Natural volatiles preventing mosquito biting: an integrated screening platform for accelerated discovery of ORco antagonists.

Insect olfactory receptors are heteromeric ligand-gated cation channels composed of an obligatory receptor subunit, ORco, and one of many variable subunits, ORx, in as yet undefined molar ratios. When expressed alone ex vivo, ORco forms homotetrameric channels gated by ORco-specific ligands acting as channel agonists. Using an insect cell-based system as a functional platform for expressing mosquito odorant receptors ex vivo, we identified small molecules of natural origin acting as specific ORco channel antagonists, orthosteric or allosteric relative to a postulated ORco agonist binding site, which cause severe inhibition of olfactory function in mosquitoes. In the present communication, we have compiled common structural features of such orthosteric antagonists and developed a ligand-based pharmacophore whose properties are deemed necessary for binding to the agonist binding site and causing inhibition of ORco's biological function. In silico screening of an available collection of natural volatile compounds with the pharmacophore resulted in identification of several ORco antagonist hits. Cell-based functional screening of the same compound collection resulted in the identification of several compounds acting as orthosteric and allosteric antagonists of ORco channel function ex vivo and inducing anosmic behaviors to Aedes albopictus mosquitoes in vivo. Comparison of the in silico screening results with those of the functional assays revealed that the pharmacophore predicted correctly 7 out of the 8 confirmed orthosteric antagonists and none of the allosteric ones. Because the pharmacophore screen produced additional hits that did not cause inhibition of the ORco channel function, we also generated a Support Vector Machine (SVM) model based on two descriptors of all pharmacophore hits. Training of the SVM on the ex vivo validated compound collection resulted in the selection of the confirmed orthosteric antagonists with a very low cross-validation out-of-sample misclassification rate. Employment of the combined pharmacophore-SVM platform for in silico screening of a larger collection of olfaction-relevant volatiles produced several new hits. Functional validation of randomly selected hits and rejected compounds from this screen confirmed the power of this virtual screening platform as a convenient tool for accelerating the pace of discovery of novel vector control agents. To the best of our knowledge, this study is the first one that combines a pharmacophore with a SVM model for identification of AgamORco antagonists and specifically orthosteric ones.

Volatile allosteric antagonists of mosquito odorant receptors inhibit human-host attraction.

Odorant-dependent behaviors in insects are triggered by the binding of odorant ligands to the variable subunits of heteromeric olfactory receptors. Previous studies have shown, however, that specific odor binding to ORco, the common subunit of odorant receptor heteromers, may allosterically alter olfactory receptor function and profoundly affect subsequent behavioral responses. Using an insect cell-based screening platform, we identified and characterized several antagonists of the odorant receptor coreceptor of the African malaria vector Anopheles gambiae (AgamORco) in a small collection of natural volatile organic compounds. Because some of the identified antagonists were previously shown to strongly repel Anopheles and Culex mosquitoes, we examined the bioactivities of the identified antagonists against Aedes, the third major genus of the Culicidae family. The tested antagonists inhibited the function of Ae. aegypti ORco ex vivo and repelled adult Asian tiger mosquitoes (Ae. albopictus). Binary mixtures of specific antagonists elicited higher repellency than single antagonists, and binding competition assays suggested that this enhanced repellence is due to antagonist interaction with distinct ORco sites. Our results also suggest that the enhanced mosquito repellency by antagonist mixtures is due to additive rather than synergistic effects of the specific antagonist combinations on ORco function. Taken together, these findings provide novel insights concerning the molecular aspects of odorant receptor function. Moreover, our results demonstrate that a simple screening assay may be used for the identification of allosteric modifiers of olfactory-driven behaviors capable of providing enhanced personal protection against multiple mosquito-borne infectious diseases.

Sublethal effects of oregano essential oil and its major compound carvacrol on biological parameters of Aedes albopictus (Diptera: Culicidae).

Mosquito management programs rely basically on the use of conventional synthetic larvicides. However, frequent applications and misuse of some synthetic insecticides have led to problems related to mosquito resistance development, harmful effects on human health and unacceptable environmental effects on non-target organisms. Recently, a growing number of phytochemicals has been tested as more eco-friendly larvicides against various mosquito species, exerting high efficacy with multiple modes of action. In the laboratory, we investigated for the first time the sublethal effects of oregano oil and its major compound carvacrol, against Aedes albopictus (Asian tiger mosquito), a mosquito of great medical importance. We determined the effects of short term (24h) exposure of 3rd- 4th larvae to LC50 concentrations on survival and development of survived larvae until adulthood, as well as on fecundity, fertility, longevity and wing length of emerged adults. Only half of 24h survived larvae from oregano oil and carvacrol treatment finally reached adulthood. Abnormal shapes of dead larvae and pupae, and failed adult emergence were also observed, indicating a potential growth inhibitory activity of the tested materials. No particular effects from exposure to larvicidal LC50 concentrations were recorded on life cycle parameters of successfully emerged adults. These findings suggest the tested oregano oil and carvacrol as sufficiently effective larvicides against Ae. albopictus at lower than the acutely toxic concentrations, promoting a more eco-friendly and less costly profile for these biopesticides.

Systematic review: Yellow fever control through environmental management mechanisms.

