Involving citizen scientists in monitoring arthropod vectors of human and zoonotic diseases: The case of Mosquito Alert in Italy.

Citizen science has been particularly effective in gathering reliable, timely, large-scale data on the presence and distributions of animal species, including mosquito vectors of human and zoonotic pathogens. This involves the participation of citizen scientists in research projects, with success strongly dependent on the capacity to disseminate project information and engage citizen scientists to contribute their time. Mosquito Alert is a citizen science that aids in the system surveillances of vector mosquitoes. It involves citizen scientists providing expert-validated photos of targeted mosquitoes, along with records of bites and breeding sites. Since 2020 the system has been disseminated throughout Europe. This article uses models to analyze the effect of promotion activities carried out by the Mosquito Alert ITALIA team from October 2020 to December 2022 on the number of citizen scientists recruited and engaged in the project, and their performance in mosquito identification. Results show a high level of citizen scientist recruitment (N > 18.000; 37 % of overall European participants). This was achieved mostly through articles generated by ad hoc press releases detailing the app's goals and functioning. Press releases were more effective when carried out at the beginning and end of the mosquito season and when mosquito's public health significance was emphasized. Despite the high number of records received (N > 20.000), only 30 % of registered participants sent records, and the probability of a participant sending a record dropped off quickly over time after first registering. Among participants who contributed, ∼50 % sent 1 record, ∼30 % ≥3 and 4 % >10 records. Participants showed good capacity to identify mosquitoes and improve identification skills with app usage. The results will be valuable for anyone interested in evaluating citizen science, as participation and engagement are seldom quantitatively assessed. Our results are also useful for designing dissemination and education strategies in citizen science projects associated with arthropod vector monitoring.

Speciation within the Anopheles gambiae complex: high-throughput whole genome sequencing reveals evidence of a putative new cryptic taxon in 'far-west' Africa.

The two main Afrotropical malaria vectors - Anopheles coluzzii and An. gambiae - are genetically distinct and reproductively isolated across West Africa. However, populations at the western extreme of their range are assigned as "intermediate" between the two species by whole genome sequence (WGS) data, and as hybrid forms by conventional molecular diagnostics. By exploiting WGS data from 1,190 specimens collected across west Africa via the Anopheles gambiae 1000 Genomes network, we identify a novel putative taxon in the far-west (provisionally named Bissau molecular form), which did not arise by admixture but rather originated at the same time as the split between An. coluzzii and An. gambiae. Intriguingly, these populations lack insecticide resistance mechanisms commonly observed in the two main species. These findings lead to a change of perspective on malaria vector species in the far-west region with potential for epidemiological implications, and a new challenge for genetic-based mosquito control approaches.

A global association between Covid-19 cases and airborne particulate matter at regional level.

Evidences of an association between air pollution and Covid-19 infections are mixed and inconclusive. We conducted an ecological analysis at regional scale of long-term exposure to air-borne particle matter and spread of Covid-19 cases during the first wave of epidemics. Global air pollution and climate data were calculated from satellite earth observation data assimilated into numerical models at 10 km resolution. Main outcome was defined as the cumulative number of cases of Covid-19 in the 14 days following the date when > 10 cumulative cases were reported. Negative binomial mixed effect models were applied to estimate the associations between the outcome and long-term exposure to air pollution at the regional level (PM10, PM2.5), after adjusting for relevant regional and country level covariates and spatial correlation. In total we collected 237,749 Covid-19 cases from 730 regions, 63 countries and 5 continents at May 30, 2020. A 10 µg/m3 increase of pollution level was associated with 8.1% (95% CI 5.4%, 10.5%) and 11.5% (95% CI 7.8%, 14.9%) increases in the number of cases in a 14 days window, for PM2.5 and PM10 respectively. We found an association between Covid-19 cases and air pollution suggestive of a possible causal link among particulate matter levels and incidence of COVID-19.

Wide-scale analysis of protein expression in head and thorax of Aedes albopictus females.

The recently available genome of Aedes albopictus - the most worldwide-spread human arbovirus vector - has revealed a large genome repertory and a great plasticity which are believed to have contributed to the species success as an invasive species and opened the way to genomic, transcriptomic and proteomic studies. We carried out the first wide-scale quantitative proteomic analysis of Ae. albopictus female head and thorax by means of a 'shotgun' approach based on nano liquid chromatography-high resolution mass spectrometry associated to protein Label Free Quantification (LFQ) which allows to assess differences in protein expression between tissues and different physiological stages. We identified 886 and 721 proteins in heads and thoraxes respectively, 5 of which were exclusively expressed in thoraxes and 170 in heads, consistently with the more complex head physiology. Head-protein expression was found to be highly divergent between virgin and mated females and limited before and after blood-feeding and oviposition. The large repertoire of proteins identified represents an instrumental source of data for genome annotation and gene-expression studies, and may contribute to studies aimed at investigating the molecular bases of physiological processes of this successful invasive species.

