Evaluation of vector competence for West Nile virus in Italian Stegomyia albopicta (=Aedes albopictus) mosquitoes.

West Nile virus (WNV) is a zoonotic arboviral pathogen transmitted by mosquitoes in a cycle that involves wild birds as reservoir hosts. The virus is responsible for outbreaks of viral encephalitis in humans and horses. In Europe, Culex pipiens (Diptera: Culicidae) is considered to be the main vector of WNV, but other species such as Stegomyia albopicta (=Aedes albopictus) (Diptera: Culicidae) may also act as competent vectors of this virus. Since 2008 human cases of WNV disease have been reported in northeast Italy. In 2011, new areas of southern Italy became involved and a first outbreak of WNV lineage 1 occurred on the island of Sardinia. On the assumption that a potential involvement of St. albopicta in WNV transmission cannot be excluded, and in order to evaluate the competence of this species for the virus, an experimental infection of an St. albopicta laboratory colony, established from mosquitoes collected in Sardinia, was carried out. The results were compared with those obtained in a colony of the main vector Cx. pipiens. The study showed St. albopicta collected on Sardinia to be susceptible to WNV infection, which suggests this Italian mosquito species is able to act as a possible secondary vector, particularly in urban areas where the species reaches high levels of seasonal abundance.

Probable autochthonous introduced malaria cases in Italy in 2009-2011 and the risk of local vector-borne transmission.

We describe two cases of probable autochthonous introduced Plasmodium vivax malaria that occurred in 2009 and 2011 in two sites of South-Central Italy. Although the sources of the infections were not detected, local transmission could not be disproved and therefore the cases were classified as autochthonous. Sporadic P. vivax cases transmitted by indigenous vectors may be considered possible in some areas of the country where vector abundance and environmental conditions are favourable to malaria transmission.

Impact of environmental changes and human-related factors on the potential malaria vector, Anopheles labranchiae (Diptera: Culicidae), in Maremma, Central Italy.

The Maremma Plain (central Italy) was hyper-endemic for malaria until the mid-20th century, when a national campaign for malaria elimination drastically reduced the presence of the main vector Anopheles labranchiae Falleroni. However, the introduction of rice cultivation over 30 yr ago has led to an increase in the An. labranchiae population and concern over possible malaria reemergence. We studied the impact of anthropogenic environmental changes on the abundance and distribution of An. labranchiae in Maremma, focusing on rice fields, the main breeding sites. Adults and larvae were collected in three main areas with diverse ecological characteristics. Data were collected on human activity, land use, and seasonal climatic and demographic variations. We also interviewed residents and tourists regarding their knowledge of malaria. Our findings showed that the most important environmental changes have occurred along the coast; An. labranchiae foci are present throughout the area, with massive reproduction strictly related to rice cultivation in coastal areas. Although the abundance of this species has drastically decreased over the past 30 yr, it remains high and, together with climatic conditions and the potential introduction of gametocyte carriers, it may represent a threat for the occurrence of autochthonous malaria cases. Our findings suggest the need for the continuous monitoring of An. labranchiae in the study area. In addition to entomological surveillance, more detailed knowledge of human-induced environmental changes is needed, so as to have a more complete database that can be used for vector-control plans and for properly managing emergencies related to autochthonous introduced cases.

[Assessment of malaria resurgence risk in Morocco. Study of the vectorial capacity of Anopheles labranchiae in a rice cultivation area in the north of the country]. / Risque de réémergence du paludisme au Maroc. Etude de la capacité vectorielle d'Anopheles labranchiae dans une zone rizicole au nord du pays.

To assess the malaria reintroduction risk in Morocco, we analyzed the malariogenic potential of a rice cultivation area in the north of the country. Our results showed that the receptivity of this area is very high during all the period of the rice cultivation, from May to October, the vectorial capacity of An. labranchiae, malaria vector in Morocco, is considerably high during the summer which corresponds to the rice cultivation period. The risk of autochthonous malaria resumption is important because of the possible presence of gametocytes carriers in the last malaria focus which is bordering the study area. The risk of a tropical malaria introduction is unimportant seen the low vulnerability of the area and the uncertain competence of its vectors considered. However, this risk must be considered with a more attention.

