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Mario Coluzzi is well known to readers of this Journal as an outstanding medical entomologist, malariologist, epidemiologist, and perhaps less well known as an evolutionary biologist. He made important advances in a number of sub-disciplines and, equally importantly, inspired a large number of researchers who continue active research enterprises on the forefront of confronting tropical diseases. After a long struggle with Parkinson’s Disease, Mario Coluzzi died in Rome on October 20, 2012.

One approach to investigate if human genetic variation influences the selection of Plasmodium falciparum drug resistance is to compare the frequency of resistant infections among human populations differing in their genetic background and living in the same epidemiological context. A further complementary approach consists in comparing drug resistance among subjects differing for genes involved in drug metabolism. Here we report, from malariological surveys performed in Burkina Faso, that the prevalence of P. falciparum chloroquine-resistant infections (pfcrt 76T and/or pfmdr1 86Y alleles) differs among sympatric ethnic groups, being higher in the Mossi and Rimaibe groups than in the Fulani group (odds ratio [OR], 2.24; 95% confidence interval [CI], 1.27-3.92; P = .007). The association analysis revealed that the human CYP2C8*2 variant, known to determine a poor drug metabolizer phenotype, was associated with P. falciparum chloroquine-resistant infections (OR, 1.66; 95% CI, 1.13-2.43; P = .008). This variant is more frequent in the Mossi-Rimaibe group (23.7% ± 1.4%) than in the Fulani group (9.9% ± 2.5%; P = .0003). This study provides an example of how host genetic variation may influence the selection dynamics of a pathogen's drug resistance.

The patchy geographical distributions of classic Kaposi's sarcoma (KS) and human herpesvirus type 8 (HHV-8), better known as Kaposi's sarcoma-associated herpesvirus (KSHV) remain unexplained. It has been proposed that certain species of bloodsucking insects (‘promoter arthropods') promote the reactivation of HHV-8/KSHV and facilitate both HHV-8/KSHV transmission and KS development. This hypothesis was tested by sampling the presence and density of human-biting Diptera with CDC light traps in two areas of Sardinia with contrasting incidence rates of classic KS. In total, 11 030 specimens (99.9% sandflies and 0.1% mosquitoes) belonging to 10 species were collected from 40 rural sites. Five of these species are considered to be possible promoter arthropods because of the irritation their bites cause: Phlebotomus perniciosus Newstead; Phlebotomus perfiliewi Parrot (Diptera: Psychodidae); Aedes berlandi Seguy; Culiseta annulata (Schrank) and Culex theileri Theobald (Diptera: Culicidae). Five species are probable ‘non-promoters' because their bites are not particularly irritating: Culiseta longiareolata (Macquart); Culex pipiens s.l.; Anopheles algeriensis Theobald; Anopheles maculipennis s.l., and Anopheles plumbeus Stephens. A significant correlation was found between the geographical distribution of promoter arthropods and incidence rates of KS (Spearman's r = 0.59,P < 0.01). Promoter arthropods were more likely to be caught in areas with cutaneous leishmaniasis and a past high prevalence of malaria, and in areas of limestone, acid volcanic soil and cereal cultivation. The study supports the association between promoter arthropods and classic KS, which may explain the geographic variability of KS and HHV-8/KSHV, and highlights the links with a number of variables previously associated with the incidence of KS.

Patterns of endemicity of human herpesvirus 8 (HHV8) are still undefined in some European populations, such as those from Western Balkan countries. Serum samples from 605 human immunodeficiency virus-seronegative subjects (299 Albanians and 306 Kosovars) were tested for the presence of HHV8 antibodies to a capsid-related open reading frame (ORF65)-encoded protein and a latency-associated nuclear antigen (LANA) to determine HHV8 seroprevalence in populations from Albania and from the Kosovo region of former Yugoslavia. Levels of co- circulation with hepatitis A (HAV) and hepatitis B (HBV) viruses were also determined. HHV8 antibodies to at least one of the two antigens were detected in 28.8% of Albanians and 18% of Kosovars. The seroprevalence of HHV8 was found to be 25.0 and 16.8% in Albanian and Kosovar children (

Field-collected specimens of all known taxa in the Anopheles gambiae complex were analyzed on the basis of chromosome inversions with reference to a standard polytene chromosome map. The phylogenetic relationships among the seven described species in the complex could be inferred from the distribution of fixed inversions. Nonrandom patterns of inversion distribution were observed and, particularly on chromosome arm 2R, provided evidence for genetically distinct populations in A. gambiae , A. arabiensis , and A. melas . In A. gambiae from Mali, stable genetic differentiation was observed even in populations living in the same region, suggesting a process of incipient speciation which is being confirmed by studies with molecular markers. The possible role of chromosome differentiation in speciation of the A. gambiae complex and in the emergence of distinct chromosomal forms within the nominal species is discussed in relation to human malaria.

Anopheles gambiae is the principal vector of malaria, a disease that afflicts more than 500 million people and causes more than 1 million deaths each year. Tenfold shotgun sequence coverage was obtained from the PEST strain of A. gambiae and assembled into scaffolds that span 278 million base pairs. A total of 91% of the genome was organized in 303 scaffolds; the largest scaffold was 23.1 million base pairs. There was substantial genetic variation within this strain, and the apparent existence of two haplotypes of approximately equal frequency (“dual haplotypes”) in a substantial fraction of the genome likely reflects the outbred nature of the PEST strain. The sequence produced a conservative inference of more than 400,000 single-nucleotide polymorphisms that showed a markedly bimodal density distribution. Analysis of the genome sequence revealed strong evidence for about 14,000 protein-encoding transcripts. Prominent expansions in specific families of proteins likely involved in cell adhesion and immunity were noted. An expressed sequence tag analysis of genes regulated by blood feeding provided insights into the physiological adaptations of a hematophagous insect.

