Beniamino Caputo

Study of Aedes albopictus dispersal in Rome, Italy, using sticky traps in mark-release-recapture experiments

We report the results of three mark–release–recapture experiments carried out in an urban area in Rome, Italy, to study the active dispersal of Aedes albopictus (Diptera: Culicidae). The 4.3% recapture rate obtained supports the use of sticky traps in MRR experiments to study the dispersal of Ae. albopictus females. Most fluorescent dust‐marked females were recaptured at the gravid stage at 50–200 m from the release sites during the first 9 days after release. The average of daily‐MDTs (Mean Distance Traveled) was 119 m and the maximum observed distance travelled ranged from 199 m to 290 m in the three replicates. These data provide the first information about the dispersal of Ae. albopictus in a temperate European area and appear to be consistent with the few data available on this subject from other urban areas, where dispersal was constrained by physical barriers. Although caution should be taken in generalizing these results, they should be considered when planning control activities in urban areas in Italy, as well as in other European countries. This is particularly relevant if control is intended to interrupt pathogen transmission in cases of possible arbovirus epidemics, such as the Chikungunya outbreak that occurred in Ravenna, Italy in 2007.

Anopheles gambiae complex along The Gambia river, with particular reference to the molecular forms of An. gambiae s.s.

BackgroundThe geographic and temporal distribution of M and S molecular forms of the major Afrotropical malaria vector species Anopheles gambiae s.s. at the western extreme of their range of distribution has never been investigated in detail.Materials and methodsCollections of indoor-resting An. gambiae s.l. females were carried out along a ca. 400 km west to east transect following the River Gambia from the western coastal region of The Gambia to south-eastern Senegal during 2005 end of rainy season/early dry season and the 2006 rainy season. Specimens were identified to species and molecular forms by PCR-RFLP and the origin of blood-meal of fed females was determined by ELISA test.ResultsOver 4,000 An. gambiae s.l. adult females were collected and identified, 1,041 and 3,038 in 2005 and 2006, respectively. M-form was mainly found in sympatry with Anopheles melas and S-form in the western part of the transect, and with Anopheles arabiensis in the central part. S-form was found to prevail in rural Sudan-Guinean savannah areas of Eastern Senegal, in sympatry with An. arabiensis. Anopheles melas and An. arabiensis relative frequencies were generally lower in the rainy season samples, when An. gambiae s.s. was prevailing. No large seasonal fluctuations were observed for M and S-forms. In areas where both M and S were recorded, the frequency of hybrids between them ranged from to 0.6% to 7%.DiscussionThe observed pattern of taxa distribution supports the hypothesis of a better adaptation of M-form to areas characterized by water-retaining alluvial deposits along the Gambia River, characterized by marshy vegetation, mangrove woods and rice cultivations. In contrast, the S-form seems to be better adapted to free-draining soil, covered with open woodland savannah or farmland, rich in temporary larval breeding sites characterizing mainly the eastern part of the transect, where the environmental impact of the Gambia River is much less profound and agricultural activities are mainly rain-dependent. Very interestingly, the observed frequency of hybridization between the molecular forms along the whole transect was much higher than has been reported so far for other areas.ConclusionThe results support a bionomic divergence between the M and S-forms, and suggest that the western extreme of An. gambiae s.s. geographical distribution may represent an area of higher-than-expected hybridization between the two molecular forms.

Host-feeding patterns of aedes albopictus (Diptera: Culicidae) in urban and rural contexts within Rome province, Italy

Knowledge of the frequency of contact between a mosquito species and its different hosts is essential to understand the role of each vector species in the transmission of diseases to humans and/or animals. However, no data are so far available on the feeding habits of Aedes albopictus in Italy or in other recently colonized temperate regions of Europe, due to difficulties in collecting blood-fed females of this diurnal and exophilic species. We analyzed Ae. albopictus host-feeding patterns in two urban and two rural sites within the area of Rome (Italy). Ae. albopictus was collected using sticky-traps and the blood-meal origin of 303 females was determined by direct dot-ELISA. The blood-fed sample, although representing only 4% of the total Ae. albopictus collected, demonstrates the useful application of sticky-trap in studying the feeding behavior of the species. The human blood index was significantly different among sites, ranging from 79-96% in urban sites to 23-55% in rural sites, where horses and bovines represented the most bitten hosts. The results obtained confirm the plastic feeding behavior shown by Ae. albopictus in its original range of distribution and highlights the high potential of this species as a vector of human pathogens in urban areas of Italy, where both humans and the mosquito itself may reach very high densities.

The "auto-dissemination" approach: a novel concept to fight Aedes albopictus in urban areas.