Chromosomal evidence of incipient speciation in the Afrotropical malaria mosquito Anopheles funestus.

The analysis of chromosomal polymorphism of paracentric inversions in anopheline mosquitoes has often been instrumental to the discovery of sibling species complexes and intraspecific genetic heterogeneities associated with incipient speciation processes. To investigate the population structure of Anopheles funestus Giles (Diptera: Culicidae), one of the three most important vectors of human malaria in sub-Saharan Africa, a three-year survey of chromosomal polymorphism was carried out on 4,638 karyotyped females collected indoors and outdoors from two villages of central Burkina Faso. Large and temporally stable departures from Hardy-Weinberg equilibrium due to significant deficits of heterokaryotypes were found irrespective of the place of capture, and of the spatial and temporal units chosen for the analysis. Significant linkage disequilibrium was observed among inversion systems on independently assorting chromosomal arms, indicating the existence of assortative mating phenomena. Results were consistent with the existence of two chromosomal forms characterized by contrasting degrees of inversion polymorphism maintained by limitations to gene flow. This hypothesis was supported by the reestablishment of Hardy-Weinberg and linkage equilibria when individual specimens were assigned to each chromosomal form according to two different algorithms. This pattern of chromosomal variability is suggestive of an incipient speciation process in An. funestus populations from Burkina Faso.

A microsatellite map of the African human malaria vector Anopheles funestus.

Microsatellite markers and chromosomal inversion polymorphisms are useful genetic markers for determining population structure in Anopheline mosquitoes. In Anopheles funestus (2N = 6), only chromosome arms 2R, 3R, and 3L are known to carry polymorphic inversions. The physical location of microsatellite markers with respect to polymorphic inversions is potentially important information for interpreting population genetic structure, yet none of the available marker sets have been physically mapped in this species. Accordingly, we mapped 32 polymorphic A. funestus microsatellite markers to the polytene chromosomes using fluorescent in situ hybridization (FISH) and identified 16 markers outside of known polymorphic inversions. Here we provide an integrated polytene chromosome map for A. funestus that includes the breakpoints of all known polymorphic inversions as well as the physical locations of microsatellite loci developed to date. Based on this map, we suggest a standard set of 16 polymorphic microsatellite markers that are distributed evenly across the chromosome complement, occur predominantly outside of inversions, and amplify reliably. Adoption of this set by researchers working in different regions of Africa will facilitate metapopulation analyses of this primary malaria vector.

[Note on Anopheles maculipennis complex in Morocco]. / Note sur le complexe Anopheles maculipennis au Maroc.

Anopheles belonging to Anopheles maculipennis complex, collected from February to June 2002 in eight provinces of Morocco (Khouribga, Taounate, Alhouceima, Chefchaouen, Fes, Khemisset, Kalaa Sraghna and Benslimane), were identified with characterization of the ribosomal DNA by PCR and ITS2 sequence analysis. The results of this study showed that all the identified specimens belong to the Anopheles labranchiae species.

[Systematics and biology of Anopheles vectors of Plasmodium in Africa, recent data]. / Systématique et biologie des anophèles vecteurs de Plasmodium en Afrique, données récentes.

Renewed interest in research on Plasmodium vectors in Africa and development of genetic and molecular biology techniques has been spearheaded by the WHO and the PAL+ program of the French research ministry. New findings have led to a better understanding of the systematics and biology of the main vector groups. The purpose of this article is to describe the newest data on the Anopheles gambiae complex and the M and S forms of An. gambiae s.s., on species in the An. funestus group and genetic polymorphism of An. funestus, on the two probable species in the An. moucheti complex, and on An. mascarenesis.

