MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION OF AMBLYOMMA SCUTATUM (ACARI: IXODIDAE) ACCIDENTALLY INTRODUCED IN ITALY.

Eight ticks were found in Comacchio (FE), Italy parasitizing a young black iguana (Ctenosaura similis) that had been accidentally transported in a commercial plant container from Costa Rica. Specimens were identified morphologically as Amblyomma scutatum and then confirmed by the barcoding of the mitochondrial cytochrome c oxidase subunit 1 gene. Amblyomma scutatum is a common tick known to infest reptiles in Central America, Mexico, and Venezuela, but not in Europe. In Italy, the possibility for this tick to become endemic is unlikely because of the absence of its principal hosts. Nevertheless, this finding confirms the high risk of introducing exotic species that is linked with global commerce and therefore the need for veterinary control of shipments.

Arbovirus screening of mosquitoes collected in 2022 in Emilia-Romagna, Italy, with the implementation of a real-time PCR for the detection of Tahyna virus.

Several Arboviruses (Arthropod-borne virus) are a concrete health risk. While some arboviruses, such as the West Nile virus (WNV) and the Usutu virus (USUV) are actively surveyed, others are neglected, including the Tahyna virus (TAHV). In this work, we tested - searching for all the three viruses - 37,995 mosquitoes collected in 95 attractive traps, baited by carbon dioxide, distributed in the lowlands of Emilia-Romagna, northern Italy, between 19 July and 12 August 2022. Among the 668 pools obtained, WNV was detected in 45 pools of Culex (Cx.) pipiens and USUV was recorded in 24 pools of the same mosquito; ten of these Cx. pipiens pools tested positive for both WNV and USUV. Interestingly, we recorded a significant circulation of both WNV lineage 1 (WNV-L1) and lineage 2 (WNV-L2): WNV-L1 strains were detected in 40 pools, WNV-L2 strains in three pools and both lineages were detected in two pools. TAHV was detected in 8 different species of mosquitoes in a total of 37 pools: Aedes (Ae.) caspius (25), Ae. albopictus (5), Ae. vexans (3), Cx. pipiens (2), Ae. cinereus (1) and Anopheles maculipennis sl (1). The significant number of Ae. caspius-pools tested positive and the estimated viral load suggest that this mosquito is the principal vector in the surveyed area. The potential involvement of other mosquito species in the TAHV cycle could usefully be the subject of further experimental investigation. The results obtained demonstrate that, with adequate sampling effort, entomological surveillance is able to detect arboviruses circulating in a given area. Further efforts must be made to better characterise the TAHV cycle in the surveyed area and to define health risk linked to this virus.

Detection of West Nile and Usutu Virus RNA in Autumn Season in Wild Avian Hosts in Northern Italy.

West Nile (WNV) and Usutu (USUV) viruses are two mosquito-borne viruses belonging to the family Flaviviridae and genus Flavivirus. The natural transmission cycle of WNV and USUV involves mosquitoes and birds, while mammals are thought to be accidental hosts. The goal of this study was to report-in the context of "off-season monitoring" and passive surveillance-the detection of WNV and USUV RNA in wild birds. To this end, we analyzed biological samples of wild birds in Northern Italy, from October to May, hence outside of the regional monitoring period (June-September). The virological investigations for the detection of USUV and WNV RNA were performed using real-time PCR on frozen samples of the brain, myocardium, kidney, and spleen. In a total sample of 164 wild birds belonging to 27 different species, sequences of both viruses were detected: four birds (2.44%) were positive for WNV and five (3.05%) for USUV. Off-season infections of WNV and especially USUV are still widely discussed and only a few studies have been published to date. To the best of our knowledge, this study is the first report on the detection of USUV RNA until December 22nd. Although further studies are required, our results confirm the viral circulation out-of-season of Flavivirus in wild birds, suggesting reconsidering the epidemiological monitoring period based on each individual climate zone and taking into consideration global warming which will play an important role in the epidemiology of vector-borne diseases.

