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 (unpublished) and G. Dimopoulos et al. (unpublished).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.