Background The main constraint to the fight against container-breeding mosquito vectors of human arboviruses is the difficulty in targeting the multiplicity of larval sources, mostly represented by small man-made water containers. The aim of this work is to assess the feasibility of the “auto-dissemination” approach, already tested for Aedes aegypti, as a possible alternative to traditional, inefficient control tools, against Ae. albopictus in urban areas. The approach is based on the possibility that wild adult females, exposed to artificial resting sites contaminated with pyriproxyfen, can disseminate this juvenile hormone analogue to larval habitats, thus interfering with adult emergence. Methodology We carried out four field experiments in two areas of Rome that are typically highly infested with Ae. albopictus, i.e. the main cemetery and a small green area within a highly urbanised neighbourhood. In each area we used 10 pyriproxyfen “dissemination” stations, 10 “sentinel” sites and 10 covered, control sites. The sentinel and control sites each contained 25 Ae. albopictus larvae. These were monitored for development and adult emergence. Principal Findings When a 5% pyriproxyfen powder was used to contaminate the dissemination sites, we observed significantly higher mortality at the pupal stage in the sentinel sites (50–70%) than in the controls (<2%), showing that pyriproxyfen was transferred by mosquitoes into sentinel sites and that it had a lethal effect. Conclusions The results support the potential feasibility of the auto-dissemination approach to control Ae. albopictus in urban areas. Further studies will be carried out to optimize the method and provide an effective tool to reduce the biting nuisance caused by this aggressive species and the transmission risk of diseases such as Dengue and Chikungunya. These arboviruses pose an increasing threat in Europe as Ae. albopictus expands its range.

Chromosomal plasticity and evolutionary potential in the malaria vector Anopheles gambiae sensu stricto: insights from three decades of rare paracentric inversions

BackgroundIn the Anopheles gambiae complex, paracentric chromosomal inversions are non-randomly distributed along the complement: 18/31 (58%) of common polymorphic inversions are on chromosome arm 2R, which represents only ~30% of the complement. Moreover, in An. gambiae sensu stricto, 6/7 common polymorphic inversions occur on 2R. Most of these inversions are considered markers of ecological adaptation that increase the fitness of the carriers of alternative karyotypes in contrasting habitats. However, little is known about the evolutionary forces responsible for their origin and subsequent establishment in field populations.ResultsHere, we present data on 82 previously undescribed rare chromosomal inversions (RCIs) recorded during extensive field sampling in 16 African countries over a 30 year period, which may shed light on the dynamics of chromosomal plasticity in An. gambiae. We analyzed breakpoint distribution, length, and geographic distribution of RCIs, and compared these measures to those of the common inversions. We found that RCIs, like common inversions, are disproportionately clustered on 2R, which may indicate that this arm is especially prone to breakages. However, contrasting patterns were observed between the geographic distribution of common inversions and RCIs. RCIs were equally frequent across biomes and on both sides of the Great Rift Valley (GRV), whereas common inversions predominated in arid ecological settings and west of the GRV. Moreover, the distribution of RCI lengths followed a random pattern while common inversions were significantly less frequent at shorter lengths.ConclusionBecause 17/82 (21%) RCIs were found repeatedly at very low frequencies – at the same sampling location in different years and/or in different sampling locations – we suggest that RCIs are subject mainly to drift under unperturbed ecological conditions. Nevertheless, RCIs may represent an important reservoir of genetic variation for An. gambiae in response to environmental changes, further testifying to the considerable evolutionary potential hidden within this pan-African malaria vector.

The "far-west" of Anopheles gambiae molecular forms.

The main Afrotropical malaria vector, Anopheles gambiae sensu stricto, is undergoing a process of sympatric ecological diversification leading to at least two incipient species (the M and S molecular forms) showing heterogeneous levels of divergence across the genome. The physically unlinked centromeric regions on all three chromosomes of these closely related taxa contain fixed nucleotide differences which have been found in nearly complete linkage disequilibrium in geographic areas of no or low M-S hybridization. Assays diagnostic for SNP and structural differences between M and S forms in the three centromeric regions were applied in samples from the western extreme of their range of sympatry, the only area where high frequencies of putative M/S hybrids have been reported. The results reveal a level of admixture not observed in the rest of the range. In particular, we found: i) heterozygous genotypes at each marker, although at frequencies lower than expected under panmixia; ii) virtually all possible genotypic combinations between markers on different chromosomes, although genetic association was nevertheless detected; iii) discordant M and S genotypes at two X-linked markers near the centromere, suggestive of introgression and inter-locus recombination. These results could be indicative either of a secondary contact zone between M and S, or of the maintenance of ancestral polymorphisms. This issue and the perspectives opened by these results in the study of the M and S incipient speciation process are discussed.