Sulla base di una revisione della letteratura su human herpesvirus 8 (HHV8) e sarcoma di Kaposi (KS) e sulla distribuzione del KS in Italia (in particolare in Veneto), ipotizziamo che la puntura di insetti ematofagi sia un cofattore nella sieroconversione per HHV8 e quindi nella trasmissione del virus e forse nella patogenesi del KS. Potenti sostanze con azione immunomodulante, anticoagulante e vasodiatante, rilasciate con la saliva degli artropodi, sono responsabili di un microambiente tessutale idoneo alla replicazione virale. HHV8 in seguito elude il sistema di immunosorveglianza dell’ospite persistendo in maniera latente nell’organismo infettato. Nell’eventualita di deregolazione del sistema immune (invecchiamento), la risposta locale a nuove punture d’insetti potrebbe indurre l’attivazione del virus e preluderer all’insorgenza del KS. sebbene la sieroconversione per HHV8 dipenda dall’intimo contatto tra bambino e genitori o familiari sieropositivi, questa condizione da sola non e sufficiente a spiegare il pattern epidemiologico della distribuzione del KS. Il patogeno non verrebbe pero trasmesso dall’artropode, Il quale unicamente prepara il microambiente cutaneo per il virus. Il virus verrebbe tramesso con la saliva dei soggetti sieropositivi, in considerazione della frequente abitudine di leccare e succhiare la cute del bambino in corrispondenza delle sedi del prurito e delle lesioni da grattamento dovute alle punture. Proponiamo quindi di introdurre una nuova categoria di artropode importante in medicina, oltre a quelle gia definite di vettore biologico e meccanico, che potrebbe essere denominata «artropode promotre». L’artropode promotre dovrebbe apparenere ad una specie capace di indurre una reazione di lunga durata di piersensibilita immediata o ritardata, dovuta alle sostanze iniettate con la saliva. Questo tipo di reazione infiammatoria e attribuibile a molti artropodi ematofagi, non necessariamente associati all’uomo, come specie di flebotomi (Phlebotomus spp.), simulidi (Simulium spp.), moscerini pungenti (Culicoides spp.,Leptoconops spp.) e culicini (Ochlerotatus, Coquillettidia, eAedes). D’altra parte, i dati epidemiologici non supportano il coinvolgimento della comune e ubiquitaria zanzara domesticaCulex pipiens, ne della vicaria tropicaleCx quinquefasciatus, verosimilmente perche le loro punture rarmente inducono risposte infiammatorie di lunga durata. E altresi improbabile che i vettori afrotropicali di malaria (Anopheles gambiae eAn. funestus), le cimici, o i pidocchi svolgano alcun ruolo. La peculiare variabilita del gene di HHV8orf-K1 potrebbe essere dovuta all’adattamento del virus allo specifico microambiente creato dalla risposta immune dell’opsite agli antigeni salivari delle specie di artropodi ematofagi prevalenti in una data area geografica. L’ipotesi dell7rsartropode promotre potrebbe essere testata in modelli animali dato che esistono virus filogeneticamente molto vicini ad HHV8 negli scimpanze, gorilla, macachi e saimiri. Ulteriori informazioni potrebbero essere ottenute da indagini epidemiologiche, ad esempio comparando la sieroconversione di bambini con genitori sieropositivi nati e residenti rispettivamente in Africa e in Italia e ipotizzando una minore frequenza di punture di artropodi nel secondo gruppo. La prova piu convincente potrebbe essere ottenuta da esperimenti sul campo in un’area ad alta prevalenza di HHV8 reclutando bambini sieronegativi nati e allevati da genitori o familiari sieropositivi, e monitorando la sieroconversione in due gruppi randomizzati: il primo naturalmente esposto alle punture di insetti e l’altro protetto dalle punture con ogni mezzo disponibile (zanzariere impregnate di insetticida, uso di insetticidi nelle abitazioni, e repellenti). Questo secondo gruppo potrebbe essere ulteriormente suddivisio in due sottogruppi uno dei quali dovrebbe essere oggetto di un intervento di educazione sanitaria esortando le madri e altri familiari ad evitare di contaminare con la loro saliva la cute dei figli. Si dovrebbe infine considerare la possibilita che altri virus possano utilizzare, piu o meno sporadicamente, questa stressa tipologia di trasmissione non sessurale (per esempio il virus dell7rsepatite B).

Here we address the issue of the possible interplay between host genetic variation and the risk of acquiring Plasmodium falciparum drug-resistant strains. The involvement of human genetic variation as a possible co-factor in the selection and spread of P. falciparum drug resistance is a new tool in the study of malaria and possibly of other infectious diseases. The driving hypothesis of this approach is that parasite drug resistance could be affected both by ethnicity and human variability in the genes encoding for enzymes that metabolise antimalarials (cytochrome P450 liver enzymes). Understanding if parasite drug sensitivity is influenced and possibly modulated by human diversity can contribute to a better knowledge and control of the spread of drug resistance. So far, few studies have addressed this strategic issue. To explore this hypothesis we carried out an association analysis on 506 human/P. falciparum DNA samples from adult asymptomatic subjects belonging to three sympatric ethnic groups of Burkina Faso, an area of hyperendemic malaria in West Africa. Here we report that the prevalence of chloroquine-resistant infections (pfcrt 76T and/or pfmdr1 86Y) differs among sympatric ethnic groups, being higher in Mossi and Rimaibe compared to Fulani (OR: 2.24; 1.27-3.92; P = 0.007). Moreover, the human CYP2C8*2 variant, known to determine a poor drug metaboliser phenotype, is associated with P. falciparum chloroquine-resistant infections (OR: 1.66; 1.13-2.43; P = 0.008). The results strongly suggest that human genetic variation affects the dynamics of selection of parasite drug-resistance. We strongly believe that these observations are of general interest and may have important implications in public health.

Afrotropical malaria vectors of the Anopheles gambiae complex (Diptera: Culicidae), particularly An. gambiae sensu stricto, are attracted mainly to human hosts. A major source of human volatile emissions is sweat, from which key human-specific components are the carboxylic acids (E)- and (Z)-3-methyl-2-hexenoic acid and 7-octenoic acid. Electrophysiological studies on the antennae of An. gambiae s.s. showed selective sensitivity to these compounds, with a threshold at 10(-6) g comparable to that of known olfactory stimulants 1-octen-3-ol, p-cresol, isovaleric acid, and lower than threshold sensitivity to L-lactic acid and the synthetic mosquito repellent N,N-diethyltoluamide (DEET). A combination of the acids released at concentrations > 10(-5) g in wind tunnel bioassays significantly reduced the response to CO2, the major attractant released by human hosts, for strains of An. gambiae s.s. originating from East and West Africa. Field trials with odour-baited entry traps (OBETs) in Burkina Faso showed that 7-octenoic acid significantly increased (by 1.7-fold) the catch of females of An. gambiae sensu lato (comprising two sibling species: An. arabiensis Patton and An. gambiae s.s.) in OBETs baited with CO2, whereas combinations of the acids significantly reduced the catch in CO2-baited traps (by 2.1-fold) and in whole human odour-baited traps (by 1.5-fold). The pure (E) and (Z) geometric isomers of 3-methyl-2-hexenoic acid gave comparable results to the (EIZ) isomer mixture. These results provide the first experimental evidence that human-specific compounds affect the behaviour of highly anthropophilic An. gambiae s.l. mosquitoes. The compounds appear to inhibit the upwind flight' response to known long-range attractants, and may serve either to mask' the attractants present or, more probably, to 'arrest' upwind flight when mosquitoes arrive at a host under natural conditions. In the final approach to hosts, vectors are known to reduce their flight speed and increase their turning rate, to avoid overshooting the source. In our experimental apparatus, these changes in flight behaviour would reduce the number of mosquitoes entering the ports of the collection devices.

