Abstract Mosquito-borne diseases represent an escalating global health threat, driven by climate change, urbanization, and the spread of invasive vectors into new territories. Effective surveillance is constrained by a critical ‘taxonomic impediment’: the rate of specimen collection far outpaces the capacity of expert entomologists to process and identify trap catches. To address this bottleneck we developed MosAICo, an integrated AI-powered ecosystem for automated mosquito species identification designed for real-world, national-scale entomological surveillance. The system combines a standardized benchtop imaging device with MosAICo-Net, a deep learning pipeline enabling efficient and principled open-set recognition and uncertainty quantification. Trained and evaluated on 12, 499 specimens spanning 15 species collected across Italy, the model identifies seven priority vector species while explicitly rejecting out-of-distribution specimens. On a geographically stratified held-out test set, MosAICo-Net achieved over 90% accuracy on target species, and an AUROC of 0.96 for out-of-distribution detection. Field validation across 20 Italian surveillance sites confirmed these results: 94% micro accuracy on 1, 470 field-collected target specimens and strong agreement with expert manual counts ( = 0.66). To assess cross-geographic generalizability, the system was further evaluated on 118 Aedes albopictus specimens collected at the fringe of the species invasion front in Ghana: a 97.4% accuracy with only a single specimen escalated to expert review, suggests that MosAICo is well-suited for deployment in distant and epidemiologically critical regions. The system processes up to 82 specimens per image, matching expert throughput at constant speed regardless of taxonomic complexity. By embedding uncertainty-aware AI within a standardized hardware-software pipeline, MosAICo acts as a scalable force multiplier for public health entomology, freeing expert attention for rare, invasive, or ambiguous specimens that require human validation.

Anopheles superpictus (subgenus Cellia) plays an efficient role in malaria transmission in countries of the Mediterranean basin, Eastern Europe, and the Caucasus region, where it has been involved in the transmission of both Plasmodium falciparum and P. vivax. In Italy, this species was historically considered a secondary malaria vector, primarily recorded in the South including Sicily, along small rivers. It was less frequently observed in central Italy, only rarely reported in the North. Between 2022 and 2024, as part of routine investigations on residual anophelism, An. superpictus specimens were collected for the first time in the southern Maremma Plain (Magliano in Toscana, Grosseto, Tuscany). Adult mosquitoes were collected in animal shelters and tool premises of two farms using traps and manual aspiration methods. Larval sampling was performed by exploring potential breeding sites along the Albegna riverbed. Anophelinae adults and larvae were morphologically identified at the species level, and subsamples were molecularly confirmed by sequencing the cytochrome c oxidase subunit 1 gene and the ribosomal DNA internal transcribed spacer 2 region. A total of 1,106 adult mosquitoes were collected from July to October 2022. Anopheles superpictus was the most abundant species recorded (n = 512; 46

In recent years, globalization and climate change have led to a rise in the number of imported cases of Aedes-diseases in Europe, resulting in increased frequency and magnitude of local transmissions due to the presence of competent vectors. Recently, Italy has experienced the establishment of three exotic Aedes mosquitoes relevant to human health, Aedes albopictus, Aedes koreicus and Aedes japonicus. Aedes aegypti, the primary vector of dengue and yellow fever, distributed in tropical and subtropical regions, has recently reappeared in Europe and the risk for its re-introduction in Italy is high given the climatic conditions suitable for the species. To address the risk of introduction and spread of Aedes-diseases, the Health Authorities recommend the strengthening of entomological surveillance at regional level, particularly in strategic areas and Points of Entry, such as ports and airports. In 2021, a Korean research team developed a multiplex-PCR assay for the identification of six Aedini species, not including Ae. aegypti. In the present study, the previous diagnostic test was improved by designing reverse primers for the identification of Ae. aegypti and Aedes geniculatus. This latter native mosquito lays eggs morphologically similar to those of invasive species with which it can sometimes be found in sympatry. Furthermore, a ten-minute DNA extraction method was implemented. The results obtained demonstrate a perfect diagnostic capacity and sensitivity of the method in discriminating the five species tested. Here, findings of a sensitive, rapid and cost-effective molecular assay developed for the early identification of invasive species at high-risk sites are shown.

