Investigation on key aspects of mating biology in the mosquito Aedes koreicus.

Aedes koreicus Edwards, 1917 (Hulecoetomyia koreica) is a mosquito (Diptera: Culicidae) from Northeast Asia with a rapidly expanding presence outside its original native range. Over the years, the species has been discovered in several new countries, either spreading after first introduction or remaining localised to limited areas. Notably, recent studies have demonstrated the ability of the species to transmit zoonotic parasites and viruses both in the field and in laboratory settings. Combined with its invasive potential, the possible role of Ae. koreicus in pathogen transmission highlights the public health risks resulting from its invasion. In this study, we used a recently established population from Italy to investigate aspects of biology that influence reproductive success in Ae. koreicus: autogeny, mating behaviour, mating disruption by the sympatric invasive species Aedes albopictus Skuse, 1894, and the presence of the endosymbiont Wolbachia pipientis Hertig, 1936. Our laboratory population did not exhibit autogenic behaviour and required a bloodmeal to complete its ovarian cycle. When we exposed Ae. koreicus females to males of Ae. albopictus, we observed repeated attempts at insemination and an aggressive, disruptive mating behaviour initiated by male Ae. albopictus. Despite this, no sperm was identified in Ae. koreicus spermathecae. Wolbachia, an endosymbiotic bacterium capable of influencing mosquito reproductive behaviour, was not detected in this Ae. koreicus population and, therefore, had no effect on Ae. koreicus reproduction.

Early start of seasonal transmission and co-circulation of West Nile virus lineage 2 and a newly introduced lineage 1 strain, northern Italy, June 2022.

In spring 2022, Europe faced an unprecedented heatwave, increasing the risk of West Nile virus (WNV) outbreaks. As early as 7 June 2022, WNV was detected in Culex mosquitoes in northern Italy, and - in the following days - in two blood donors, a patient with encephalitis, wild birds and additional mosquito pools. Genome sequencing demonstrated co-circulation of WNV lineage 2 and a newly introduced WNV lineage 1, which was discovered in the region in 2021.

[The CCM Project "Preventing vector-borne diseases through the development and pilot implementation of new operational supporting tools"]. / Il progetto CCM "Prevenzione delle malattie a trasmissione vettoriale: sviluppo e implementazione pilota di strumenti di supporto operativo".

The relevant ecological changes due to the anthropological pressure on the planet is causing reactions in the biological systems, which we must understand and characterize at the local scale to assist the adoption of suitable decisions in the public health risk management. The project presented in this paper is focused on the vector-borne diseases, mainly transmitted by mosquitoes. The species of mosquito currently playing an active role of public health importance in Italy are Aedes albopictus, as a vector of Chikungunya, Dengue and Zika viruses, as well as Culex pipiens, as a vector of West Nile and Usutu viruses. Predictive models are indicating the risk of introduction and establishment in the Mediterranean basin of Aedes aegypti, which is causing a mayor concern. The project has been conducted by the Emilia-Romagna, Piemonte, Veneto, Marche and Sicilia Regions, together with the Italian National Health Institute. The aim was to develop and pilot testing new approaches and technological tools possibly assisting the public health system in the management of the risk. The activities have been organized on six specific objectives: 1. Development and field testing of guidelines for the surveillance of invasive mosquito species; 2. Creation of risk maps for Chikungunya, Dengue and Zika viruses based on the quantitative monitoring of Aedes albopictus populations; 3. Shaping web platform models assisting the sharing of data from/to actors involved in the integrated surveillance of West Nile at the regional level; 4. Developing communication formats dealing with vector borne diseases addressing citizen and specific categories; 5. Approaching the cost-benefit analysis in the view to assist decision makers; 6. Sero-surveillance on arboviruses not included in the Ministry directive (Tick Borne Encephalitis and Toscana virus). The achieved results are presented and the acquired experience is made available to promote the adoptions on a larger scale of the approaches and methods to be certainly adapted in the specific contexts. The one health approach seems suitable to address the necessities emerging in this field by taking into consideration the different triggers shaping the dynamics we are observing.

First autochthonous dengue outbreak in Italy, August 2020.

In August 2020, during the coronavirus disease (COVID-19) pandemic, five locally acquired cases of dengue virus type 1 were detected in a family cluster in Vicenza Province, North-East Italy where Aedes albopictus mosquitoes are endemic. The primary case was an importation from West Sumatra, Indonesia. This is the first outbreak of autochthonous dengue reported in Italy. During the COVID-19 pandemic, screening of febrile travelers from endemic countries is crucial in areas where competent vectors are present.