Fulani of Burkina Faso (West Africa) are a particularly interesting ethnic group because of their lower susceptibility to Plasmodium falciparum malaria as compared to sympatric populations, Mossi and Rimaibe. Moreover, the occurrence of a Caucasoid component in their genetic make-up has been suggested on the basis of their physical traits and cultural traditions even though this view was not supported by genetic studies. A total of 149 unrelated subjects (53 Mossi, 47 Rimaibe and 49 Fulani) have been typed for 97 HLA class I alleles with the amplification refractory mutation system/polymerase chain reaction (ARMS/PCR) technique. Mossi and Rimaibe data were pooled since none of the 42 statistically testable alleles exhibited a significant heterogeneity. These pooled gene frequencies were found to be very different from those of Fulani: a certain (P

We karyotyped and identified by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis Anopheles gambiae s.s. samples collected in several African countries. The data show the existence of two non-panmictic molecular forms, named S and M, whose distribution extended from forest to savannahs. Mosquitoes of the S and M forms are homosequential standard for chromosome-2 inversions in forest areas. In dry savannahs, S is characterized mainly by inversion polymorphisms typical of Savanna and Bamako chromosomal forms, while M shows chromosome-2 arrangements typical of Mopti and/or Savanna and/or Bissau, depending on its geographical origin. Chromosome-2 inversions therefore seem to be involved in ecotypic adaptation rather than in mate-recognition systems. Strong support for the reproductive isolation of S and M in Ivory Coast comes from the observation that the kdr allele is found at high frequencies in S specimens and not at all in chromosomal identical M specimens. However, the kdr allele does not segregate with molecular forms in Benin.

Patterns of DNA sequence variation in the ribosomal DNA (rDNA) second internal transcribed spacer (ITS2) and five unlinked single-copy nuclear loci were examined for evidence of reproductive isolation among four chromosomally recognized taxa of Anopheles gambiae from West Africa: Savanna, Bamako, Mopti and Forest, as well as sibling species An. arabiensis and An. merus. Included among the single-copy loci were three sequence-tagged random amplified polymorphic DNA (RAPD) loci, two of which (R15 and R37) had been reported as discriminating between Mopti and other chromosomal forms. Each of the five single-copy sequences were highly polymorphic in most samples. However, the R15 and R37 loci had no diagnostic value, and therefore are not recommended as tools in recognition of field-collected An. gambiae chromosomal forms. Although pairwise comparisons between species generally revealed significant levels of differentiation at all five loci, variation was not partitioned by chromosomal form within An. gambiae at any single-copy locus examined. The few exceptions to these trends appear related to a location either inside or nearby chromosomal inversions. At the tryptophan oxygenase locus inside inversion 2Rb, variation was structured only by inversion orientation and not by taxonomic designation even between An. gambiae and An. arabiensis, providing the first molecular evidence that the 2Rb inversion was transferred between species by introgressive hybridization. By contrast, the rDNA showed fixed differences between species and a difference diagnostic for Mopti, consistent with effective, if not complete, reproductive isolation. The apparent disagreement between the data from this locus and multiple single-copy loci within An. gambiae may be explained by the much lower effective population size of rDNA, owing to concerted evolution, which confers increased sensitivity at much shorter divergence times. Taken together with the accompanying reports by della Torre et al. (2001), Favia et al. (2001) and Gentile et al. (2001), our data suggest that neutral molecular markers may not have the sensitivity required to detect isolation between these recently established taxa.

Previous interethnic comparative studies on the susceptibility to malaria performed in West Africa showed that Fulani are more resistant to Plasmodium falciparum malaria than are sympatric ethnic groups. This lower susceptibility is not associated to classic malaria-resistance genes, and the analysis of the immune response to P. falciparum sporozoite and blood stage antigens, as well as non-malaria antigens, revealed higher immune reactivity in Fulani. In the present study we compared the expression profile of a panel of genes involved in immune response in peripheral blood mononuclear cells (PBMC) from Fulani and sympatric Mossi from Burkina Faso. An increased expression of T helper 1 (TH1)-related genes (IL-18, IFNγ, and TBX21) and TH2-related genes (IL-4 and GATA3) and a reduced expression of genes distinctive of T regulatory activity (CTLA4 and FOXP3) were observed in Fulani. Microarray analysis on RNA from CD4 + CD25 + (T regulatory) cells, performed with a panel of cDNA probes specific for 96 genes involved in immune modulation, indicated obvious differences between the two ethnic groups with 23% of genes, including TGFβ, TGFβRs, CTLA4, and FOXP3, less expressed in Fulani compared with Mossi and European donors not exposed to malaria. As further indications of a low T regulatory cell activity, Fulani showed lower serum levels of TGFβ and higher concentrations of the proinflammatory chemokines CXCL10 and CCL22 compared with Mossi; moreover, the proliferative response of Fulani to malaria antigens was not affected by the depletion of CD25 + regulatory cells whereas that of Mossi was significantly increased. The results suggest that the higher resistance to malaria of the Fulani could derive from a functional deficit of T regulatory cells.

We analyzed the humoral immune response to the amino- (amino acids 22-125) and carboxy-terminal (amino acids 289-390) non-repetitive domains of the Plasmodium falciparum circumsporozoite protein (Pf CSP) in individuals belonging to three west African ethnic groups (the Fulani, Mossi, and Rimaibe ´) living in the same conditions of hyperendemic transmission in a Sudan savanna area of Burkina Faso. Previous surveys conducted in the same area showed obvious interethnic differences in the susceptibility and immune reactivity to malaria, with the Fulani showing lower infection and disease rates and higher humoral responses to various P. falciparum antigens than sympatric ethnic groups. A total of 764 subjects (311 Mossi, 273 Rimaibe ´, and 180 Fulani) of all age classes were tested. The total mean 6 SE anti-(CSPf-N-term) and anti-(CSPf-C-term) seroprevalences were 65.6 6 1.7% and 57.0 6 1.8%, respectively. These seroprevalences were lower than that recorded in the same sample for the central (NANP)40 repetitive domain (88.3 6 1.2%). As previously reported for other P. falciparum antigens (Pf CSP- (NANP)40, thrombospondin-related anonymous protein, merozoite surface protein-1, Pf 155-ring-infected eryhtrocyte surface antigen, and Pf 332), in spite of similar exposure to malaria, the Fulani showed higher immune reactivity than sympatric populations for both antigens tested. Our results confirm the presence of B cell epitopes in the non-repetitive regions of the Pf CSP; moreover a further evidence of interethnic differences in the capacity to mount humoral responses against P. falciparum malaria was obtained. The assessment of the biological basis of interethnic hetero- geneities in the susceptibility and in the humoral immune responses to malaria appears relevant in the development of anti-malaria vaccines. The circumsporozoite protein (CSP) is considered to be the major surface antigen of malaria sporozoites. The main structural and antigenic properties of CSP are identical in all Plasmodium species; the molecule contains a species-specif- ic repetitive domain encompassing about 40% of the primary structure of the protein, which corresponds to the B cell im- munodominant epitope. 1 In Plasmodium falciparum,the cen- tral domain contains about 40 repeats of the tetrapeptide NANP plus 3-4 NVDP repeats. The central domain is flanked on both sides by sequences containing conserved regions in different Plasmodium species. 2 The carboxy-ter- minal conserved region (region II) bears a striking homology to a cell adhesion domain of thrombospondin 3,4 and to re- gions of several other adhesive proteins. 5-8 The involvement of CSP in the invasion of sporozoites into hepatocytes has been shown by Cerami and others. 9 The flanking non-repeat