The global expansion of Aedes albopictus from Southeast Asia to various regions worldwide poses a significant public health concern due to its role as a vector for several pathogens, including chikungunya virus (CHIKV), which infects over one million people annually. In this study, aimed at understanding the molecular interactions between Ae. albopictus and CHIKV, we analyzed by RNA sequencing CHIKV-infected and uninfected control mosquitoes. We focused our attention on key mosquito organs at one- and five-days post-blood meal ingestion, which correspond to viral entry and dissemination, and found specific transcriptional changes involving various pathways during the CHIKV lifecycle. The mosquito midgut plays a crucial role in the early stages, when the virus enters along with human blood components, encounters the resident microbiota, interacts with the developing peritrophic matrix, and counteracts the mosquito's digestive enzymes. We found that RNA interference (RNAi) was predominantly activated in the midgut during the initial virus invasion. Additionally, several key enzymes involved in autophagy and ubiquitination were also more abundant in infected midguts compared to controls. At later time points, after viral dissemination into the hemocoel, key immune responses are triggered in the hemolymph and, accordingly, immune mechanisms such as the activation of leucine-rich repeats (LRRs) proteins, secretion of antimicrobial peptides (e.g., holotricin), and melanization (mediated by phenoloxidase, PO) were the most prominent. RNA-seq results were validated by RT-qPCR on selected candidates in different tissues and a catalogue of Ae. albopictus immune genes (891 contigs) grouped into 24 different immune and immune-related families was compiled. This study explores the molecular interactions between Ae. albopictus and CHIKV across developmental stages, providing key insights into arbovirus transmission dynamics and mosquito vector competence.

In 2024, Italy reported its first five cases of Oropouche fever in travelers returning from Cuba and Brazil. The Oropouche virus (OROV), an emerging Orthobunyavirus of the Peribunyaviridae family, is a zoonotic arbovirus responsible for febrile illnesses in humans, often misdiagnosed owing to its clinical similarity to those caused by dengue and Zika virus infections. Originally endemic to the Amazon region and first detected in Trinidad and Tobago in 1955, OROV has since spread throughout South and Central America. Recent outbreaks in Brazil and Cuba have been linked to the newly identified genotype V. Although Culicoides paraensis midges are recognized as the primary vector in the urban transmission cycle, their presence in Cuba was documented only after the outbreak. This late detection, coupled with a possibly low population density of this species, has raised concerns that other, more abundant arthropod species may have played a role in transmitting OROV to humans on the island. While some mosquito species have been implicated as potential OROV secondary vectors, their role remains uncertain. This study evaluates the vector competence of Italian populations of Aedes albopictus and Culex pipiens for the newly circulating OROV strain, which was introduced into the country by infected travelers. Experimental infections were conducted under biosafety level 3 conditions, exposing adult female mosquitoes to an infectious blood meal containing OROV. Viral presence was assessed using a real-time RT-PCR in mosquito bodies, legs + wings, saliva, and the first gonotrophic progeny (F1). Results indicated that, while both species successfully ingested infectious virus particles, Cx. pipiens exhibited no signs of infection, dissemination, or transmission at any time point. In Ae. albopictus, viral RNA was detected in two body samples but not in saliva or disseminated tissues, confirming an absence of transmission capability. In addition, no evidence of transmission to F1 generation was detected in either species. These findings are consistent with previous research on American mosquito populations, indicating that barriers to OROV transmission likely occur at the midgut level. Despite the apparent lack of vector competence in Italian Ae. albopictus and Cx. pipiens, the ongoing geographic expansion of OROV highlights the need for continued surveillance, particularly as the 2025 vector activity season approaches in Europe. The increasing circulation of the virus, along with its potential for adaptive evolution, highlights the importance of further research into the virus–vector interactions that could enable OROV to establish itself in new ecological niches.