Clinical and virological findings in patients with Usutu virus infection, northern Italy, 2018.

BackgroundUsutu virus (USUV) is a mosquito-borne flavivirus, which shares its transmission cycle with the phylogenetically related West Nile virus (WNV). USUV circulates in several European countries and its activity has increased over the last 5 years.AimTo describe human cases of USUV infection identified by surveillance for WNV and USUV infection in the Veneto Region of northern Italy in 2018.MethodsFrom 1 June to 30 November 2018, all cases of suspected autochthonous arbovirus infection and blood donors who had a reactive WNV nucleic acid test were investigated for both WNV and USUV infection by in-house molecular methods. Anti-WNV and anti-USUV IgM and IgG antibodies were detected by ELISA and in-house immunofluorescence assay, respectively; positive serum samples were further tested by WNV and USUV neutralisation assays run in parallel.ResultsEight cases of USUV infection (one with neuroinvasive disease, six with fever and one viraemic blood donor who developed arthralgia and myalgia) and 427 cases of WNV infection were identified. A remarkable finding of this study was the persistence of USUV RNA in the blood and urine of three patients during follow-up. USUV genome sequences from two patients shared over 99% nt identity with USUV sequences detected in mosquito pools from the same area and clustered within lineage Europe 2.ConclusionsClinical presentation and laboratory findings in patients with USUV infection were similar to those found in patients with WNV infection. Cross-reactivity of serology and molecular tests challenged the differential diagnosis.

Assessing the potential risk of Zika virus epidemics in temperate areas with established Aedes albopictus populations.

Based on 2015 abundance of Aedes albopictus in nine northern Italian municipalities with temperate continental/oceanic climate, we estimated the basic reproductive number R0 for Zika virus (ZIKV) to be systematically below the epidemic threshold in most scenarios. Results were sensitive to the value of the probability of mosquito infection after biting a viraemic host. Therefore, further studies are required to improve models and predictions, namely evaluating vector competence and potential non-vector transmissions.

Avian malaria parasites in the last supper: identifying encounters between parasites and the invasive Asian mosquito tiger and native mosquito species in Italy.

BACKGROUND: The invasive Asian tiger mosquito Aedes albopictus has dramatically expanded its distribution range, being catalogued as one of the world's 100 worst invasive alien species. As vectors of pathogens, Ae. albopictus may create novel epidemiological scenarios in the invaded areas. METHODS: Here, the frequency of encounters of Ae. albopictus with the avian malaria parasite Plasmodium and the related Haemoproteus was studied in an area with established populations in northeastern Italy and compared with those from four native mosquito species, Anopheles maculipennis s.l., Culex hortensis, Culex pipiens, and Ochlerotatus caspius. The abdomens of mosquitoes with a recent blood meal were used to identify both the blood meal source and the parasites harboured. RESULTS: Aedes albopictus had a clear antropophilic behaviour while An. maculipennis and Oc. caspius fed mainly on non-human mammals. Birds were the most common hosts of Cx. pipiens and reptiles of Cx. hortensis. Parasites were isolated from three mosquito species, with Cx. pipiens (30%) showing the highest parasite prevalence followed by Cx. hortensis (9%) and Ae. albopictus (5%). CONCLUSIONS: These results are the first identifying the avian malaria parasites harboured by mosquitoes in Italy and represent the first evidence supporting that, although Ae. albopictus could be involved in the transmission of avian malaria parasites, the risk of avian malaria parasite spread by this invasive mosquito in Europe would be minimal.

Seasonal and Daily Activity Patterns of Mosquito (Diptera: Culicidae) Vectors of Pathogens in Northeastern Italy.