Anopheles arabiensis , one of the two most potent malaria vectors of the gambiae complex, is characterized by the presence of chromosomal paracentric inversions. Elucidation of the nature and the dynamics of these inversions is of paramount importance for the understanding of the population genetics and evolutionary biology of this mosquito and of the impact on malaria epidemiology. We report here the cloning of the breakpoints of the naturally occurring polymorphic inversion 2Rd′ of A. arabiensis . A cDNA clone that cytologically mapped on the proximal breakpoint was the starting material for the isolation of a cosmid clone that spanned the breakpoint. Analysis of the surrounding sequences demonstrated that adjacent to the distal breakpoint lies a repetitive element that exhibits distinct distribution in different A. arabiensis strains. Sequencing analysis of that area revealed elements characteristic of transposable element terminal repeats. We called this presumed transposable element Odysseus . The presence of Odysseus at the junction of the naturally occuring inversion 2Rd′ suggests that the inversion may be the result of the transposable element’s activity. Characteristics of Odysseus ’ terminal region as well as its cytological distribution in different strains may indicate a relatively recent activity of Odysseus .

The efficiency of miniature CDC light-traps in catching West African malaria vectors was evaluated during two rainy seasons in a village near Ouagadougou, Burkina Faso. Traps were employed both indoors and outdoors using human baits protected by an insecticide-free mosquito-net and different sources of light. Indoors, light from incandescent bulbs increased the catch of Anopheles gambiae s.l. (mainly A. arabiensis Patton and the Mopti chromosomal form of A. gambiae s.s. Giles) and A. funestus Giles c. 2.5 times as compared to traps whose light bulb was removed. Conversely, the difference was not significant when a UV ‘Blacklight-blue’ fluorescent tube was compared to the incandescent bulb. Protecting the bait with a mosquito-net increased the catch c. 3 times for A. gambiae s.l. and c. 3.5 times for A. funestus. A prototype model of double bednet gave intermediate yields. Outdoors, the addition of incandescent bulbs to unlighted traps did not significantly increase the number of vectors caught, but the addition of the mosquito-net to the unprotected human bait did so by c. 1.5–4 times. Thus, the CDC light-trap hung close to a human sleeping under a bednet and fitted with an incandescent bulb, was considered the most practical and efficient in terms of numbers of vectors caught, consequently its indoor efficiency was compared to human landing catches on single collectors and estimated to be 1.08 times and density-independent. Outdoor light-trap catches were either not significantly correlated to biting collections (as for A. gambiae s.l.), or density-dependent in their efficiency (as for A. funestus); thus, they were not considered a reliable means for estimating malaria vector outdoor biting densities in this area. No difference was found in the parous rate of A. gambiae s.l. samples obtained with CDC light-traps and human landing collections.

It has been shown that insecticide-treated bed nets or curtains may reduce morbidity and mortality from malaria in hyper-holoendemic areas of sub-Saharan Africa. This protection could partially depend on the transitory imbalance between the anti-malaria immunity acquired by the population before the intervention and the lowered sporozoite load resulting from the anti-vector measure. To verify if the efficacy of the intervention is influenced by the baseline immune status of the population, we compared the protective effect of permethrin-impregnated curtains (PIC) against malaria infection among groups with different baseline levels of anti-malaria immunity. We analyzed the impact of PIC on the Plasmodium falciparum infection rate in two rural villages of Burkina Faso inhabited by three ethnic groups: the Fulani, Mossi, and Rimaibé. These have been previously shown to differ for several malariologic indices, with the Fulani being characterized by lower infection and disease rates and by higher immune response to P. falciparum with respect to the other ethnic groups. The PIC were distributed in June 1996 and their impact on malaria infection was evaluated in groups whose baseline levels of immunity to malaria differed because of their age and ethnic group. Age- and ethnic-dependent efficacy of the PIC was observed. Among the Mossi and Rimaibé, the impact (parasite rate reduction after PIC installation with respect to the pre-intervention surveys) was 18.8% and 18.5%, respectively. A more than two-fold general impact (42.8%) was recorded in the Fulani. The impact of the intervention on infection rates appears positively correlated with the levels of anti-malaria immunity. Since decreased transmission entails a reduction of immunity, the efficacy of the intervention in the long term cannot be taken for granted. The expected complementary role of a hypothetical vaccine is stressed by these results, which also emphasize the importance of the genetic background of the population in the evaluation and application of malaria control strategies.

The humoral immune response against synthetic peptides of two Plasmodium falciparum blood-stage antigens, Pf155/ring-infected erythrocyte surface antigen (RESA) (EENV)6 and Pf332 (SVTEEIAEEDK)2, in individ- uals belonging to three sympatric ethnic groups (Mossi, Rimaibe, and Fulani) living in the same conditions of hyperendemic transmission in a Sudan savanna area northeast of Ouagadougou, Burkina Faso were examined. The Mossi and Rimaibe are Sudanese Negroid populations with a long tradition of sedentary farming, while the Fulani are nomadic pastoralists partly settled and characterized by non-Negroid features of possible Caucasoid origin. A total of 764 subjects (311 Mossi, 273 Rimaibe, and 180 Fulani) were tested. A lower P. falciparum prevalence was observed in the Fulani of all age groups. The serologic results clearly indicate the existence of interethnic differences in the capacity to respond to these two P. falciparum antigens. The Mossi and Rimaibe showed similar responses, whereas the Fulani displayed consistently higher prevalences and levels of antibodies against both epitopes tested. The anti-(EENV) 6 and anti-(SVTEEIAEEDK)2 seroprevalences were 29.9% and 38.9% in Mossi, 29.7% and 39.2% in Rimaibe, 86.1% and 76.1% in Fulani (all P values of Fulani-Mossi and Fulani-Rimaibe comparisons K 0.001). Anti-RESA and anti-Pf332 antibody levels were approximately 65% (P K 0.001) and 45% (P K 0.001), respectively, higher in seropositive Fulani than in seropositive Mossi and Rimaibe, who showed very similar values. The observed differences cannot be explained in terms of interethnic heterogeneity of malaria exposure since these communities have lived in the same area for more than 30 years and the P. falciparum inoculation rate, measured during two consecutive years, was substantially uniform for the three ethnic groups. The possibility of remarkable heterogeneities in the capacity to mount immune responses against P. falciparum antigens among populations with different genetic backgrounds must be taken into account in the development of anti-malaria vaccines. The comparison of the response to Plasmodium falcipa- rum antigens among populations with different genetic back- grounds exposed to high and uniform transmission of the same parasite strains is one of the possible approaches to detect genetically based heterogeneities in the immune re- sponse to malaria. This approach has been recently applied to the study of the anti-circumsporozoite protein (CSP), anti-thrombospondin-related anonymous protein (TRAP), and anti-merozoite surface antigen-1 (MSA-1) immune re- sponses