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.

The frequency of locally transmitted dengue virus (DENV) infections has increased in Europe in recent years, facilitated by the invasive mosquito species Aedes albopictus, which is well established in a large area of Europe. In Italy, the first indigenous dengue outbreak was reported in August 2020 with 11 locally acquired cases in the Veneto region (northeast Italy), caused by a DENV-1 viral strain closely related to a previously described strain circulating in Singapore and China. In this study, we evaluated the vector competence of two Italian populations of Ae. albopictus compared to an Ae. aegypti lab colony. We performed experimental infections using a DENV-1 strain that is phylogenetically close to the strain responsible for the 2020 Italian autochthonous outbreak. Our results showed that local Ae. albopictus is susceptible to infection and is able to transmit the virus, confirming the relevant risk of possible outbreaks starting from an imported case.

Abstract Background Disease-vector mosquito monitoring is an essential prerequisite to optimize control interventions and evidence-based risk predictions. However, conventional entomological monitoring methods are labor- and time-consuming and do not allow high temporal/spatial resolution. In 2022, a novel system coupling an optical sensor with machine learning technologies (VECTRACK) proved effective in counting and identifying Aedes albopictus and Culex pipiens adult females and males. Here, we carried out the first extensive field evaluation of the VECTRACK system to assess: (i) whether the catching capacity of a commercial BG-Mosquitaire trap (BGM) for adult mosquito equipped with VECTRACK (BGM + VECT) was affected by the sensor; (ii) the accuracy of the VECTRACK algorithm in correctly classifying the target mosquito species genus and sex; (iii) Ae. albopictus capture rate of BGM with or without VECTRACK. Methods The same experimental design was implemented in four areas in northern (Bergamo and Padua districts), central (Rome) and southern (Procida Island, Naples) Italy. In each area, three types of traps—one BGM, one BGM + VECT and the combination of four sticky traps (STs)—were rotated each 48 h in three different sites. Each sampling scheme was replicated three times/area. Collected mosquitoes were counted and identified by both the VECTRACK algorithm and operator-mediated morphological examination. The performance of the VECTRACK system was assessed by generalized linear mixed and linear regression models. Aedes albopictus capture rates of BGMs were calculated based on the known capture rate of ST. Results A total of 3829 mosquitoes (90.2% Ae. albopictus) were captured in 18 collection-days/trap/site. BGM and BGM + VECT showed a similar performance in collecting target mosquitoes. Results show high correlation between visual and automatic identification methods (Spearman Ae. albopictus: females = 0.97; males = 0.89; P < 0.0001) and low count errors. Moreover, the results allowed quantifying the heterogeneous effectiveness associated with different trap types in collecting Ae. albopictus and predicting estimates of its absolute density. Conclusions Obtained results strongly support the VECTRACK system as a powerful tool for mosquito monitoring and research, and its applicability over a range of ecological conditions, accounting for its high potential for continuous monitoring with minimal human effort. Graphical Abstract

INTRODUCTION:Mosquitoes represent a way of spreading infectious diseases, as vectors of pathogens. Many types of ultrasonic devices have recently been promoted as effective and suitable alternatives to the use of biocides known as toxic to humans and environment. MATERIALS AND METHODS:Four ultrasonic mosquito repellers have been analysed and tested on females of two species, Culex pipiens and Aedes albopictus, in laboratory conditions. The behavior of the mosquitoes of reaching the attractant, with the repellers both at ON and OFF, was observed. RESULTS:The total mean number of Cx. pipiens interested to attractant when the repellers were ON was 2.8 versus 2.9 at OFF. The total mean number of Ae. albopictus interested to attractant when the repellers were ON was 5.5 versus 6.1 at OFF. CONCLUSIONS:The repellence efficacy resulted not significant (P>0.05) in all the electronic ultrasound repellers tested. The number of mosquitoes of both species, displaying the attractant's search behavior appeared independent from the switch ON/OFF mode. Open questions remain: the need of conducting further research to establish a relationship between ultrasonic emission and mosquito effective disturbance.