Anopheles sacharovi (Diptera: Culicidae): a reemerging malaria vector in the Ararat Valley of Armenia.

In 1994, the first indigenous case of malaria since the 1960s was reported in Armenia, and the number of cases quickly increased in the ensuing years. In 1998, a roll-back malaria program was developed to eliminate the recently established foci of malaria infection and to prevent the reestablishment of malaria in Armenia. As part of this program, we carried out entomological surveys to identify the potential malaria vectors in the Ararat Valley, the area where most of the indigenous malaria cases had been reported. In particular, we attempted to ascertain the presence of Anopheles sacharovi Favre, which is historically the most important malaria vector in Armenia yet which had not been reported since 1965. In 1998-2000, we collected adult mosquitoes and larvae in the city of Masis and in three rural villages of the Ararat Valley. Species identification of the members of the Anopheles maculipennis complex was performed through egg and larval morphology, heteroduplex analysis, and sequencing of the second internal transcribed spacer of ribosomal DNA. Two species of the complex were identified: An. sacharovi, found in all of the study sites, andAn. maculipennis s.s. Meigen, the most common species in the area. The reemergence of An. sacharovi in the study sites shows that the receptivity for malaria is still high in the Ararat Valley and, likely, in other regions of Armenia.

Malaria incidence and mortality in Italy in 1999-2000.

In 1999-2000, a total of 2060 malaria cases were reported by the ISS. Most of the patients took inappropriate treatments or did not have any prophylaxis. Ninety-three per cent became infected in African malarious countries, 4% in Asian countries, and 3% in Latin America. P. falciparum accounted for 84% of the cases, followed by P. vivax (8%), P. ovale (5%), and P. malariae (2%). Deaths corresponded to an annual case fatality rate of 0.3% in 1999 and 0.5% in 2000. In general, imported malaria cases reflect the number of Italian travellers who underestimate the infection risk in Asian and Latin American malarious countries and permanent residents of African origin who visit their relatives in their native countries.

Spatial and temporal variations of the chromosomal inversion polymorphism of Anopheles funestus in Senegal.

The polymorphism of paracentric inversions of An. funestus polytene chromosomes was studied along a transect in Senegal in order to assess their variations at the spatial and temporal level. There was an increase in the degree of chromosomal polymorphism from the West to South-East. At the geographical level the variations in inversion frequencies were highly significant whatever the chromosomal arm considered. However, the variations in the chromosomal inversion frequencies did not change significantly over either seasons or years, except for inversion 3b in the village of Dielmo. Such geographical variability within a relatively limited area, associated to temporal stability, suggest a restricted gene flow between the populations studied, probably due to discontinuities in the An. funestus distribution and to its bioecology.

Identification of the sibling species of the Anopheles maculipennis complex by heteroduplex analysis.

The group of anopheline mosquitoes referred to as 'Anopheles maculipennis complex' includes the most important malaria vectors of the Palearctic Western region. The species belonging to this complex, however, are difficult or impossible to distinguish by morphological characters. To differentiate sibling palearctic species belonging to this complex, interspecific differences in the ITS2 sequences were used to set up a rapid and sensitive diagnostic tool based on heteroduplex analysis. The relative heteroduplex mobility allowed the following seven species to be readily distinguished: An. atroparvus, An. labranchiae, An. maculipennis s.s. , An. martinius, An melanoon, An. messeae and An. sacharovi.

Chromosomal inversion polymorphism of Anopheles funestus from forest villages of South Cameroon.

The polymorphism of paracentric inversions of Anopheles funestus polytene chromosomes was studied in three villages (Nkoteng, Obala, and Simbock) located in a forest area of South Cameroon in order to analyse the genetic structure of these populations. A total of 146-210 chromatids could be scored from specimens collected over about two years. A low degree of chromosomal polymorphism was observed with two floating inversions on chromosomal arm 2 (2h and 2d), and three fixed arrangements on arms 3 (3a and 3b), and 5 (5a). Such arrangement of inversions has never been recorded elsewhere so far. The chromosome analysis indicated that the population from Obala was in Hardy-Weinberg equilibrium, whereas the samples from Nkoteng and Simbock showed a significant excess and deficit of heterokaryotypes, respectively. Significant differences in inversion frequencies on chromosomal arm 2 among villages lying in contrasting eco-climatic settings suggested an adaptive role of these inversions.