The role of odors in mosquito host preferences was studied in a village near Ouagadougou, Burkina Faso. Two odor-baited entry-traps were put beside one another and a choice of host odor-laden air was blown out of them. Odors of a human and a calf (of similar mass) were drawn from two tents in which each was separately concealed. Allowances were made for trap position, differences in human-subject attractiveness, CO2 levels, and trap contamination with alternative host odors. Choices for the human-baited trap greater than the 0.5 random expectation were made by Anopheles gambiae s.l. (0.96) and An. pharoensis (0.68). The choices for the human-baited trap of Culex antennatus were significantly lower than 0.5 (0.25), whereas for the Cx. decens species group (0.56), the difference was not significant. Interpretation of the latter result was complicated by the significant effect of CO2 levels on the index. Species caught in low numbers but whose trap distribution showed a bias towards the human-baited trap were An. funestus (total numbers in the human-baited trap to the calf-baited trap = 9:0), Mansonia africana (17: 1), Aedes dalzieli (22:4), and Ae. hirsutus (13:1); species showing bias towards the calf-baited trap were An. rufipes (0:11), Cx. duttoni (0:17), and Cx. nebulosus (2:35). Mansonia uniformis was the only species distributed randomly between the two traps. Molecular identification of the An. gambiae s.l. samples revealed a marked difference in trap distribution: for the human-baited trap the ratio was 52% An. arabiensis to 48% An. gambiae s.s.; for the calf-baited trap, it was 92% An. arabiensis to 8% An. gambiae s.s.

Three chromosomal forms of Anopheles gambiae s.s., designated as Bamako, Mopti and Savanna, were studied for diagnostic PCR assays based on the analysis of the X-linked ribosomal DNA (rDNA). The study was performed on a 1.3 kb fragment containing part of the 28S coding region and part of the intergenic spacer region. The amplified material was cut with fourteen restriction enzymes to detect Restriction Fragment Length Polymorphisms (RFLPs). The enzymes Tru9I and HhaI produced patterns of DNA bands which differentiated Mopti from Savanna and Bamako; moreover, a distinct 'hybrid' pattern was recognized in the F1 female progeny from the cross of Mopti with either one of the other two chromosomal forms. The diagnostic significance of the PCR-RFLP assay was verified on 203 karyotyped females from field samples collected in two villages in Mali and one village in Burkina Faso. Agreement was observed between the chromosomal and the molecular identifications. No 'hybrid' molecular patterns were detected even among carriers of rare heterokaryotypes hypothetically produced by crosses between Mopti and Savanna. The results confirm previous observations indicating barriers to gene flow within An. gambiae s.s. and supporting the specific status of the taxonomic units proposed on cytogenetic ground.

The Anopheles gambiae complex includes the major vectors of malaria in sub-Saharan Africa where >80% of all world-wide cases occur. These mosquitoes are characterized by chromosomal inversions associated to the speciation process and to intraspecific ecological and behavioral flexibility. It has been postulated that introgressive hybridization has selectively transferred inversions on the second chromosome between A. gambiae and A. arabiensis, the two most important vectors of malaria. Here we directly test this hypothesis with laboratory experiments in which hybrid populations were established and the fate of chromosomal inversions were followed. Consistent with the hypothesis, “foreign” X chromosomes were eliminated within two generations, while some “foreign” second chromosomes persisted for the duration of the experiments and, judging from the excess of heterozygotes, established stable heterotic polymorphisms. Only those second chromosome inversions found naturally in the species could be introgressed.

Mosquito responses to carbon dioxide were investigated in Noungou village, 30 km northeast of Ouagadougou in the Sudan savanna belt of Burkina Faso, West Africa. Species of primary interest were the main malaria vectors Anopheles gambiae s.s. and An.arabiensis, sibling species belonging to the An.gambiae complex. Data for An.funestus, An.pharoensis, Culex quinquefasciatus and Mansonia uniformis were also analysed. Carbon dioxide was used at concentrations of 0.04-0.6% (cf. 0.03% ambient concentration) for attracting mosquitoes to odour-baited entry traps (OBETs). The "attractiveness' of whole human odour was also compared with CO2 emitted at a rate equivalent to that released by the human bait. In a direct choice test with two OBETs placed side-by-side, the number of An.gambiae s.l. entering the trap with human odour was double the number trapped with CO2 alone (at the human equivalent rate), but there was no significant difference between OBETs for the other species of mosquitoes. When OBETs were positioned 20 m apart, again CO2 alone attracted half as many An.gambiae s.l. and only 40% An.funestus, 65% Ma.uniformis but twice as many An.pharoensis compared to the number trapped with human odour. The dose-response for all mosquito species was essentially similar: a linear increase in catch with increasing dose on a log-log scale. The slopes of the dose-response curves were not significantly different between species, although there were significant differences in the relative numbers caught. If the dose-response data are considered in relation to a standard human bait collection (HBC), however, the behaviour of each species was quite different. At one extreme, even the highest dose of CO2 did not catch more An.gambiae s.l. than one HBC. At the other extreme, the three highest doses of CO2 caught significantly more Ma.uniformis than did one HBC. An.pharoensis and Cx quinquefasciatus showed a threshold response to CO2, responding only at doses above that normally released by one man. An.funestus did not respond to CO2 alone at any dose in sufficient numbers to assess the dose response. Within the An.gambiae complex, An.arabiensis "chose' the CO2-baited trap with a higher probability than An.gambiae s.s. Also An.arabiensis, the less anthropophilic of the two species, was more abundant in CO2-baited OBETs than in human bait collections.