Migratory birds reach Europe from sub-Saharan Africa, and some avian species may harbor and transport infected ectoparasites, mainly ticks, native to the territories of departure. In 2022, a project focused on identifying the introduction of pathogens in Italy from Africa via migratory birds represented an important opportunity to investigate this particular route of tick dispersal. Among ticks collected from migratory birds on the island of Ventotene, Latium Region, we found one larva of a soft tick on a common whitethroat (Curruca communis) that was morphologically and molecularly identified to the species level as Argas (Persicargas) persicus (Oken 1818). This study reports the fifth detection of this species in Italy; therefore, it is considered very rare. Further monitoring programmes should be implemented to better monitor the spread of this species and possibly update its distribution.

Abstract Background Anopheles sacharovi, a member of the Anopheles maculipennis complex, was a historical malaria vector in Italy, no longer found since the last report at the end of 1960s. In September 2022, within the Surveillance Project for the residual anophelism, a single specimen of An. maculipennis sensu lato collected in Lecce municipality (Apulia region) was molecularly identified as An. sacharovi. This record led to implement a targeted entomological survey in September 2023. Methods Investigation was conducted in the areas around the first discovery, focusing on animal farms, riding stables and potential breeding sites. Adult and immature mosquitoes were collected, using active search or traps, in several natural and rural sites. Mosquitoes belonging to An. maculipennis complex were identified morphologically and molecularly by a home-made routine quantitative polymerase chain reaction (qPCR) assay, developed specifically for the rapid identification of An. labranchiae, and, when necessary, by amplification and sequencing of the ITS-2 molecular marker. Results Out of the 11 sites investigated, 6 were positive for Anopheles presence. All 20 An. maculipennis s.l. (7 adults, 10 larvae and 3 pupae) collected in the areas were identified as An. sacharovi by ITS-2 sequencing. Conclusions The discovery of An. sacharovi, considered to have disappeared from Italy for over 50 years, has a strong health relevance and impact, highlighting an increase in the receptivity of the southern areas. As imported malaria cases in European countries are reported every year, the risk of Plasmodium introduction by gametocyte carriers among travellers from endemic countries should be taken into greater consideration. Our findings allow rethinking and building new models for the prediction and expansion of introduced malaria. Furthermore, to prevent the risk of reintroduction of the disease, the need to strengthen the surveillance of residual anophelism throughout the South should be considered. Graphical Abstract

Migratory birds play an important role in transporting ixodid ticks and tick-borne pathogens between continents. During the Boreal spring, migratory birds reach Europe, mainly from sub-Saharan Africa or from northern African countries but not much is known about the diversity and ecology of the ticks they spread. From 2017 to 2022, in the framework of two consecutive projects focused on sampling migratory birds from Africa to Europe, a total of 27 immature Amblyomma ticks were collected from migratory birds, belonging to 8 species, captured on the Island of Ventotene, an important stop-over site in the Mediterranean Sea. In the absence of adult specimens, morphological identification was limited to assigning these ticks to the Amblyomma genus. In this study, sequencing and comparative analysis of three mitochondrial molecular markers (12S rDNA, 16S rDNA, COI) were performed to achieve taxonomic identification. Sequences obtained from Ventotene specimens matched at 100% identity with Amblyomma sparsum. In conclusion, this study documented that immature stages of this species belonging to the Amblyomma marmoreum complex reached the Pontine Islands for six consecutive years. The entry of alien tick species and their potentially transmitted pathogens deserves further study, also in light of the globally ongoing climate change.