Insect Colonisation and the Decomposition Process in Aerated versus Watertight Burial Systems.

In recent years, burial systems have covered increasingly higher costs due to the pollution caused by decomposition products. These products are understood as chemicals and microorganisms in the surrounding soil and groundwater and represent a topical issue. The purpose of this research was to ascertain the extent of decomposition when pig carcasses are buried in two different burial systems ("aerated" vs. "watertight") and catalogue the arthropods associated with burials at different time-points of removal from niches (after 6, 12, 24, 36, and 60 months). Thirteen taxa were collected in aerated niches, whereas five were collected in watertight niches. The initial access or exclusion of insect colonisers affected overall functional activity. Two Diptera species, Hydrotaea capensis and Megaselia scalaris, were the most abundant, supporting the hypothesis that insects can colonise carcasses in aerated burial systems. Furthermore, some species of bacteria have been documented as facilitators of the initial decomposition process of the carcass. Most bacterial colonies develop only in aerated niches. The trial showed that the first enzymatic-bacterial and insect actions helped promote the process of cadaveric decomposition and later skeletonisation, mainly when associated with aeration modes of the tomb/mound. The results obtained provide essential information on the process of human decomposition and taphonomy in cemeteries. Moreover, these data could benefit forensic science by adding information on insect colonisation and body modification in medico-legal investigations concerning the post-mortem interval in exhumed bodies and illegal burials.

Monitoring of Insecticide Resistance Mutations and Pathogen Circulation in Sand Flies from Emilia-Romagna, a Leishmaniasis Endemic Region of Northern Italy.

The continuously expanding distribution of sand flies, proven vectors of Leishmania and of several phleboviruses, is a growing public health issue in Europe. Especially in Italy, visceral leishmaniasis (VL) is occurring with increasing incidence northward, in previously non-endemic provinces. Around the globe, disease elimination efforts largely focus on sand fly vector insecticidal control, often leading to the development of resistance. In Emilia-Romagna (ER), northern Italy, insecticides are heavily applied for agricultural and mosquito control, but not specifically against sand flies. Here, we investigated the sand fly species composition in certain environmental settings in ER provinces and monitored the presence of pyrethroid resistance mutations and pathogen circulation. Phlebotomus perfiliewi, a dominant vector of Leishmania infantum, was detected almost exclusively in the region. No mutations in the voltage-gated sodium channel gene, e.g., knock-down resistance mutations I1011M, L1014F/S, V1016G, or F1020S, were recorded. Pathogen monitoring revealed that almost 40% of the tested sand fly pools were positive for Leishmania, while the presence of Toscana and Fermo phleboviruses was also observed in much lower frequencies (≤3% positive pools). Regular epidemiological and entomological monitoring, alongside resistance surveillance, is highly recommended to ensure the sustainability and efficiency of vector control interventions.

Arbovirus Screening in Mosquitoes in Emilia-Romagna (Italy, 2021) and Isolation of Tahyna Virus.