Cooperative interactions between odorant-binding proteins of Anopheles gambiae

To understand olfactory discrimination in Anopheles gambiae, we made six purified recombinant OBPs and investigated their ligand-binding properties. All OBPs were expressed in bacteria with additional production of OBP47 in the yeast Kluveromyces lactis. Ligand-binding experiments, performed with a diverse set of organic compounds, revealed marked differences between the OBPs. Using the fluorescent probe N-phenyl-1-naphthylamine, we also measured the binding curves for binary mixtures of OBPs and obtained, in some cases, unexpected behaviour, which could only be explained by the OBPs forming heterodimers with binding characteristics different from those of the component proteins. This shows that OBPs in mosquitoes can form complexes with novel ligand specificities, thus amplifying the repertoire of OBPs and the number of semiochemicals that can be discriminated. Confirmation of the likely role of heterodimers was demonstrated by in situ hybridisation, suggesting that OBP1 and OBP4 are co-expressed in some antennal sensilla of A. gambiae.

Control methods against invasive Aedes mosquitoes in Europe: a review.

Five species of invasive Aedes mosquitoes have recently become established in Europe: Ae. albopictus, Ae. aegypti, Ae. japonicus japonicus, Ae. koreicus and Ae. atropalpus. These mosquitoes are a serious nuisance for people and are also competent vectors for several exotic pathogens such as dengue and chikungunya viruses. As they are a growing public health concern, methods to control these mosquitoes need to be implemented to reduce their biting and their potential for disease transmission. There is a crucial need to evaluate methods as part of an integrated invasive mosquito species control strategy in different European countries, taking into account local Aedes infestations and European regulations. This review presents the control methods available or in development against invasive Aedes mosquitoes, with a particular focus on those that can be implemented in Europe. These control methods are divided into five categories: environmental (source reduction), mechanical (trapping), biological (e.g. copepods, Bacillus thuringiensis var. israelensis, Wolbachia), chemical (insect growth regulators, pyrethroids) and genetic (sterile insect technique and genetically modified mosquitoes). We discuss the effectiveness, ecological impact, sustainability and stage of development of each control method.

Ligand-Binding Study of Anopheles gambiae Chemosensory Proteins

Chemosensory proteins (CSPs) are a class of small proteins expressed only in arthropods and endowed with heterogeneous functions. Some of them are involved in chemical communications, others in development or other physiological roles. The numbers of CSPs in different species of insects range from 4 in Drosophila to at least 70 in locusts, whereas in other arthropods such as crustaceans and millipedes, only 2-3 very similar sequences have been reported in each species. We have expressed, in a bacterial system, 5 of the 8 CSPs predicted by the genome of the malaria mosquito Anopheles gambiae, 4 identified at the protein level (SAP1, SAP2, SAP3, and CSP3) and a fifth annotated as part of this work, obtaining the proteins with high yields and in their soluble forms. Purified CSPs have been used to study their ligand-binding properties, both using competitive binding assays and quenching of intrinsic tryptophan fluorescence, in order to get insights into their physiological functions. The agreement between the 2 sets of data supports the assumptions that the ligands, including the fluorescent reporter, bind within the core of the proteins. Their different affinities toward a set of pure chemicals suggest specific roles in chemical communication.

A proteomic investigation of soluble olfactory proteins in Anopheles gambiae

Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) are small soluble polypeptides that bind semiochemicals in the lymph of insect chemosensilla. In the genome of Anopheles gambiae, 66 genes encode OBPs and 8 encode CSPs. Here we monitored their expression through classical proteomics (2D gel-MS analysis) and a shotgun approach. The latter method proved much more sensitive and therefore more suitable for tiny biological samples as mosquitoes antennae and eggs. Females express a larger number and higher quantities of OBPs in their antennae than males (24 vs 19). OBP9 is the most abundant in the antennae of both sexes, as well as in larvae, pupae and eggs. Of the 8 CSPs, 4 were detected in antennae, while SAP3 was the only one expressed in larvae. Our proteomic results are in fairly good agreement with data of RNA expression reported in the literature, except for OBP4 and OBP5, that we could not identify in our analysis, nor could we detect in Western Blot experiments. The relatively limited number of soluble olfactory proteins expressed at relatively high levels in mosquitoes makes further studies on the coding of chemical messages at the OBP level more accessible, providing for few specific targets. Identification of such proteins in Anopheles gambiae might facilitate future studies on host finding behavior in this important disease vector.