Mitochondrial and ribosomal internal transcribed spacer (ITS2) diversity of the African malaria vector Anopheles funestus.

The pattern of sequence variation in the mitochondrial DNA cytochrome b gene (cyt-b) and ribosomal DNA internal transcribed spacer 2 (ITS2) was examined in Anopheles funestus from Senegal and Burkina Faso in West Africa and Kenya in East Africa. From both West African countries, samples included individuals hypothesized to represent reproductively isolated taxa based upon different karyotypes and behaviours. Analysis of the cyt-b data revealed high haplotypic diversity (86%) and an average pairwise difference per site of 0.42%. Sequence variation was not partitioned by geographical origin or karyotype class. The most common haplotype was sampled across Africa (approximately 6000 km). Analysis of the ITS2 data revealed one of the longest spacers yet found in anophelines (approximately 704 bp). In common with other anopheline ITS2 sequences, this one had microsatellites and frequent runs of individual nucleotides. Also in common with data from other anopheline ITS2 studies, the An. funestus sequences were almost monomorphic, with only two rare polymorphisms detected. The results from both markers are congruent and do not support the hypothesis of reproductively isolated chromosomal taxa within An. funestus. Whether the lack of support by mitochondrial DNA (mtDNA) and ribosomal DNA (rDNA) sequences is a result of the recent origin of the presumptive taxa, or of the absence of barriers to gene flow, remains to be elucidated, using more rapidly evolving markers such as microsatellites.

Chromosomal and bionomic heterogeneities suggest incipient speciation in Anopheles funestus from Burkina Faso.

Sampling of day-resting Anopheles funestus was carried out in September-November 1991, October-December 1992, and November 1994 at two sites near Ouagadougou, Burkina Faso: the small village of Noungou where humans outnumber cattle, and the nearby Fulani settlement of Loumbila where cattle outnumber humans. Collections made inside human dwellings were supplemented in 1992 by outdoor-resting samples from artificial pit-shelters. Indoor-resting An. funestus were also collected in November 1992 and November 1994 in four villages of the Banfora area (southern Burkina Faso) and in a sudanese-sahelian village in northern Burkina Faso (Tougouri). Half-gravid female sub-samples were preserved in carnoy's fixative and processed for polytene chromosome analysis. The material from the two villages near Ouagadougou was analysed by ELISA to know (i) the human/animal origin of the blood meal; (ii) the infectivity for Plasmodium falciparum malaria; and (iii) the possible correlation of these parameters with chromosomal variants. A total of 1416 An. funestus could be scored for the whole polytenic complement, while the origin of the blood meal and circumsporozoite protein (CSP) positivity were asserted from 1076 and 1154 specimens, respectively. With a few exceptions, four polymorphic paracentric inversions (3a, 3b, 2a and 5a in decreasing order of frequency) were observed in all populations. Inversion 2s, whose breaking points include those of inversion 2a, was found only as the heterokaryotype 2s/+ floating at an overall frequency of 3.7% in two villages of the Banfora area and in the two sites near Ouagadougou. Two heterokaryotypes 2a/t out of 186 scored specimens were observed in different years from one village of the Banfora area. Wide variations in inversion frequencies were observed among the samples without consistent geographical or temporal clines. Highly significant departures from Hardy-Weinberg equilibrium were recorded for inversions 3a and 3b in most samples, with the alternative homokaryotypes (standard and inverted) significantly more frequent than expected. Conversely, inversion 5a was in Hardy-Weinberg equilibrium in most samples, whereas the 2a-s inversion system was intermediate between these extremes. However, a deficit of heterokaryotypes was apparent practically in all samples. Significantly higher frequencies of the standard homokaryotypes were recorded (i) in the exophilic samples collected in Loumbila for arrangement 3a; (ii) in the animal-fed sub-sample collected outdoors in Noungou vs. the parallel human-fed sub-sample for arrangements 2a-s, 3a, and 3b, or vs. the samples obtained from indoor catches in both Loumbila and Noungou in the case of inversion 3a; (iii) in the December CSP-negative sub-sample from Loumbila vs. the parallel CSP-positive sub-sample for arrangement 2a. A plausible working hypothesis is that An. funestus in Burkina Faso includes two taxonomic units, one of which is mainly monomorphic standard with most inverted arrangements floating at very low frequencies, and probably uniquely characterised by arrangement 2s, while the other taxon is nearly fixed for arrangement 3ab and polymorphic for all the other inversions at intermediate to high frequencies. The latter would be characterised by a higher vectorial capacity and would probably correspond to An. funestus s.s. from East Africa. About the former hypothetical taxon, its endophily and anthropophily appear less marked and its relationship with other members of the An. funestus subgroup will require specific investigations.