Candidate biomarkers for mosquito age-grading identified by label-free quantitative analysis of protein expression in Aedes albopictus females.

We applied a "shotgun" approach based on nanoliquid chromatography-high resolution mass spectrometry associated to label free quantification (LFQ) to identify proteins varying with age, independently from the physiological state, in Aedes albopictus, a mosquito species which in the last decades invaded temperate regions in North America and Europe, creating concerns for associated high nuisance and risk of arbovirus transmission. The combined "shotgun" and LFQ approach was shown to be highly suitable to simultaneously compare several biological samples, as needed in a study aimed to analyze different age-groups and physiological states of adult mosquito females. The results obtained represent the first wide-scale analysis of protein expression in Ae. albopictus females: >1000 and 665 proteins were identified from few micrograms of crude protein extracts of mosquito heads and thoraxes, respectively. Six of these proteins were shown to significantly vary from 2- to 16-day-old females, independently from their physiological state (i.e. virgin, mated, host-seeking, blood-fed, and gravid). BIOLOGICAL SIGNIFICANCE: Mosquito-borne diseases, such as malaria, dengue and other arboviroses, are a persistent cause of global mortality and morbidity, affecting hundreds of thousands of people. Billions of people living in tropical areas are at risk of being bitten every day by an infective mosquito female and the spread of tropical species such as Aedes albopictus to temperate areas is creating alarm in the northern hemisphere. Mosquito longevity is a critical factor affecting mosquito-borne pathogen transmission cycles and the mosquito capacity to transmit pathogens. However, large scale analyses of the age structure of mosquito field populations is hampered by the lack of optimal age-grading approaches. Our findings open new perspectives for the development of reliable, simple and cheap protein-based assays to age-grade Ae. albopictus females and, most likely, other mosquito species of higher medical relevance, such as the main dengue vector, Aedes aegypti, and the major Afrotropical malaria vectors. These assays would greatly contribute to epidemiological studies aimed at defining the actual vectorial capacity of a given mosquito species. Moreover, they would be very valuable in assessing the effectiveness of mosquito control interventions based on the relative ratio between young and old individuals before and after the intervention.

Prominent intraspecific genetic divergence within Anopheles gambiae sibling species triggered by habitat discontinuities across a riverine landscape.

The Anopheles gambiae complex of mosquitoes includes malaria vectors at different stages of speciation, whose study enables a better understanding of how adaptation to divergent environmental conditions leads to evolution of reproductive isolation. We investigated the population genetic structure of closely related sympatric taxa that have recently been proposed as separate species (An. coluzzii and An. gambiae), sampled from diverse habitats along the Gambia river in West Africa. We characterized putatively neutral microsatellite loci as well as chromosomal inversion polymorphisms known to be associated with ecological adaptation. The results revealed strong ecologically associated population subdivisions within both species. Microsatellite loci on chromosome-3L revealed clear differentiation between coastal and inland populations, which in An. coluzzii is reinforced by a unusual inversion polymorphism pattern, supporting the hypothesis of genetic divergence driven by adaptation to the coastal habitat. A strong reduction of gene flow was observed between An. gambiae populations west and east of an extensively rice-cultivated region apparently colonized exclusively by An. coluzzii. Notably, this 'intraspecific' differentiation is higher than that observed between the two species and involves also the centromeric region of chromosome-X which has previously been considered a marker of speciation within this complex, possibly suggesting that the two populations may be at an advanced stage of differentiation triggered by human-made habitat fragmentation. These results confirm ongoing ecological speciation within these most important Afro-tropical malaria vectors and raise new questions on the possible effect of this process in malaria transmission.

Estimating mosquito population size from mark-release-recapture data.

Accurate estimation of population size is key to understanding the ecology of disease vectors, as well as the epidemiology of the pathogens they carry and to plan effective control activities. Population size can be estimated through mark-release-recapture (MRR) experiments that are based on the assumption that the ratio of recaptured individuals to the total captures approximates the ratio of marked individuals released to the total population. However, methods to obtain population size estimates usually consider pooled data and are often based on the total number of marked and unmarked captures. We here present a logistic regression model, based on the principle of the well-known Fisher-Ford method, specific for MRR experiments where the information available is the number of marked mosquitoes released, the number of marked and unmarked mosquitoes caught in each trap and on each day, and the geographic coordinates of the traps. The model estimates population size, taking into consideration the distance between release points and traps, the time between release and recapture, and the loss of marked mosquitoes to death or dispersal. The performance and accuracy of the logistic regression model has been assessed using simulated data from known population sizes. We then applied the model to data from MRR experiments with Aedes albopictus Skuse performed on the campus of "Sapienza" University in Rome (Italy).

