Daniela Boccolini

Experimental studies of susceptibility of Italian Aedes albopictus to Zika virus.

We report a study on vector competence of an Italian population of Aedes albopictus for Zika virus (ZIKV). Ae. albopictus was susceptible to ZIKV infection (infection rate: 10%), and the virus could disseminate and was secreted in the mosquitos saliva (dissemination rate: 29%; transmission rate: 29%) after an extrinsic incubation period of 11 days. The observed vector competence was lower than that of an Ae. aegypti colony tested in parallel.

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.

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 (≈ 6000 km). Analysis of the ITS2 data revealed one of the longest spacers yet found in anophelines (≈ 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.

Incidence of malaria and risk factors in Italian travelers to malaria endemic countries

BACKGROUND Imported malaria has been an increasing problem in Italy in the last three decades of the 1900s, representing the main risk for travelers visiting tropical and sub-tropical countries where malaria is endemic. Even though the total number of imported cases has been declining since 2000, malaria still represents the most frequent notifiable imported disease in Italy. The present study analyzes all the malaria cases reported in Italy in 2000-2006 in order to assess the trend of incidence over the time and reviewing the risk factors for travelers visiting malaria endemic countries. METHODS All 2000-2006 case report forms were analyzed. The incidence of malaria in Italian travelers was calculated by continent and by countries most visited, using data provided by the Ministry of Transportation. RESULTS Out of the 5219 malaria cases reported and confirmed in the study period five were autochthonous and 5214 imported, 1518 of which occurred in Italian citizen and 3696 in foreigners. Between 2000 and 2006 imported malaria cases fell from 977 to 630 respectively, with a total reduction of about 36%. Most of the cases were contracted in Africa (93%) and Plasmodium falciparum was the etiological agent in 83% of the cases, with an annual average fatality rate of about 0.5%. The average of the crude incidence rate (CIR) among Italians was calculated by continent for both global cases (gCIR) and for P. falciparum cases (pfCIR) resulting of 1.2/1000 and 0.9 for Africa, 0.08/1000 and 0.02 for Asia, 0.03/1000 and 0.003 for Central and South America, respectively. The gCIR by continent slightly but decreased constantly over the study period. DISCUSSION The different factors which may influence the risk of contracting malaria for travelers visiting endemic countries and the strategy to reduce completely the number of fatal cases were considered and discussed.

Experimental investigation of the susceptibility of Italian Culex pipiens mosquitoes to Zika virus infection.

We investigated the susceptibility of an Italian population of Culex pipiens mosquitoes to Zika virus (ZIKV) infection, tested in parallel with Aedes aegypti, as a positive control. We analysed mosquitoes at 0, 3, 7, 10, 14, 20 and 24 days after an infectious blood meal. Viral RNA was detected in the body of Cx. pipiens up to three days post-infection, but not at later time points. Our results indicate that Cx. pipiens is not susceptible to ZIKV infection.

Intrapopulation Polymorphism in Anopheles messeae (An. maculipennis Complex) Inferred by Molecular Analysis

Abstract We evaluated the internal transcribed spacer two (ITS2) sequence to detect intraspecific polymorphism in the Palearctic Anopheles maculipennis complex, analyzing 52 populations from 12 countries and representing six species. For An. messeae, two fragments of the cytochrome oxidase I (COI) gene were also evaluated. The results were compared with GenBank sequences and data from the literature. ITS2 analysis revealed evident intraspecific polymorphism for An. messeae and a slightly less evident polymorphism for An. melanoon, whereas for each of the other species, 100% identity was found among populations. ITS2 analysis of An. messeae identified five haplotypes that were consistent with the geographical origin of the populations. ITS2 seems to be a reliable marker of intraspecific polymorphism for this complex, whereas the COI gene is apparently uninformative.

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

Abstract.  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 4638 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.

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.

Genetic and phenotypic variation of the malaria vector Anopheles atroparvus in southern Europe.

BackgroundThere is a growing concern that global climate change will affect the potential for pathogen transmission by insect species that are vectors of human diseases. One of these species is the former European malaria vector, Anopheles atroparvus. Levels of population differentiation of An. atroparvus from southern Europe were characterized as a first attempt to elucidate patterns of population structure of this former malaria vector. Results are discussed in light of a hypothetical situation of re-establishment of malaria transmission.MethodsGenetic and phenotypic variation was analysed in nine mosquito samples collected from five European countries, using eight microsatellite loci and geometric morphometrics on 21 wing landmarks.ResultsLevels of genetic diversity were comparable to those reported for tropical malaria vectors. Low levels of genetic (0.004 <FST<0.086) and phenotypic differentiation were detected among An. atroparvus populations spanning over 3,000 km distance. Genetic differentiation (0.202 <FST<0.299) was higher between the sibling species An. atroparvus and Anopheles maculipennis s.s. Differentiation between sibling species was not so evident at the phenotype level.ConclusionsLevels of population differentiation within An. atroparvus were low and not correlated with geographic distance or with putative physical barriers to gene flow (Alps and Pyrenées). While these results may suggest considerable levels of gene flow, other explanations such as the effect of historical population perturbations can also be hypothesized.

Assessment of the risk of malaria re-introduction in the Maremma plain (Central Italy) using a multi-factorial approach

AbstractKeywords: Mosquito-borne diseases, Residual anophelism, Anopheles labranchiae, Vectorial capacity, Climatechange, Plasmodium falciparum, Experimental infectionBackgroundIn recent years, the increase in globalization [1], the risein the average temperature of the earth together with anincreasing frequency and intensity of extreme weatherevents, as storms, floods and droughts [2,3], and theenvironmental changes induced by human activities [4],have raised the concern about the possible introductionor reintroduction of Vector Borne Diseases in Countrieswhere these were absent or eradicated [5]. These con-siderations, coupled with the recent spread of somemosquito vector borne diseases in Europe [6,7] and theincreasing number of imported malaria cases recordedin the Continent [8] have renewed interest in the possi-ble reintroduction of malaria in Southern Europe [7-9],particularly in the countries facing the Western Medi-terranean Basin, where potential Anopheline vectors arestill present [10-13]. Moreover, in recent years auto-chthonous malaria cases have been sporadically reportedin Italy, France, Spain and Greece [14-20].In 2005, a five-year study was implemented in Italy, aswell as in other South European countries, with the aim toassess the status of the local potential malaria vectors andthe possible re-introduction of malaria transmission[21-25]. In Italy, the selected study area was the Maremmaplain, a region that was hyperendemic for malaria until60 years ago [26-28] and that more recently was recog-nized as the major “at risk” area for the malaria reintro-duction into Italy [14,29,30].In Maremma, after the malaria eradication campaign(1947-1951), Anopheles labranchiae, the main endophilicvector of the Anopheles maculipennis complex was dra-matically reduced in abundance. However, in subsequentyears, the species has progressively re-colonized most ofthe area coming back to substantial densities [31-33].This was mainly due to the introduction of intensive ricecultivation in the early 1970s. Since then, Maremma hasbeen subjected to continuous entomological surveillancethat was intensified after1997, when an autochthonousPlasmodium vivax malaria case, transmitted byAn. lab-ranchiae, occurred in the Province of Grosseto [14]. Thestudies carried out in the area since eradication, providesa database that allowed a follow-up the history of malariaand its vectors in Maremma over the past 60 years. Start-ing from the findings of the most recent entomologicaland environmental studies [23,34], the present study waschosen to evaluate the malariogenic potential of the areausing a multifactorial approach.Methods