Malaria surveillance in Italy: 1997 analysis and 1998 provisional data.

Malaria is the commonest imported infectious disease in Italy. Malaria was endemic throughout much of the country until it was eradicated nearly 50 years ago. Since then, a malaria surveillance system has been set up to detect locally acquired cases that

Bionomical and cytogenetic heterogeneities of Anopheles funestus in Senegal.

Malaria transmission by Anopheles funestus was investigated from May 1994 to September 1997 in different locations from western to eastern Senegal along the northern border of The Gambia. 10515 A. funestus were captured on human volunteers or by indoor pyrethrum spraying. Circumsporozoite protein rates showed that A. funestus had a high infection rate, 2-7%, in the whole of the study area. Analysis of feeding behaviour showed great variation of anthropophilic rates from western Senegal, where populations were highly anthropophilic, to eastern Senegal, where they were much more zoophilic. In eastern Senegal many females captured in bedrooms had fed outside on horses. Polytene chromosome analysis showed that the general pattern of karyotype distribution is consistent with the hypothesis of 3 chromosomally differentiated populations of A. funestus. In samples from a central part of the study area, analysis showed lack of karyotype intergradation with a deficit of heterokaryotypes, suggesting the presence of 2 genetically differentiated populations in an area of sympatric.

Field studies on inversion polymorphism in Anopheles superpictus from southern Italy.

Polytene chromosome studies were carried out on various population samples of Anopheles superpictus from different localities in Southern Italy. More than 7,000 female specimens, mostly obtained from daytime collections of indoor resting mosquitoes, were successfully scored for nurse cell polytene chromosomes. Night biting samples were also examined in some localities. Only one chromosomal polymorphism, due to a paracentric inversion involving the central third of the 2L arm, was recorded in all samples. In all localities, the inverted 2La arrangement showed remarkably stable frequency although the populations examined were isolated from each other and at least some of them have presumably been subject to bottle neck in recent years because of insecticide treatments or ecological changes affecting the availability of breeding places. Departures from the Hardy-Weinberg expectations, indicating an excess of heterokaryotypes, were noted and critically analysed by comparing samples obtained simultaneously in the same locality from different cow sheds, from different sections of the same cow shed and from night and day catches in the same cow shed. The phenomenon was not found uniformly distributed among indoor resting samples: significant departures from the Hardy-Weinberg expectations were observed in some cow sheds but not in others situated nearby or even adjacent to them. These results did not support the hypothesis that the excess of heterokaryotypes is due to their greater longevity or to differential mortality in the preimaginal stages. It is suggested that different karyotypes may react differently to microclimatic specific conditions, since the Hardy-Weinberg disequilibrium was mostly observed in samples from resting sites that were more lit and subject to wider climatic variation.