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.

Blood-feeding preferences of Aedes albopictus (Diptera: Culicidae) in urban and rural settings within the province of Rome, Italy.

We here report the results of field trials carried out in Rome with the aim to obtain data on the feeding behaviour of Aedes albopictus, in relation to different availability and abundance of putative hosts. Human Blood Index values were found higher than 75% in urban areas, where humans represented the most abundant hosts, and lower than 60% in rural areas, where host alternative to humans were frequent. The overall results confirm the generalist feeding-behaviour shown by this species in its original range of distribution and highlighting its high potential as vector of human pathogens in urban areas of Italy.

Comparative analysis of epicuticular lipid profiles of sympatric and allopatric field populations of Anopheles gambiae s.s. molecular forms and An. arabiensis from Burkina Faso (West Africa).

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.

A new kernel method for microcalcification detection: Spin Glass-Markov Random Fields.

Mammography associated with clinical breast examination is the only effective method for mass breast screening. Microcalcifications are one of the primary signs for early detection of breast cancer. In this paper we propose a new kernel method for classification of difficult-to-diagnose regions in mammographic images. It consists of a novel class of Markov Random Fields, using techniques developed within the context of statistical mechanics. This method is used for the classification of positive Region of Interest (ROI's) containing clustered microcalcifications and negative ROI's containing normal tissue. The obtained results show that the proposed approach can be successfully employed for detection of microcalcifications

Toward a quantitative analysis of skin lesion images.

Melanoma is the most dangerous form of skin cancer. Skin cancers, and particularly melanomas, can be easily cured if detected early. The regularity of the pigment network structure is a significant sign for early diagnosis of melanoma. We present here a quantitative analysis of the morphology of the pigment network structure. Our method is based on the extraction of the pigment network structure using digital image processing techniques. Once the network is extracted, it is possible to derive statistical indicators relative to its regularity or irregularity. We report experiments on a database of 14 images, showing the effectiveness of our method.

Analysis of periapical lesion using statistical textural features.

In this work we present a quantitative study on different regions of periapical images with a series of textural features, extracted using cooccurrence matrices; those features are used for a pattern recognition analysis by means of an artificial neural network. The obtained results show that it is possible to recognize in an objective way changes in bone pattern.

Clinical characteristics of corneal foreign bodies and their associated culture results.

A prospective study was performed to investigate the clinical characteristics of corneal foreign bodies and to determine if these characteristics were related to the culture results of the foreign body. The clinical characteristics included the type of foreign body, the mechanism of injury, the time present in the cornea, and the presence of a rust ring or an infiltrate. Sixty-three foreign bodies were removed from corneas and cultured for bacteria, and 14.3% cultured positive for bacteria. The major pathogen was coagulase-negative Staphylococcus. The majority of foreign bodies were metallic, and mechanisms of injury were similar for both culture-positive and culture-negative groups. There was no significant correlation between the mean time present in the cornea and the culture results. The presence of a rust ring or an infiltrate was not found to be significant when predicting culture results. No clinical features readily distinguished culture-negative foreign bodies from culture-positive foreign bodies.

[Circulating immune complexes in a population of subjects seropositive for rheumatoid factor]. / Immunocomplessi circolanti in una popolazione di soggetti sieropositivi per il fattore reumatoide.

The level of immune complexes has been detected with the C1q binding test in 179 subjects positive by Latex agglutination test (titre greater than 1/20). The average of C1q binding was 5.36%, significantly higher than in 30 normal blood donors. A positive correlation was observed between the percent of C1q binding and, respectively, the titre of the latex test and the erythrocyte sedimentation rate. Of the 36 patients with rheumatoid arthritis, 16 (45.7%) showed an high level of C1q binding activity (greater than 5%), as well as 4 of the 8 patients with chronic hepatitis (50%) and 11 of the 31 patients with infectious diseases. No immune complexes were detected among the 11 healthy latex positive subjects.

Remote sensing of subsurface water temperature by Raman scattering.

The application of Raman scattering to remote sensing of subsurface water temperature and salinity is considered, and both theoretical and experimental aspects of the technique are discussed. Recent experimental field measurements obtained in coastal waters and on a trans-Atlantic/Mediterranean research cruise are correlated with theoretical expectations. It is concluded that the Raman technique for remote sensing of subsurface water temperature has been brought from theoretical and laboratory stages to the point where practical utilization can now be developed.