-dependent manner. Further study of these genes and their involvement in the development of P. falciparum in An. gambiae may lead to novel strategies to control the transmission of this deadly parasite.

Experimental procedures

Mosquitoes

Two laboratory-adapted strains of An. gambiae were used: the G3 strain and a Cameroon strain. Mosquitoes were reared at 26°C and 80% relative humidity under conventional procedures as reported elsewhere (Bonnet et al., 2000).

Experimental feeding on human blood samples with and without P. falciparum

Batches of 50 4-day-old female mosquitoes (Cameroon strain), starved of sugar for at least 12 h, were fed by membrane feeding, either on parasite-free blood from European donors or on blood containing P. falciparum collected from volunteers recruited during epidemiological studies in Cameroon (Bonnet et al., 2000). The parasitological data for the blood samples are described in Table 3.

Table 3.  Parasitological data for the blood samples used.
 Parasitological dataMosquito infection
Blood sampleGametocytes µl−1Asexual forms µl−1% InfectionOocyst means
  1. Samples A, B, C and D were used for the differential display analysis and samples B, C, E and F for the RT–PCR analysis. Mosquito infection was tested on separate batches of a minimum of 20 surviving mosquitoes 7 days after feeding by determining the percentage of infected mosquitoes per batch (% infection) and mean oocyst densities per infected midgut (oocyst means).

A4135146.6612.1
B820344.4410.3
C015 45500
D010 47500
E4427331.614.7
F028 50000

Midgut preparation and RNA extraction

Mosquito midguts were dissected in phosphate-buffered saline (PBS) at 4°C. They were opened by a longitudinal cut and extensively washed in PBS to remove the gut content and peritrophic matrix remnants. For the first DDRT–PCR, midguts (G3 strain mosquitoes) were collected from unfed females and from females fed on parasite-free blood. Twenty midguts were dissected every hour from 14 h to 28 h after feeding and pooled. Total RNA was extracted using the Micro-scale total RNA separator kit (Clontech) according to the manufacturer's instructions. Next, five pools of RNA were made by mixing an equal amount of RNA from three consecutive times after blood feeding. For the second DDRT–PCR, midguts were isolated from 20 mosquitoes 14 h after feeding on blood samples A, B, C or D (Table 3) or on parasite-free blood. RNA was extracted using the Tri Reagent kit (MRC) according to the manufacturer's instructions. Some control RNA samples were extracted using the Tri Reagent kit from whole midguts without removal of the contents. All RNA samples were treated with RNAse-free DNase I (Boehringer Mannheim).

Differential display

The technique was used as described by Liang and Pardee (1992) using a kit from Display Systems (Tandil). Reverse transcription was performed with M-MLV reverse transcriptase (Gibco BRL). After PCR amplification, the products were resolved on 6% polyacrylamide gels in non-denaturing conditions. Bands of interest were eluted from the gel (Reeves et al., 1995) and resuspended in 10 µl of Diethylpyrocarbonate (DEPC)-treated water. To isolate cDNAs regulated by a blood meal, the display was performed with 300 ng of RNA from each RNA pool, reverse transcribed with four downstream primers (dT11AA, dT11AC, dT11AG and dT11CA), and cDNA was amplified with these primers in combination with 24 upstream 10-mer primers. cDNA fragments found at two or more consecutive times after feeding but not in unfed midguts were selected. To isolate cDNA regulated by the presence of P. falciparum in the blood meal, 300 ng of RNA extracted from midguts of mosquitoes fed with parasite-carrying or parasite-free blood were reverse transcribed with three downstream primers (dT11VA, dT11VC and dT11VG), and cDNA was amplified with these primers and 11 upstream 10-mer primers. To improve the reproducibility of the display, each PCR reaction was performed in duplicate, and the display was performed for each of the three conditions (i.e. parasite-free blood, parasite-containing blood, with or without gametocytes) with two different batches of mosquitoes fed on different individuals. We selected bands showing a reproducible expression profile in the two PCRs and for the two sets of RNA used for each condition.

