Felix Gregor Sauer

Until recently, Anopheles petragnani , a member of the Anopheles claviger complex, was only known from the western Mediterranean region. In Germany, a stable population has been reported to occur at least since 2015 in the southwest, with further isolated specimens being collected in the federal state of Bavaria. Starting in 2021, a mosquito breeding site survey and adult trapping were initiated in northern Rhineland-Palatinate. Following the observation that two breeding sites from that area in 2025 produced unusually dark adult An. claviger complex specimens emerging from the collected larvae and pupae, we morphologically analysed fourth instar larvae and identified not only An. claviger sensu stricto but predominantly An. petragnani . Further field sampling and subsequent molecular analyses revealed An. petragnani to occur at six additional sites, along with several sites harbouring An. claviger s.s. only. The morphological examinations unveiled previously undescribed colour differences between the two species, namely of the larval antennae and of the adult halteres. It remains open whether the new localities must be attributed to new introduction events, range expansion or the possibility that these An. petragnani breeding sites were simply overlooked in the past. The updated species’ northern distribution limit in Germany shifts by some 200 km to ca. 50° northern latitude.

The globally distributed arbovirus West Nile virus (WNV) is expanding across Europe, with rising numbers of human cases and an increasingly broad geographic distribution. Transmitted primarily by Culex mosquitoes, WNV lineages 1 and 2 are pathogenic to humans, yet their differences in transmission dynamics remain unclear. As vertebrate hosts develop varying viremia, mosquitoes encounter a wide range of viral doses, which may influence transmission. Here, we investigated the dose-response differences between the lineages in Cx. pipiens biotype pipiens , Cx. torrentium , and Aedes albopictus . Mosquitoes were fed increasing titers of one strain of either WNV lineage, and infection and transmission were assessed after 14 days at 24 °C. Lineage 2 required lower infectious doses while causing higher transmission. Notably, Cx. torrentium was identified as a highly competent vector for lineage 2. These findings highlight important differences between WNV lineages, improve our understanding of dose-dependent transmission, and therefore support refined risk assessments in Europe.

Background: Mosquitoes are well known for their ability to transmit pathogens, including various arthropod-borne viruses (arboviruses) of veterinary and medical interest. In Europe, the increasing public health relevance of mosquito-borne pathogens highlights the need to understand how environmental drivers shape mosquito population dynamics relevant to transmission risk. Culex pipiens sensu stricto (s.s.) and Culex torrentium (collectively referred to here as Cx. pipiens s.s./ Cx. torrentium ) are the primary vectors of Usutu virus and West Nile virus in Europe and are commonly found in and around human settlements. The prediction of their spatial-temporal abundance supports early assessment of arbovirus transmission risk and the planning of effective intervention methods, such as vector control. Methods: A process-based model was developed to simulate the spatial-temporal occurrence of Cx. pipiens s.s./ Cx. torrentium in Germany, with a particular focus on depicting realistic overwintering behaviour, including, diapause induced through photoperiod and temperature in the larval stage. The model output is driven by local temperature and rainfall data provided by the European re-analysis and observations for monitoring data set and evaluated with field data from 106 sampling sites in Germany. Results: A significant association between relative simulated and observed mosquito abundance was found for 75% of the sampling sites, using site-specific beta regression models. An overall beta mixed-effects model across all sites was also significant (estimate = 2.17, standard error = 0.062, Z -value = 35.03, p -value < 0.0001, marginal R ² = 0.4). Conclusions: This model offers a robust framework for the depiction of the mosquito population dynamics of Cx. pipiens s.s./ Cx. torrentium under current and future climate scenarios, thereby supporting vector surveillance and control strategies across Europe. Graphical