The seasonal and daily activity of mosquito vectors of pathogens affecting animals and humans were studied in northeastern Italy at a site within the Po River Delta Park. A CDC-CO2 trap and a gravid trap were operated at 2-h intervals for 24 h every 15 d from May to October 2010. Overall, 5,788 mosquitoes comprising six species were collected, namely Culex pipiens L. (75.1% of total), Aedes caspius (Pallas) (15.2%), Aedes vexans (Meigen) (6.9%), Anopheles maculipennis s.l. Meigen (2.6%), Culiseta annulata (Schrank) (0.2%), and Culex modestus Ficalbi (<0.1%). The relative abundance of these species increased from May until the beginning of July and then decreased, disappearing at the beginning of October. The diel host-seeking patterns and oviposition site-seeking patterns were species specific and were differentially affected by the ecological variables recorded at the day and hour of mosquito collection or two weeks before collection. Knowledge of the seasonal and daily host-seeking patterns of mosquitoes highlights the time periods of the day and the seasons of potential exposure for animals and humans to mosquito-borne pathogens, therefore delineating the best time for the application of preventive measures. Furthermore, knowledge of the oviposition site-seeking activity of the mosquitoes optimizes the capture of gravid females, thereby enhancing the likelihood of detecting pathogens.

Human and entomological surveillance of West Nile fever, dengue and chikungunya in Veneto Region, Italy, 2010-2012.

BACKGROUND: Since 2010 Veneto region (North-Eastern Italy) planned a special integrated surveillance of summer fevers to promptly identify cases of West Nile Fever (WNF), dengue (DENV) and chikungunya (CHIKV). The objectives of this study were (i) To increase the detection rate of imported CHIKV and DENV cases in travellers from endemic areas and promptly identify potential autochthonous cases.(ii) To detect autochthonous cases of WNF, besides those of West Nile Neuroinvasive Disease (WNND) that were already included in a national surveillance. METHODS: Human surveillance: a traveler who had returned within the previous 15 days from endemic countries, with fever >38°C, absence of leucocytosis (leukocyte count <10,000 µL), and absence of other obvious causes of fever, after ruling out malaria, was considered a possible case of CHIKV or DENV. A possible autochthonous case of WNF was defined as a patient with fever >38°C for <7 days, no recent travel history and absence of other obvious causes of fever. Entomologic surveillance: for West Nile (WNV) it was carried out from May through November placing CDC-CO2 traps in five provinces of Veneto Region, while for DENV and CHIKV it was also performed around residences of viremic cases. RESULTS: Human surveillance: between 2010 and 2012, 234 patients with fever after travelling were screened, of which 27 (11,5%) were found infected (24 with DENV and 3 with CHIKV). No autochthonous case of DENV or CHIKV was detected. Autochthonous patients screened for WNF were 408, and 24 (5,9%) were confirmed cases. Entomologic surveillance: the WNV was found in 10, 2 and 11 pools of Culex pipiens from 2010 to 2012 respectively, in sites of Rovigo, Verona, Venezia and Treviso provinces). No infected Aedes albopictus with DENV or CHIKV was found. CONCLUSIONS: Veneto is the only Italian region reporting WNV human cases every year since 2008. WNV is likely to cause sporadic cases and unforeseeable outbreaks for decades. Including WNF in surveillance provides additional information and possibly an early alert system. Timely detection of DENV and CHIKV should prompt vector control measures to prevent local outbreaks.

Italian peninsula as a hybridization zone of Ixodes inopinatus and I. ricinus and the prevalence of tick-borne pathogens in I. inopinatus, I. ricinus, and their hybrids.

BACKGROUND: Ixodes inopinatus was described from Spain on the basis of morphology and partial sequencing of 16S ribosomal DNA. However, several studies suggested that morphological differences between I. inopinatus and Ixodes ricinus are minimal and that 16S rDNA lacks the power to distinguish the two species. Furthermore, nuclear and mitochondrial markers indicated evidence of hybridization between I. inopinatus and I. ricinus. In this study, we tested our hypothesis on tick dispersal from North Africa to Southern Europe and determined the prevalence of selected tick-borne pathogens (TBPs) in I. inopinatus, I. ricinus, and their hybrids. METHODS: Ticks were collected in Italy and Algeria by flagging, identified by sequencing of partial TROSPA and COI genes, and screened for Borrelia burgdorferi s.l., B. miyamotoi, Rickettsia spp., and Anaplasma phagocytophilum by polymerase chain reaction and sequencing of specific markers. RESULTS: Out of the 380 ticks, in Italy, 92 were I. ricinus, 3 were I. inopinatus, and 136 were hybrids of the two species. All 149 ticks from Algeria were I. inopinatus. Overall, 60% of ticks were positive for at least one TBP. Borrelia burgdorferi s.l. was detected in 19.5% of ticks, and it was significantly more prevalent in Ixodes ticks from Algeria than in ticks from Italy. Prevalence of Rickettsia spotted fever group (SFG) was 51.1%, with significantly greater prevalence in ticks from Algeria than in ticks from Italy. Borrelia miyamotoi and A. phagocytophilum were detected in low prevalence (0.9% and 5.2%, respectively) and only in ticks from Italy. CONCLUSIONS: This study indicates that I. inopinatus is a dominant species in Algeria, while I. ricinus and hybrids were common in Italy. The higher prevalence of B. burgdorferi s.l. and Rickettsia SFG in I. inopinatus compared with that in I. ricinus might be due to geographical and ecological differences between these two tick species. The role of I. inopinatus in the epidemiology of TBPs needs further investigation in the Mediterranean Basin.