Reamplification, cloning, sequencing and sequence analysis

All selected cDNAs were reamplified either by direct reamplification (Liang and Pardee, 1992) or by transient ligation and thermal asymmetric PCR (Bonnet et al., 1998). Reamplification products were inserted into PCR-Script SK(+) (Stratagene), pCR2.1-TOPO or pZero-2.1 (Invitrogen) and used to transform Top10 Escherichia coli electrocompetent cells. Plasmid DNA was isolated using Quiaprep spin (Quiagen). Sequencing was performed on both strands, directly or after cloning, using manual sequencing (Pharmacia T7 polymerase kit) or automated sequencing (ABI automated sequencer; Perkin-Elmer). Algorithms blastx and blastn were used to determine nucleic acid and protein sequence similarity to sequences available in public databases. The gcg program (Wisconsin package) using the Prosit database was used to identify sequence motifs. Sequence data have been submitted to the GenBank database under the following accession numbers: AF348121AF348134.

RT-PCR

RT-PCR analysis was used to verify the expression profile. RT-PCR was performed using 1 µl of 50 µl total RNA, extracted from pools of 10 midguts and the PCR access kit (Promega) in a final volume of 25 µl. Amplification conditions were: a 45 min reverse transcription at 48°C followed by 2 min at 94°C, five touch-down cycles (30 s at 94°C, 45 s at 5°C above the Tm of the specific primers, 1 min at 68°C) followed by a number of cycles in the linear range of the amplification (30 s at 94°C, 45 s at the Tm of the specific primers, 1 min at 68°C) and a final 7 min elongation step (68°C). Products were analysed on 2% agarose gels in Tris borate buffer. Actin1D RNA was amplified in each sample as a standard (Salazar et al., 1994). All experiments were carried out at least twice with a few exceptions, as mentioned in the figure legends. Reaction products were quantified using the imagequant program (Molecular Dynamics) after scanning photography of gels. The 14 pairs of specific primers used were as follows:

8P1 (5′-TGTCTCAAGGAACCGAATAG) and (5′-ACGTAA TGAAGCCGAACAGC); 17P1 (5′-TGTCAGCCTGTAACTT CTCA) and (5′-ACACGCTAATATTGGGTCAC); 20P2 (5′-G GGCCTATACATGCTTGCTA) and (5′-AAAATATGAATTCT GGTTGC); 20P2b (5′-GCTGGTTGGTGTTGTTTC) and (5′-GCGATTGCTTTCGTTTGA); 23P2 (5′-CATTCATAAACTCC GTCCAA) and (5′-TAAGGATCACGCAACACTTC); 24P2 (5′-CACAACATCCCTCGCTCAGA) and (5′-TTACTGTACG GTGGTGATTG); 29P1 (5′-GCTAATATGAAGCAGACAAG) and (5′-CGATACAGGGAGGAACAAAT); 34P2 (5′-AGGG CAATAGAGCGAAGAGT) and (5′-TTAACCCACCCCCAAC AAGG); 36P2 (5′-AGTAGTGGCATTGGGTTGG) and (5′-TT TACTCCACCAAAGACAGT); 14Yde: (5′-CGTGGAAGGAGT TGATTATG) and (5′-TGGGATCGTAATTGCTTGTG); 55 Yde: (5′-AAGGGAAGGGTGTGTGTGTGT) and (5′-ATTGC GACTGCTCAATAG); 58Yde: (5′-GGGGGATGATATAGATA GGAA) and (5′-CCAGCGCAGAGCCATCCT); 67Yde: (5′-G GGGGAGAGAACAACAAC) and (5′-GCGGTCTGAATGGAT GGT); 70Yde: (5′-CGCAATCATAAAACCAAAATAC) and (5′-GCATCCGTATGTGTGTTCT).

Acknowledgements

We thank F. Rodhain in whose laboratory this work was performed. We are indebted to G. Milon and G. Langsley for their critical reading of the manuscript, and we are grateful to N. Ayad, I. Tchikangwa and R. Beyene for mosquito rearing. This project was supported by fellowships to S. Bonnet (MRT, Pasteur-Weizmann, CANAM), G. Prévot (MRT, Pasteur-Weizmann, FRM, CRG) and research funds from the Pasteur Institute and UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases.

Footnotes

  1. Present address: Institut Pasteur de la Guyane, 23 Avenue Pasteur, BP 6010, 97306 Cayenne Cedex, Guyane Française.

  2. The first two authors contributed equally to this work.

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