1. Diptera represent a diverse insect order, including vectors of human and animal pathogens. Their accurate species identification remains a major bottleneck in ecological and epidemiological studies. Morphological identification requires taxonomic expertise, while molecular methods are costly and not universally reliable. Wing geometric morphometrics offers an alternative, but manual landmark annotation is time-consuming and introduces observer bias. 2. We developed ITHILDIN, an automated pipeline for landmark and semilandmark annotation of Diptera wings, combining UNet++ segmentation and an Hourglass landmark prediction model. Using mosquitoes as the primary model system, we extended an existing repository with 5,793 additional images. Models were trained on 5991 annotations of landmarks and segmentations and then evaluated on 12,522 images across 34 taxa. We assessed landmark prediction accuracy against human observers and ML-morph, evaluated species identification using Linear Discriminant Analysis on 17 homologous landmarks and 52 semilandmarks, and tested out-of-distribution generalisation by reproducing an independent study. Transferability was demonstrated by adapting the pipeline to the Dipteran families Drosophilidae and Glossinidae. 3. The Hourglass model achieved a mean landmark error of 4.5 pixels (95% CI: 4.3–4.6), within human observer variability (4.7 pixels, 95% CI: 4.4–5.0) and substantially outperforming ML-Morph (12.7 pixels, 95% CI: 11.1–14.2). The semilandmark-based approach for species identification achieved 91% balanced accuracy across 34 taxa, comparable to CNN performance (94%). On out-of-distribution data, the landmark pipeline generalised substantially better than the CNN and a soft-voting ensemble of the landmark and CNN classifiers achieved 88% balanced accuracy on a replicated study. 4. Combining geometric morphometrics with deep learning provides a reproducible, interpretable, and generalisable alternative to black-box CNN classifiers for Diptera wing analysis. By acting as a consistent single observer comparable to human annotation, the system eliminates inter-observer bias, enabling large-scale and cross-study morphometric analyses of Dipteran wings. The system is publicly available at www.ithildin.bnitm.de and transferable to other Diptera families with moderate retraining effort. Data availability: Images used in this study are accessible under CC BY 4.0 license at https://doi.org/10.6019/S-BIAD1478 . Downloadable and installable docker application can be accessed on the applications’ git page: https://anonymous.4open.science/r/ITHILDIN-4313/

Accurate species identification is crucial to assess the medical and veterinary relevance of a mosquito specimen, but it requires high experience of the observers and well-equipped laboratories. This study aimed to evaluate whether low-cost imaging in combination with geometric wing morphometrics can provide accurate identification of invasive, morphologically similar Aedes species. The right wings of 670 female specimens covering 184 Ae. aegypti , 156 Ae. albopictus , 166 Ae. j. japonicus and 164 Ae. koreicus , were removed, mounted and photographed with a professional stereomicroscope (Olympus SZ61, Olympus, Tokyo, Japan) and a macro lens (Apexel-24XMH, Apexel, Shenzhen, China) attached to a smartphone. The coordinates of 18 landmarks on the vein crosses were digitalized by a single observer for each image. In addition, the landmarks of 20 specimens per species and imaging device were digitalized by six different observers to assess the degree of the observer error. The superimposed shape variables were used to compare the species classification accuracy of linear discriminant analysis (LDA), support vector machine (SVM), Random Forest (RF), and XGBoost. In the single-observer landmark data, the LDA achieved the best classification results with a mean accuracy of 95 % for landmarks from microscope images and 92 % for those obtained from smartphone images. In the multi-observer landmark data, LDA consistently performed worse than the other three classifiers, and the reduction in the accuracy was more pronounced for smartphone images than for microscope images. This pattern was associated with a higher degree of observer error for smartphone images, as confirmed by a landmark-wise comparison across all landmarks. Geometric wing morphometrics provides a reliable method to distinguish the most common invasive Aedes species in Europe. Thereby, the image quality obtained by smartphones equipped with a macro lens is sufficient and represents a cost-effective alternative to professional microscopes. However, due to the greater degree of observer variation for smartphone images, landmark coordinates for such images should ideally be collected by a single observer.