Assessing the Risk of Dengue Virus Local Transmission: Study on Vector Competence of Italian Aedes albopictus.

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.

Estimating the potential risk of transmission of arboviruses in the Americas and Europe: a modelling study.

BACKGROUND: Estimates of the spatiotemporal distribution of different mosquito vector species and the associated risk of transmission of arboviruses are key to design adequate policies for preventing local outbreaks and reducing the number of human infections in endemic areas. In this study, we quantified the abundance of Aedes albopictus and Aedes aegypti and the local transmission potential for three arboviral infections at an unprecedented spatiotemporal resolution in areas where no entomological surveillance is available. METHODS: We developed a computational model to quantify the daily abundance of Aedes mosquitoes, leveraging temperature and precipitation records. The model was calibrated on mosquito surveillance data collected in 115 locations in Europe and the Americas between 2007 and 2018. Model estimates were used to quantify the reproduction number of dengue virus, Zika virus, and chikungunya in Europe and the Americas, at a high spatial resolution. FINDINGS: In areas colonised by both Aedes species, A aegypti was estimated to be the main vector for the transmission of dengue virus, Zika virus, and chikungunya, being associated with a higher estimate of R0 when compared with A albopictus. Our estimates highlighted that these arboviruses were endemic in tropical and subtropical countries, with the highest risks of transmission found in central America, Venezuela, Colombia, and central-east Brazil. A non-negligible potential risk of transmission was also estimated for Florida, Texas, and Arizona (USA). The broader ecological niche of A albopictus could contribute to the emergence of chikungunya outbreaks and clusters of dengue autochthonous cases in temperate areas of the Americas, as well as in mediterranean Europe (in particular, in Italy, southern France, and Spain). INTERPRETATION: Our results provide a comprehensive overview of the transmission potential of arboviral diseases in Europe and the Americas, highlighting areas where surveillance and mosquito control capacities should be prioritised. FUNDING: EU and Ministero dell'Università e della Ricerca, Italy (Piano Nazionale di Ripresa e Resilienza Extended Partnership initiative on Emerging Infectious Diseases); EU (Horizon 2020); Ministero dell'Università e della Ricerca, Italy (Progetti di ricerca di Rilevante Interesse Nazionale programme); Brazilian National Council of Science, Technology and Innovation; Ministry of Health, Brazil; and Foundation of Research for Minas Gerais, Brazil.

VectAbundance: a spatio-temporal database of Aedes mosquitoes observations.

Modelling approaches play a crucial role in supporting local public health agencies by estimating and forecasting vector abundance and seasonality. However, the reliability of these models is contingent on the availability of standardized, high-quality data. Addressing this need, our study focuses on collecting and harmonizing egg count observations of the mosquito Aedes albopictus, obtained through ovitraps in monitoring and surveillance efforts across Albania, France, Italy, and Switzerland from 2010 to 2022. We processed the raw observations to obtain a continuous time series of ovitraps observations allowing for an extensive geographical and temporal coverage of Ae. albopictus population dynamics. The resulting post-processed observations are stored in the open-access database VectAbundance.This initiative addresses the critical need for accessible, high-quality data, enhancing the reliability of modelling efforts and bolstering public health preparedness.

Development of microsatellite markers for the invasive mosquito Aedes koreicus.