Several kinds of traps are available for the collection of Culicidae species creating nuisance problems and/or a potential risk of pathogen transmission. The choice of the most appropriate sampling device should take into consideration the objective of the monitoring activity (e.g., faunistic research, vector control evaluation, arbovirus surveillance, etc.), the ecological and behavioral characteristics of the target mosquito species, and the ecology of the sampling areas. However, there are few factual criteria technical personnel can rely on to choose the most suitable sampling method, particularly when the targets are represented by mosquito species in temperate areas. We carried out a Latin square experiment in three ecologically different settings in Mantua municipality (northern Italy) to compare the performance of four different traps targeting host-seeking mosquitoes: two traps specifically designed for mosquito monitoring purposes (Centers for Disease Control and Prevention CO(2) trap and Biogents BG Eisenhans de Luxe trap) and two designed to reduce mosquito densities in outdoor domestic settings (Activa Acti Power Trap PV 440 and Activa Acti Power Trap MT 250 Plus). Overall, 1,930 specimens belonging to nine species were collected and differences in the performance of the four traps with reference to their ability to detect overall species diversity, as well as to collect single species, were highlighted. These observations, coupled with an analysis of the costs associated with the trap's purchase, operation, and servicing, provide useful indications for the implementation of mosquito monitoring in temperate areas.

Background The aim of this study was to investigate cytochrome P450 2C8*2 (CYP2C8*2) distribution and allele frequency in three populations from West and East Africa exposed to Plasmodium falciparum malaria. CYP2C8 enzyme is involved in the metabolism of the anti-malarials amodiaquine and chloroquine. The presence of the CYP2C8*2 defective allele has been recently associated to higher rate of chloroquine-resistant malaria parasites. Methods A total of 503 young subjects were genotyped for the single nucleotide polymorphism rs11572103 (A/T). Eighty-eight were from southern Senegal, 262 from eastern Uganda and 153 from southern Madagascar. The PCR-RFLP technique was used to discriminate the wild-type (A) from the defective allele (T). Results A CYP2C8*2 (T) allele frequency of 0.222 ± 0.044 was detected in Senegal, 0.105 ± 0.019 in Uganda and 0.150 ± 0.029 in Madagascar. Conclusions This study demonstrated that CYP2C8*2 allele is widespread in Africa. This allele occurs at different frequency in West and East Africa, being higher in Senegal than in Uganda and Madagascar. These data indicate that an important fraction of the populations analysed has a decreased enzymatic activity, thus being at higher risk for drug accumulation with two possible consequences: i) an exacerbation of drug-associated adverse side effects; ii) an increase of drug-resistance selection pressure on P. falciparum parasites.

Salivary proteins injected by blood feeding arthropods into their hosts evoke a saliva-specific humoral response which can be useful to evaluate exposure to bites of disease vectors. However, saliva of hematophagous arthropods is a complex cocktail of bioactive factors and its use in immunoassays can be misleading because of potential cross-reactivity to other antigens. Toward the development of a serological marker of exposure to Afrotropical malaria vectors we expressed the Anopheles gambiae gSG6, a small anopheline-specific salivary protein, and we measured the anti-gSG6 IgG response in individuals from a malaria hyperendemic area of Burkina Faso, West Africa. The gSG6 protein was immunogenic and anti-gSG6 IgG levels and/or prevalence increased in exposed individuals during the malaria transmission/rainy season. Moreover, this response dropped during the intervening low transmission/dry season, suggesting it is sensitive enough to detect variation in vector density. Members of the Fulani ethnic group showed higher anti-gSG6 IgG response as compared to Mossi, a result consistent with the stronger immune reactivity reported in this group. Remarkably, anti-gSG6 IgG levels among responders were high in children and gradually declined with age. This unusual pattern, opposite to the one observed with Plasmodium antigens, is compatible with a progressive desensitization to mosquito saliva and may be linked to the continued exposure to bites of anopheline mosquitoes. Overall, the humoral anti-gSG6 IgG response appears a reliable serological indicator of exposure to bites of the main African malaria vectors (An. gambiae, Anopheles arabiensis and, possibly, Anopheles funestus) and it may be exploited for malaria epidemiological studies, development of risk maps and evaluation of anti-vector measures. In addition, the gSG6 protein may represent a powerful model system to get a deeper understanding of molecular and cellular mechanisms underlying the immune tolerance and progressive desensitization to insect salivary allergens.

The Anopheles gambiae salivary gland protein 6 (gSG6) is a small protein specifically found in the salivary glands of adult female mosquitoes. We report here the expression of a recombinant form of the protein and we show that in vivo gSG6 is expressed in distal-lateral lobes and is secreted with the saliva while the female mosquito probes for feeding. Injection of gSG6 dsRNA into adult An. gambiae females results in decreased gSG6 protein levels, increased probing time and reduced blood feeding ability. gSG6 orthologs have been found so far only in the salivary glands of Anopheles stephensi and Anopheles funestus, both members of the Cellia subgenus. We report here the gSG6 sequence from five additional anophelines, four species of the An. gambiae complex and Anopheles freeborni, a member of the subgenus Anopheles. We conclude that gSG6 plays some essential blood feeding role and was recruited in the anopheline subfamily most probably after the separation of the lineage which gave origin to Cellia and Anopheles subgenera.

MALDI profiling and imaging mass spectrometry (IMS) are novel techniques for direct analysis of peptides and small proteins in biological tissues. In this work we applied them to the study of Anopheles gambiae antennae, with the aim of analysing expression of soluble proteins involved in olfaction perireceptor events. MALDI spectra obtained by direct profiling on single antennae and by the analysis of extracts, showed similar profiles, although spectra obtained through profiling had a richer ion population and higher signal to noise ratio. Male and female antennae showed distinct protein profiles. MALDI imaging experiments were also performed and differences were observed in the localization of some proteins. Two proteins were identified through high resolution measurement and top-down MS/MS experiments. A 8 kDa protein only present in the male antennae matched with an unannotated sequence of the An. gambiae genome, while the presence of odorant binding protein 9 (OBP-9) was confirmed through experiments of 2-DE, followed by MS and MS/MS analysis of digested spots. This work shows that MALDI MS profiling is a technique suitable for the analysis of proteins of small and medium MW in insect appendices, and allows obtaining data for several specimens which can be investigated for differences between groups. Proteins of interest can be identified through other complementary MS approaches.

We analysed by gas chromatography-mass spectrometry (GC-MS) and Gas Chromatography-Flame Ionization Detector (GC-FID) the epicuticular lipid profiles of field females of the major Afro-tropical malaria vector, Anopheles gambiae. The samples were collected in three villages in Burkina Faso (West Africa), where An. gambiae M and S molecular forms and An. arabiensis live sympatrically. The aim was to compare the cuticular hydrocarbon (CHC) composition of individual field specimens of these three taxa, to highlight possible differences among them. All the samples analysed by GC-MS (55 individuals and eight pools) were characterized by the same 48 CHCs and 10 oxygenated compounds. The 19 most abundant CHCs were quantified in 174 specimens by GC-FID: quantitative intra-taxon differences were found between allopatric populations of both An. arabiensis and S-form. Inter-taxa quantitative differences in the relative abundances of some hydrocarbons between pairs of sympatric taxa were also found, which appear to be mainly linked to local situations, with the possible exception of diMeC(35) between An. arabiensis and S-form. Moreover, MeC(29) shows some degree of differentiation between S- and M-form in all three villages. Possible causes of these differences are discussed.