Comprehensive risk assessment for vector-borne disease requires effective monitoring of various vector groups. However, this depends on the traps’ ability to capture multiple vector families. This study compares 3 commercially available traps, BG-Sentinel 2, BG-Trap Station as a combination of a BG-Pro and Gravid trap, and the Encephalitis Virus Surveillance trap, with the VectorCube, a newly developed prototype. Multiple openings enable multidirectional vector capture, and color cues can be easily attached and changed. All traps were tested under a uniform, carbon dioxide flow rate at 10 sampling sites (6 in Germany and 4 in Gabon) using a 4 × 4 Latin square design. Each site completed at least 1 full Latin square rotation, resulting in 48 trap comparison days. A total of 3,477 blood-seeking insects from 6 families were captured: 2,797 Culicidae, 324 Simuliidae, 268 Ceratopogonidae, 76 Muscidae, 8 Glossinidae, and 4 Psychodidae. Most Culicidae were collected with the BG-Sentinel 2 followed by the VectorCube. The VectorCube captured the majority of Simuliidae, Muscidae, and Glossinidae observed in this study, while Psychodidae were exclusively collected with BG-Sentinel 2 and BG-Trap Station in Gabon. Mean family richness per trapping session was highest in the VectorCube, followed by BG-Sentinel 2, BG-Trap Station, and Encephalitis Virus Surveillance trap. Overall, the VectorCube and BG-Sentinel 2 demonstrated the highest performance in vector abundance and diversity, driven primarily by high Culicidae catches, whereas captures of non-mosquito hematophagous Diptera occured at lower frequencies and were more site-dependent, including sporadic captures of larger-bodied vectors such as Muscidae and Glossinidae.

The globally distributed arbovirus West Nile virus (WNV) continues to expand across Europe, with rising numbers of human cases and an increasingly broad geographic distribution. WNV is primarily transmitted by mosquitoes of the genus Culex . Out of nine WNV lineages, human pathogenicity has been clearly established for lineages 1 and 2, but differences in their transmission dynamics, such as minimal infectious dose and transmission efficiency, remain poorly understood. In this study, we investigated how viral lineage, mosquito species, and blood meal titer influence the vector competence of two primary WNV vectors from Europe, Cx. pipiens biotype pipiens and Cx. torrentium , as well as the invasive mosquito species Aedes albopictus . Female mosquitoes were fed with increasing blood meal titers containing either of the WNV lineages. After an incubation period of 14 days at a mean temperature of 24°C, mosquito body titers were quantified, and the presence of infectious viral particles in the saliva was assessed. Our results revealed clear differences between the two lineages. Lineage 2 resulted in higher transmission efficiencies across all three species and required lower infectious doses to cause transmission. Among the tested species, Cx. torrentium proved to be a highly competent vector (max. transmission efficiency = 30%, minimum infectious dose = 10 ⁵ TCID ₅₀ /mL), despite its underrepresentation in research. These findings provide detailed insights into how viral lineage, mosquito species, and blood meal titer might shape WNV transmission, informing future risk assessments and efforts to mitigate WNV transmission in Europe.

Tahyna virus (TAHV) was the first mosquito-borne virus isolated in Europe, and has since been found throughout Eurasia and Africa. Infections are mostly asymptomatic but can cause “Valtice fever”, characterized by influenza-like symptoms, mainly in children, with severe cases occasionally causing neurological symptoms. The virus is maintained in an enzootic cycle between small mammals and mosquitoes. Recent and comprehensive studies of vector competence for TAHV are scarce. To fill this gap, and to better understand the transmission cycle of TAHV, we studied ten taxa (Ae. aegypti, Ae. albopictus, Ae. japonicus japonicus, Ae. koreicus, Ae. rusticus, Cx. pipiens biotype pipiens, Cx. torrentium, Cs. morsitans/fumipennis, An. daciae, and An. stephensi) by orally infecting them with TAHV. All taxa were susceptible to TAHV infection. Additionally, the invasive species Ae. albopictus, and Ae. rusticus, a species native to Europe, were able to transmit the virus at 27 ± 5 °C, with transmission efficiencies of 3.3% and 14.3%, respectively. Therefore, it is plausible for TAHV to be transmitted by Ae. albopictus and Ae. rusticus in natural settings in Europe. At a lower temperature of 24 ± 5 °C, infection and transmission decreased in Ae. albopictus. This data will allow future risk models and early warning systems to better predict TAHV transmission.