BACKGROUND: Aedes koreicus is a mosquito species native to East Asia which has recently invaded several countries in Europe. In Italy, this mosquito was first detected in the North-East in 2011 and is now widely distributed in the entire northern part of the country. The development of specific genetic markers, such as microsatellites, is necessary to uncover the dispersal routes of this mosquito from its native areas and, eventually, to plan future control interventions. METHODS: Available raw sequences of genomic DNA of Ae. koreicus were screened in silico using BLASTn to identify possible microsatellite-containing sequences. Specific primer pairs were then designed, and their efficiency was determined through polymerase chain reaction (PCR) on 32 individuals of Ae. koreicus collected in Italy. PCR conditions were optimised in three multiplex reactions. Genotyping of individual mosquitoes was performed on both single and multiplex PCR reactions. Finally, analysis of intra-population variation was performed to assess the level of polymorphism of the markers. RESULTS: Mosquito genotyping provided consistent results in both single and multiplex reactions. Out of the 31 microsatellite markers identified in the Ae. koreicus genome raw sequences, 11 were polymorphic in the examined mosquito samples. CONCLUSIONS: The results show that the 11 microsatellite markers developed here hold potential for investigating the genetic structure of Ae. koreicus populations. These markers could thus represent a novel and useful tool to infer the routes of invasion of this mosquito species into Europe and other non-native areas.

Insects and Public Health: An Overview.

Insects are, by far, the most common animals on our planet. The ubiquity and plethora of ecological niches occupied by insects, along with the strict and sometimes forced coexistence between insects and humans, make insects a target of public health interest. This article reports the negative aspects historically linked to insects as pests and vectors of diseases, and describes their potential as bioindicators of environmental pollution, and their use as food and feed. Both negative and positive impacts of insects on human and animal health need to be addressed by public health professionals who should aim to strike a balance within the wide range of sometimes conflicting goals in insect management, such as regulating their production, exploiting their potential, protecting their health and limiting their negative impact on animals and humans. This requires increased insect knowledge and strategies to preserve human health and welfare. The aim of this paper is to provide an overview of traditional and emerging topics bridging insects and public health to highlight the need for professionals, to address these topics during their work. The present and future role and activities of public health authorities regarding insects are analyzed.

A modified BG-Sentinel trap equipped with FTA card as a novel tool for mosquito-borne disease surveillance: a field test for flavivirus detection.

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.

Shallow Whole-Genome Sequencing of Aedes japonicus and Aedes koreicus from Italy and an Updated Picture of Their Evolution Based on Mitogenomics and Barcoding.

Aedes japonicus and Aedes koreicus are two invasive mosquitoes native to East Asia that are quickly establishing in temperate regions of Europe. Both species are vectors of arboviruses, but we currently lack a clear understanding of their evolution. Here, we present new short-read, shallow genome sequencing of A. japonicus and A. koreicus individuals from northern Italy, which we used for downstream phylogenetic and barcode analyses. We explored associated microbial DNA and found high occurrences of Delftia bacteria in both samples, but neither Asaia nor Wolbachia. We then assembled complete mitogenomes and used these data to infer divergence times estimating the split of A. japonicus from A. koreicus in the Oligocene, which was more recent than that previously reported using mitochondrial markers. We recover a younger age for most other nodes within Aedini and other Culicidae. COI barcoding and phylogenetic analyses indicate that A. japonicus yaeyamensis, A. japonicus amamiensis, and the two A. koreicus sampled from Europe should be considered as separate species within a monophyletic species complex. Our studies further clarify the evolution of A. japonicus and A. koreicus, and indicate the need to obtain whole-genome data from putative species in order to disentangle their complex patterns of evolution.

Surveillance for West Nile, dengue, and chikungunya virus infections, Veneto Region, Italy, 2010.

In 2010, in Veneto Region, Italy, surveillance of summer fevers was conducted to promptly identify autochthonous cases of West Nile fever and increase detection of imported dengue and chikungunya in travelers. Surveillance highlighted the need to modify case definitions, train physicians, and when a case is identified, implement vector control measures.

Updated occurrence and bionomics of potential malaria vectors in Europe: a systematic review (2000-2021).