We have examined Plasmodium falciparum gametocyte prevalence, density and their genetic complexity among children of 2 sympatric ethnic groups (Mossi and Fulani) in villages in Burkina Faso. The 2 groups are known to have distinct differences in their susceptibility and immune responses to malaria. We used RT-PCR and sequence-specific probes to detect and type RNA of the gametocyte-specific protein Pfs48/45. There were no differences in detection rates of asexual forms and gametocytes among the 2 groups, using PCR and RT-PCR, respectively. However, there were significant differences in densities of asexual forms and gametocytes, which were both higher among Mossi than Fulani. Both asexual forms and gametocyte densities were influenced by age and ethnicity. Multiple-clone infections with more than 1 gametocyte genotype were equally prevalent among Fulani and Mossi. These differences can most probably be attributed to genetic differences in malaria susceptibility in the 2 ethnic groups.

The 'promoter-arthropod' hypothesis, which postulates that exposure to the bites of certain species of haematophagous arthropods is an environmental risk cofactor linked to human herpes virus 8 (HHV-8) and Kaposi's sarcoma, was investigated in the Po River valley, northern Italy. The presence and density of adult female mosquitoes (Diptera: Culicidae) was determined by CDC light trap catches in two adjacent districts, at variance with respect to Kaposi's sarcoma incidence and HHV-8 seroprevalence. A total of 3910 specimens belonging to 11 species was collected in 34 rural sites (six municipalities) representative of the two districts. Five of these species are considered to be possible 'promoters' because of the irritation their bites cause humans: Aedes vexans (Meigen) and Ae. caspius (Pallas) (87% of sampled promoters), Culex modestus Ficalbi, Culiseta annulata (Schrank) and Coquillettidia richiardii (Ficalbi). Six are probable 'non-promoters': Cx. pipiens s.l., Cx. martinii Medschid, Anopheles claviger (Meigen), An. maculipennis s.l., An. plumbeus Stephens and Uranotaenia unguiculata Edwards. The density of promoters by site was correlated with the incidence rates of Kaposi's sarcoma at the district level (Pearson's r = 0.33, P = 0.06) and at the municipal level (r = 0.50, P< 0.01). Similar correlations emerged for non-promoters (r = 0.48, P< 0.01 and r = 0.42, P = 0.01, respectively). The density of promoters was higher than that of non-promoters in sites with livestock (odds ratio, OR = 2.8, 95% CI 2.2-3.6) and in municipalities with Kaposi's sarcoma cases (OR = 2.5, 95% CI 1.7-3.5). The study provides additional evidence of the association between the density of some mosquito species and Kaposi's sarcoma.

Chromosome rearrangements (such as inversions, fusions, and fissions) may play significant roles in the speciation between parapatric (contiguous) or partly sympatric (geographically overlapping) populations. According to the “hybrid-dysfunction” model, speciation occurs because hybrids with heterozygous chromosome rearrangements produce dysfunctional gametes and thus have low reproductive fitness. Natural selection will, therefore, promote mutations that reduce the probability of intercrossing between populations carrying different rearrangements and thus promote their reproductive isolation. This model encounters a disabling difficulty: namely, how to account for the spread in a population of a chromosome rearrangement after it first arises as a mutation in a single individual. The “suppressed-recombination” model of speciation points out that chromosome rearrangements act as a genetic filter between populations. Mutations associated with the rearranged chromosomes cannot flow from one to another population, whereas genetic exchange will freely occur between colinear chromosomes. Mutations adaptive to local conditions will, therefore, accumulate differentially in the protected chromosome regions so that parapatric or partially sympatric populations will genetically differentiate, eventually evolving into different species. The speciation model of suppressed recombination has recently been tested by gene and DNA sequence comparisons between humans and chimpanzees, between Drosophila species, and between species related to Anopheles gambiae , the vector of malignant malaria in Africa.

We compared malaria indicators among sympatric groups to study human heterogeneities in the response to Plasmodium falciparum malaria infection. Four cross-sectional surveys and two longitudinal surveys in two sympatric ethnic groups (Dogon and Fulani) in Mali were carried out from 1998 to 2000. Spleen and parasite rates were evaluated during the cross-sectional surveys and disease incidence was assessed during longitudinal surveys. In spite of similar sociocultural factors and entomologic inoculation rates between ethnic groups, the Fulani had a significantly higher spleen enlargement rate, lower parasite rate, and were less affected by the disease than the Dogon group, whose frequency of hemoglobin C was higher than that recorded among the Fulani group. The Fulani group had significantly higher levels of IgG and IgE against crude malaria antigen than the Dogon group, suggesting a role of anti-malaria antibodies in the immune protection seen in this group.

Regulatory regions driving gene expression in specific target organs of the African malaria vector Anopheles gambiae are of critical relevance for studies on Plasmodium-Anopheles interactions as well as to devise strategies for blocking malaria parasite development in the mosquito. In order to identify an appropriate salivary gland promoter we analysed the transactivation properties of genomic fragments located just upstream of the An. gambiae female salivary gland-specific genes AgApy and D7r4. An 800 bp fragment from the AgApy gene directed specific expression of the LacZ reporter gene in the salivary glands of transgenic Anopheles stephensi. However, expression levels were lower than expected and the transgene was expressed in the proximal-rather than in the distal-lateral lobes of female glands. Surprisingly, a promoter fragment from the D7r4 gene conferred strong tissue-specific expression in Drosophila melanogaster but only low transcription levels in transgenic An. stephensi. These results imply a certain conservation of gland-specific control elements between the fruit fly and the mosquito suggesting that an increased degree of complexity, probably connected to the evolution of haematophagy, underlies the regulation of tissue-specific expression in mosquito female salivary glands.

SUMMARYSalivary glands of blood-sucking arthropods contain a variety of compounds that prevent platelet and clotting functions and modify inflammatory and immunological reactions in the vertebrate host. In mosquitoes, only the adult female takes blood meals, while both sexes take sugar meals. With the recent description of the Anopheles gambiae genome, and with a set of∼3000 expressed sequence tags from a salivary gland cDNA library from adult female mosquitoes, we attempted a comprehensive description of the salivary transcriptome of this most important vector of malaria transmission. In addition to many transcripts associated with housekeeping functions, we found an active transposable element, a set of Wolbachia-like proteins, several transcription factors, including Forkhead, Hairy and doublesex, extracellular matrix components and 71 genes coding for putative secreted proteins. Fourteen of these 71 proteins had matching Edman degradation sequences obtained from SDS-PAGE experiments. Overall, 33 transcripts are reported for the first time as coding for salivary proteins. The tissue and sex specificity of these protein-coding transcripts were analyzed by RT–PCR and microarray experiments for insight into their possible function. Notably, two gene products appeared to be differentially spliced in the adult female salivary glands, whereas 13 contigs matched predicted intronic regions and may include additional alternatively spliced transcripts. Most An. gambiae salivary proteins represent novel protein families of unknown function, potentially coding for pharmacologically or microbiologically active substances. Supplemental data to this work can be found at http://www.ncbi.nlm.nih.gov/projects/omes/index.html#Ag2.