Early detection of pathogens in vectors is important in preventing the spread of arboviral diseases, providing a timely indicator of pathogen circulation before outbreaks occur. However, entomological surveillance may face logistical constraints, such as maintaining the cold chain, and resource limitations, such as the field and laboratory workload of mosquito processing. We propose an FTA card-based trapping system that aims to simplify both field and laboratory phases of arbovirus surveillance. We modified a BG-Sentinel trap to include a mosquito collection chamber and a sugar feeding source through an FTA card soaked in a long-lasting viscous solution of honey and hydroxy-cellulose hydrogel. The FTA card ensures environmental preservation of nucleic acids, allowing continuous collection and feeding activity of specimens for several days and reducing the effort required for viral detection. We tested the trap prototype during two field seasons (2019 and 2021) in North-eastern Italy and compared it to CDC-CO2 trapping applied in West Nile and Usutu virus regional surveillance. Collections by the BG-FTA approach detected high species diversity, including Culex pipiens, Aedes albopictus, Culex modestus, Anopheles maculipennis sensu lato and Ochlerotatus caspius. When used for two-days sampling, the BG-FTA trap performed equally to CDC also for the WNV-major vector Cx. pipiens. The FTA cards detected both WNV and USUV, confirming the reliability of this novel approach to detect viral circulation in infectious mosquitoes. We recommend this surveillance approach as a particularly useful alternative in multi-target surveillance, for sampling in remote areas and in contexts characterized by high mosquito densities and diversity.

In spring, migratory birds reach Europe, mainly from sub-Saharan Africa or from northern African countries. Avian species may be implicated in the spread of pathogens, either as reservoirs, hosts or carriers of infected ectoparasites. In 2021, on Ventotene Island (Latium region, Italy) within a project focused on the potential incoming pathogens via migratory birds from Africa, we found two larvae of Argas sp., on the redstart Phoenicurus phoenicurus, that shared morphological features with the African Argas (Argas) africolumbae. Comparison of the tested larval DNA sequences to the adult reference sequences showed the highest identity (> 92%) with homologous sequences of A. africolumbae collected in South Africa and in Spain. This study reports the first detection of Argas africolumbae-like specimens in Italy.

From September 2020 to April 2021 a mosquito monitoring activity was carried out in the Caffarella Valley within the Appia Antica Regional Park in Rome, a very popular urban park completely surrounded by densely populated neighborhoods. During this entomological survey, we collected 798 mosquito larvae that were identified by morphology and by molecular tools. Out of these samples, eight taxa have been identified morphologically or molecularly in case of complex of species, belonging to five genera: Culex pipiens (49.1%, n=392), Culiseta annulata (16%, n=128), Culiseta longiareolata (13.1%, n=105), Anopheles claviger (11.5%, n=92), Aedes albopictus (5.5%, n=44), Anopheles maculipennis s.s. (2%, n=16), Culex theileri (0.7%, n=6), Uranotaenia unguiculata (1.8%, n=15). The noteworthy datum of this survey is the finding of Cx. theileri, a very common species in ponds and marshes in natural and rural environments, but never recorded before in Rome, and also the record of Ur. unguiculata, previously reported in Rome but never observed in this park. Caffarella Valley confirms its high degree of naturalness and high biodiversity in terms of mosquito species together to its particular location as a very popular urban park, completely surrounded by densely populated neighborhoods. Key Words: mosquitoes, Culex theileri, Rome, Italy

Anopheles algeriensis Theobald, 1903, considered a competent vector of Plasmodium parasites, is a mosquito species widely distributed in the Mediterranean area but rare in Northern and Central Europe. The disappearance of its suitable breeding sites in Italy is having a detrimental effect on the occurrence of this species once common along the Southern coasts and on the islands. Recently, molecular investigations have renewed interest in this species, highlighting a genetic heterogeneity among European populations. In this study, An. algeriensis populations from Italy, Germany, Romania, and Sweden were analyzed by molecular typing of the intergenic transcribed spacer 2 (ITS2). The mitochondrial cytochrome c oxidase subunit I (COI) was also analyzed from specimens collected in Southern Italy. With the aim of investigating the population structure of this species, the obtained data were compared to all publicly available ITS2 and COI sequences of An. algeriensis, adding specimens from Spain and Portugal. The analyses of both markers indicate a split between Iberian populations (Spain for ITS2 and Spain/Portugal for COI) and those from the rest of Europe, revealing two cryptic species. The analysis of the COI barcode revealed a third clade representing a cryptic species present in Danube Delta (Romania). The high levels of genetic divergence among the clades of An. algeriensis indicate that this taxon represents a species complex, potentially harboring several distinct cryptic species.