Despite the eradication of malaria across most European countries in the 1960s and 1970s, the anopheline vectors are still present. Most of the malaria cases that have been reported in Europe up to the present time have been infections acquired in endemic areas by travelers. However, the possibility of acquiring malaria by locally infected mosquitoes has been poorly investigated in Europe, despite autochthonous malaria cases having been occasionally reported in several European countries. Here we present an update on the occurrence of potential malaria vector species in Europe. Adopting a systematic review approach, we selected 288 papers published between 2000 and 2021 for inclusion in the review based on retrieval of accurate information on the following Anopheles species: An. atroparvus, An. hyrcanus sensu lato (s.l.), An. labranchiae, An. maculipennis sensu stricto (s.s.), An. messeae/daciae, An. sacharovi, An. superpictus and An. plumbeus. The distribution of these potential vector species across Europe is critically reviewed in relation to areas of major presence and principal bionomic features, including vector competence to Plasmodium. Additional information, such as geographical details, sampling approaches and species identification methods, are also reported. We compare the information on each species extracted from the most recent studies to comparable information reported from studies published in the early 2000s, with particular reference to the role of each species in malaria transmission before eradication. The picture that emerges from this review is that potential vector species are still widespread in Europe, with the largest diversity in the Mediterranean area, Italy in particular. Despite information on their vectorial capacity being fragmentary, the information retrieved suggests a re-definition of the relative importance of potential vector species, indicating An. hyrcanus s.l., An. labranchiae, An. plumbeus and An. sacharovi as potential vectors of higher importance, while An. messeae/daciae and An. maculipennis s.s. can be considered to be moderately important species. In contrast, An. atroparvus and An. superpictus should be considered as vectors of lower importance, particularly in relation to their low anthropophily. The presence of gaps in current knowledge of vectorial systems in Europe becomes evident in this review, not only in terms of vector competence but also in the definition of sampling approaches, highlighting the need for further research to adopt the appropriate surveillance system for each species.

Comparative efficacy of BG-Sentinel 2 and CDC-like mosquito traps for monitoring potential malaria vectors in Europe.

BACKGROUND: Different trapping devices and attractants are used in the mosquito surveillance programs currently running in Europe. Most of these devices target vector species belonging to the genera Culex or Aedes, and no studies have yet evaluated the effectiveness of different trapping devices for the specific targeting of Anopheles mosquito species, which are potential vectors of malaria in Europe. This study aims to fill this gap in knowledge by comparing the performance of trapping methods that are commonly used in European mosquito surveillance programs for Culex and Aedes for the specific collection of adults of species of the Anopheles maculipennis complex. METHODS: The following combinations of traps and attractants were used: (i) BG-Sentinel 2 (BG trap) baited with a BG-Lure cartridge (BG + lure), (ii) BG trap baited with a BG-Lure cartridge and CO2 (BG + lure + CO2), (iii) Centers for Disease Control and Prevention-like trap (CDC trap) baited with CO2 (CDC + CO2), (iv) CDC trap used with light and baited with BG-Lure and CO2 (CDC light + lure + CO2). These combinations were compared in the field using a 4 × 4 Latin square study design. The trial was conducted in two sites in northeastern Italy in 2019. Anopheles species were identified morphologically and a sub-sample of An. maculipennis complex specimens were identified to species level by molecular analysis. RESULTS: Forty-eight collections were performed on 12 different trapping days at each site, and a total of 1721 An. maculipennis complex specimens were captured. The molecular analysis of a sub-sample comprising 254 specimens identified both Anopheles messeae/Anopheles daciae (n = 103) and Anopheles maculipennis sensu stricto (n = 8) at site 1, while at site 2 only An. messeae/An. daciae (n = 143) was found. The four trapping devices differed with respect to the number of An. messeae/An. daciae captured. More mosquitoes were caught by the BG trap when it was used with additional lures (i.e. BG + lure + CO2) than without the attractant, CO2 [ratioBG+lure vs BG+lure+CO2 = 0.206, 95% confidence interval (CI) 0.101-0.420, P < 0.0001], while no significant differences were observed between CDC + CO2 and CDC light + lure + CO2 (P = 0.321). The addition of CO2 to BG + lure increased the ability of this combination to capture An. messeae/An. daciae by a factor of 4.85, and it also trapped more mosquitoes of other, non-target species (Culex pipiens, ratioBG+lure vs BG+lure+CO2 = 0.119, 95% CI 0.056-0.250, P < 0.0001; Ochlerotatus caspius, ratioBG+lure vs BG+lure+CO2 = 0.035, 95% CI 0.015-0.080, P < 0.0001). CONCLUSIONS: Our results show that both the BG-Sentinel and CDC trap can be used to effectively sample An. messeae/An. daciae, but that the combination of the BG-Sentinel trap with the BG-Lure and CO2 was the most effective means of achieving this. BG + lure + CO2 is considered the best combination for the routine monitoring of host-seeking An. maculipennis complex species such as An. messeae/An. daciae. The BG-Sentinel and CDC traps have value as alternative methods to human landing catches and manual aspiration for the standardized monitoring of Anopheles species in Europe.