No longer a major public health concern in developed countries, malaria kills 1-3 million people annually, mostly children under the age of five in sub-Saharan Africa. In 1998, the WHO launched the Roll Back Malaria (RBM) drive to halve malaria mortality by 2010. This article contrasts the problems confronting RBM with the successful Italian drive to eradicate malaria between the late 19th and mid 20th centuries. The Italians employed education and applied socio-political will; however, ecological and socio-economic conditions in sub-Saharan Africa are more hospitable to the disease. RBM strategies should consider the Italian experience while awaiting a major scientific breakthrough necessary to achieve success.

Forty-eight cuticular hydrocarbons (CHCs) were characterized by gas chromatography-mass spectrometry from the epicuticular surface of the major Afrotropical malaria vector Anopheles gambiae. The hydrocarbons identified were 14 n-alkanes, 16 monomethyl alkanes, 13 dimethyl alkanes, 5 alkenes, with main-chain lengths ranging from C(17) to C(47), and the results are consistent with those from other Culicidae species. Qualitative differences were not observed between laboratory pools of three females and males, between different age-groups (0-16 days) and between single field specimens, whereas quantitative differences in CHC profiles were observed. Differences between sexes were more marked in individuals aged 0-2 days than in older ones. Both sexes undergo strong CHC profile changes with age, and individuals aged 0-2 days differ remarkably from the older ones. The possibility of exploiting these changes for estimating the age of mosquito was explored through multivariate analyses of the relative abundance of the compounds, using either the whole CHC profile or a subset of CHCs. Such a method allows us to assign more than 85% of females and 75% of males to the correct age-group. Although preliminary, these results show that the method is promising, as it has already been shown in Aedes aegypti and An. stephensi. The correct determination of the vector age (particularly in the case of the An. gambiae complex of sibling species) provides valuable information in malaria epidemiology and in evaluation of the effectiveness of vector control strategies. Further efforts will be made to validate this method on single specimens reared in seminatural conditions before being proposed to medical entomologists working in the Afrotropical region.

We have characterized Plasmodium falciparum genotypes among the Mossi and Fulani sympatric ethnic groups in villages in Burkina Faso during the rainy season. Differences in clinical malaria presentation and in immune responses to malaria occur between the two groups. Asexual parasite rate, density, and gametocyte rate were higher among the Mossi than the Fulani. There was no difference in frequencies of alleles of the P. falciparum merozoite surface protein 1 (msp-1), msp-2, and glutamate-rich protein (glurp) genes among the parasites in each group. However, there were significant differences in the mean number of P. falciparum clones in the two populations, with there being more in the Mossi than in the Fulani. This effect was especially marked in older children. These differences can most probably be attributed to genetic differences in immune responsiveness to malaria between the two ethnic groups.

Previous studies identified an allelic variant of the IL4 promoter region (IL4-589T) that appears to enhance the transcriptional activity of IL4, and is associated with increased IgE levels. Total serum IgE levels are elevated in malaria endemic regions, and higher in children with severe malaria. Here, we investigated the relationship of the IL4-589C/T polymorphism with severity of the disease in a case-control study of severe malaria in Burkina Faso, West Africa. No association between the IL4-589T and severe malaria was observed. No difference in Plasmodium falciparum-specific IgE was detected between severe and uncomplicated malaria patients. Among children with severe malaria, total IgE levels were significantly elevated in those carrying the IL4-589T allele (P = 0.018). In children with uncomplicated malaria, no significant difference was found. These results raise the possibility that there is a relationship between susceptibility to severe malaria, IgE production and genetic variation in the IL4 region, which merits further investigation in other epidemiological settings.

In Mali the Anopheles gambiae complex consists of An. arabiensis and Mopti, Savanna and Bamako chromosomal forms of An. gambiae s.s. Previous chromosomal data suggests a complete reproductive isolation among these forms. Sequence analysis of rDNA regions led to the characterization of two molecular forms of An. gambiae, named M-form and S-form, which in Mali correspond to Mopti and to Savanna/Bamako, respectively, while it has failed so far to show any molecular difference between Savanna and Bamako. The population structure of An. gambiae s.l. was analysed in three villages in the Bamako and Sikasso areas of Mali and the frequency of pyrethroid resistance of the knock-down resistance (kdr) type was calculated. The results show that the kdr allele is associated only with the Savanna form populations and absent in sympatric and synchronous populations of Bamako, Mopti and An. arabiensis. This is the first molecular indication of barriers to gene flow between the Bamako and Savanna chromosomal forms. Moreover, analyses of specimens collected in the Bamako area in 1987 show that the kdr allele was already present in the Savanna population at that time, and that the frequency of this allele has gradually increased since then.

The incidence of Kaposi's sarcoma was estimated in the Veneto Region, Italy (age ≥50; 1990-96). Rates were higher in the coast and alpine valleys; in the latter there was an excess of cases for both sexes combined (SIR = 191.1; CI = 113.2-302.0). The hypothesis that birthplace/residency in areas abundant with bloodsucking insects may be a risk factor is discussed.

In two formerly malarious parts of Italy, age-related seroprevalence rates of Kaposi's sarcoma-associated herpesvirus [human herpesvirus 8 (KSHV/HHV8)] were determined from local blood donors and correlated with periods of vector control during anti-malaria campaigns. In Veneto, decreased KSHV/HHV8 seroprevalence in the 1951–1955 birth cohort coincides with the peak of DDT house-spraying. In Sardinia, where larviciding augmented indoor DDT-spraying, a significant drop of KSHV/HHV8 seroprevalence between 1945 and 1950 and 1951–1955 birth cohorts (P = 0.0046) coincides with suppression of the malaria vector Anopheles labranchiae Falleroni (Diptera: Culicidae). These results are consistent with age-related association between KSHV/HHV8 seroprevalence rates in native/resident populations and the density of malaria vectors in Veneto and Sardinia. This example supports our ‘promoter arthropod’ hypothesis on the role of haematophagous insects [putatively blackflies (Simuliidae), sandflies (Phlebotominae) and biting midges (Ceratopogonidae), as well as mosquitoes] when their bites induce hypersensitivity and immunosuppression, potentiate KSHV/HHV8 transmission via human saliva (when insect bite lesions are licked by another person whose saliva carries the virus) and may facilitate Kaposi's sarcoma.

Restrictions to gene flow among molecular forms of the mosquito Anopheles gambiae sensu stricto reveal an ongoing speciation process affecting the epidemiology of malaria in sub-Saharan Africa.