Several viruses can be transmitted by mosquitoes. We searched some of these viruses in 20,778 mosquitoes, collected in 95 traps on the plains of Emilia-Romagna (North of Italy) in 2021. We detected West Nile virus (WNV) and Usutu virus (USUV) in pools of Culex (Cx.) pipiens. In addition, we detected two insect-specific flaviviruses in three pools of Aedes (Ae.) caspius and in two of Ae. vexans. Tahyna virus (TAHV) was detected in six pools, three of Ae. caspius and three of Cx. pipiens, and one isolated strain was obtained from one of the Ae. caspius pools. Moreover, we detected TAHV in pools of several mosquito species (Ae. caspius, Ae. vexans, Ae. albopictus, Anopheles maculipennis s.l.) collected in the previous year of surveillance. Our data indicate Ae. caspius as the species most infected with TAHV in the surveyed area. Together with the likely plasticity of the cycle, we reported strong genome stability of the TAHV, probably linked to a successful adaptation of the virus to its ecological niche. Interestingly, in six pools of Cx. pipiens we detected two associated viruses among USUV, WNV, TAHV and all the three viruses in two pools. This result allows us to assume the presence of particular conditions that prompt the circulation of arboviruses, creating the conditions for viral hot spots. While no human diseases related to Tahyna virus were reported in Italy, its detection over the years suggests that it is worth investigating this virus as a potential cause of disease in humans in order to assess its health burden. IMPORTANCE We reported in this work the detection of three Arboviruses (Arthropod-borne viruses) in mosquitoes collected in Emilia-Romagna in 2021. In addition to West Nile and Usutu viruses, which were reported from more than 10 years in the study area, we detected and isolated Tahyna virus (TAHV). We also reported detections of TAHV obtained in previous years of surveillance in different species of mosquitoes. TAHV is the potential causative agent of summer influenza-like diseases and also of meningitis. Even if human cases of disease referable to this virus are not reported in Italy, its relevant presence in mosquitoes suggests investigating the possibility they could.

Fate of Salmonella enterica Typhimurium and Listeria monocytogenes in Black Soldier Fly (Hermetia illucens) Larvae Reared on Two Artificial Diets.

Ensuring food security is one of the main challenges facing the world over the next 30 years. There is, thus, an urgent need to significantly increase the supply of sustainable protein that can be transformed into animal feed. Proteins from insects offer a valuable alternative. This article presents the results of challenge tests conducted to investigate the dynamics of the microbial load of Salmonella enterica Typhimurium and Listeria monocytogenes in black soldier fly (Hermetia illucens) larvae grown on contaminated substrates. Four separate challenge tests were performed on two substrates: the Gainesville diet and a homemade diet. The challenge test procedure was carried out in accordance with ISO/DIS 20976-2 (under development). The results of this study show that, when grown on contaminated substrates, BSF larvae do not eliminate Salmonella Typhimurium or L. monocytogenes, but can reduce their microbial load. Sanitation processes downstream of the breeding of BSF larvae are, however, required to reduce the microbiological risks of this novel food.

Geometric morphometrics and machine learning as tools for the identification of sibling mosquito species of the Maculipennis complex (Anopheles).

Geometric morphometrics allows researchers to use the specific software to quantify and to visualize morphological differences between taxa from insect wings. Our objective was to assess wing geometry to distinguish four Anopheles sibling species of the Maculipennis complex, An. maculipennis s. s., An. daciae sp. inq., An. atroparvus and An. melanoon, found in Northern Italy. We combined the geometric morphometric approach with different machine learning alghorithms: support vector machine (SVM), random forest (RF), artificial neural network (ANN) and an ensemble model (EN). Centroid size was smaller in An. atroparvus than in An. maculipennis s. s. and An. daciae sp. inq. Principal component analysis (PCA) explained only 33% of the total variance and appeared not very useful to discriminate among species, and in particular between An. maculipennis s. s. and An. daciae sp. inq. The performance of four different machine learning alghorithms using procrustes coordinates of wing shape as predictors was evaluated. All models showed ROC-AUC and PRC-AUC values that were higher than the random classifier but the SVM algorithm maximized the most metrics on the test set. The SVM algorithm with radial basis function allowed the correct classification of 83% of An. maculipennis s. s. and 79% of An. daciae sp. inq. ROC-AUC analysis showed that three landmarks, 11, 16 and 15, were the most important procrustes coordinates in mean wing shape comparison between An. maculipennis s. s. and An. daciae sp. inq. The pattern in the three-dimensional space of the most important procrustes coordinates showed a clearer differentiation between the two species than the PCA. Our study demonstrated that machine learning algorithms could be a useful tool combined with the wing geometric morphometric approach.