As in 2018, when a large West Nile virus (WNV) epidemic occurred, the 2022 vector season in Italy was marked by an early onset of WNV circulation in mosquitoes and birds. Human infections were limited until early July, when we observed a rapid increase in the number of cases. We describe the epidemiology of human infections and animal and vector surveillance for WNV and compare the more consolidated data of June and July 2022 with the same period in 2018.

This study reports on the health status of the edible dormouse (Glis glis) living in Nebrodi Park (Sicily, Italy), responsible for nut crop damage in the area. In the frame of a monitoring campaign for potential zoonotic risk involving 30 dormice, rectal and conjunctival swabs and fur and nest content were collected for bacteriological and parasitological examinations, respectively. A large presence of fleas belonging to Monopsyllus sciurorum was found. Necropsy of a dead dormouse revealed an infection of Mesocestoides lineatus, whose cysts were found in the abdomen cavity and on the liver; this is the first report of this in this species. Further studies are necessary to identify their role in the environment, considering the limited knowledge of this species in Italy.

BackgroundOn March 11 2020, the World Health Organization (WHO) declared COVID-19 outbreak a pandemic. As the mosquito season progressed, the understandable concern that such insects could transmit the virus began to spread. In response to this request, the vector competence for SARS-CoV-2 of Culex pipiens and Aedes albopictus , the two most common species of vector mosquitoes in Europe, was investigated. Due to the peculiar feeding behaviour, the role of Aedes albopictus in a potential mechanical transmission of the virus was also evaluated. MethodsFor the vector competence study, mosquitoes were allowed to take up an infectious blood meal. Mosquitoes were collected and analysed at 0, 3, 7 and 10 days post-feeding. For the mechanical transmission test Ae. albopictus females were allowed to feed for a short time on a feeder containing infectious blood and then on a feeder containing virus-free blood. Mosquitoes and blood were tested for viral presence.Results Cx. pipiens and Ae. albopictus tested resulted not competent for SARS-CoV-2 and Ae. albopictus was unable to mechanically transmit the virus. ConclusionsThis study shows that the most common species of vector mosquitoes in Europe don’t transmit SARS-CoV-2 and, for the first time, that Ae. albopictus is unable to mechanically transmit the virus by feeding from a positive host to a healthy host.

Malaria still represents a potential public health issue in Italy, and the presence of former Anopheles vectors and cases imported annually merit continuous surveillance. In areas no longer endemic, the concurrent presence of gametocyte carriers and competent vectors makes re-emergence of local transmission possible, as recently reported in Greece. In October 2017, due to the occurrence of four suspected introduced malaria cases in the province of Taranto (Apulia region), entomological investigations were performed to verify the involvement of local anopheline species. In 2019–2020 entomological surveys were extended to other areas historically prone to malaria between the provinces of Taranto and Matera and the province of Foggia (Gargano Promontory). Resting mosquitoes were collected in animal shelters and human dwellings, larvae were sampled in natural and artificial breeding sites, and specimens were both morphologically and molecularly identified. A total of 2228 mosquitoes were collected, 54.3% of which were anophelines. In all the investigated areas, Anopheles labranchiae was the most widespread species, while Anopheles algeriensis was predominant at the Gargano sites, and Anopheles superpictus and Anopheles plumbeus were recorded in the province of Matera. Our findings showed a potentially high receptivity in the surveyed areas, where the abundance of the two former malaria vectors, An. labranchiae and An. superpictus, is related to environmental and climatic parameters and to anthropic activities.