OBJECTIVE: Recent research on mosquito vector-borne diseases points to the possibility for a re-emergence of yellow fever. This study investigated attempts at utilising environmental methods and their efficacy for the control of yellow fever and its main vector, Aedes aegypti. METHODS: Potentially eligible studies were searched in Cochrane Library (Reviews and Trials), the Global Index Medicus (encompassing thus the African Index Medicus, the Index Medicus for the Eastern Mediterranean Region, the Index Medicus for the South-East Asia Region, the Latin America and the Caribbean Literature on Health Sciences and the Western Pacific Region Index Medicus), Google Scholar, PubMed and Science Direct. RESULTS: Of a total number of 172 eligible studies, 20 met the pre-defined inclusion criteria. Two of them provided quantitative assessment on the efficacy of the described water management and house screening methods with a reduction of cases of 98%, and of a reduction of larvae of 100%, respectively. The remaining 18 studies described or recommended the elimination of breeding sites (through water or waste management, unspecified, or house destruction), the use of screens for houses and the improvement of air circulation without providing any data to evidence control effectiveness. CONCLUSION: This systematic review provides evidence on the historical use and the perceived effectiveness of environmental management methods for combatting yellow fever. However, these methods would benefit from further investigation via controlled trials to provide data for efficacy, costs, acceptability and feasibility.

Curcumin Derivatives as Potential Mosquito Larvicidal Agents against Two Mosquito Vectors, Culex pipiens and Aedes albopictus.

Vector-borne diseases have appeared or re-emerged in many Southern Europe countries making the transmission of infectious diseases by mosquitoes (vectors) one of the greatest worldwide health threats. Larvicides have been used extensively for the control of Aedes (Stegomyia) albopictus (Skuse, 1895) (Diptera: Culicidae) and Culex pipiens Linnaeus, 1758 (Diptera: Culicidae) mosquitoes in urban and semi-urban environments, causing the increasing resistance of mosquitoes to commercial insecticides. In this study, 27 curcuminoids and monocarbonyl curcumin derivatives were synthesised and evaluated as potential larvicidal agents against Cx. pipiens and Ae. albopictus. Most of the compounds were more effective against larvae of both mosquito species. Four of the tested compounds, curcumin, demethoxycurcumin, curcumin-BF2 complex and a monocarbonyl tetramethoxy curcumin derivative exhibited high activity against both species. In Cx. pipiens the recorded LC50 values were 6.0, 9.4, 5.0 and 32.5 ppm, respectively, whereas in Ae. albopictus they exhibited LC50 values of 9.2, 36.0, 5.5 and 23.6 ppm, respectively. No conclusive structure activity relationship was evident from the results and the variety of descriptors values generated in silico provided some insight to this end.

Isolation of Volatile Compounds with Repellent Properties against Aedes albopictus (Diptera: Culicidae) Using CPC Technology.

The present work describes the use of Centrifugal Partition Chromatography (CPC) for the bio-guided isolation of repellent active volatile compounds from essential oils. Five essential oils (EOs) obtained from three Pinus and two Juniperus species were initially analyzed by gas chromatography-mass spectrometry (GC/MS) and evaluated for their repellent properties against Aedes albopictus. The essential oil from needles of P. pinea (PPI) presented the higher activity, showing 82.4% repellency at a dose of 0.2 µL/cm2. The above EO, together with the EO from the fruits of J. oxycedrus subsp. deltoides (JOX), were further analyzed by CPC using the biphasic system n-Heptane/ACN/BuOH in ratio 1.6/1.6/0.2 (v/v/v). The analysis of PPI essential oil resulted in the recovery of (-)-limonene, guaiol and simple mixtures of (-)-limonene/ß-pheladrene, while the fractionation of JOX EO led to the recovery of ß-myrcene, germacrene-D, and mixtures of α-pinene/ß-pinene (ratio 70/30) and α-pinene/germacrene D (ratio 65/45). All isolated compounds and recovered mixtures were tested for their repellent activity. From them, (-)-limonene, guaiol, germacrene-D as well the mixtures of (-)-limonene/ß-pheladrene presented significant repellent activity (>97% repellency) against Ae. albopictus. The present methodology could be a valuable tool in the effort to develop potent mosquito repellents which are environmentally friendly.

Volatile profile of Greek dried white figs (Ficus carica L.) and investigation of the role of ß-damascenone in aroma formation in fig liquors.

BACKGROUND: During an investigation of the chemical profile of Greek figs (Ficus carica L.), several aqueous ethanol extracts (liquors) were prepared from dried Smyrna fig varieties cultivated in the two major fig-producing geographical areas in Greece: Peloponnese and Evia Island. The distinctive aroma observed among the prepared fig liquors led to the investigation of the odor profile of the different fig cultivars through HS-SPME coupled with GC/MS analysis, with focus on the factors that affect it before and during the preparation of the respective liquors. RESULTS: Significant variation in volatiles was noticed among all fig cultivars, as also between each fig cultivar pulp and the respective liquor. The observed diversity was a result of chemical reactions taking place in the ethanol matrix during the preparation of the liquor. The 'key' odor compound of dried fig aroma was found to be ß-damascenone. Owing to its low detection threshold and minute quantity in fig cultivars, the presence of ß-damascenone was furthermore confirmed through GC/MS/MS and GC/TOF-MS. ß-Damascenone was identified in variable quantities among fig varieties and their liquors, predominating in Kalamon fig cultivar of Peloponnese region, while its amount was found to be dependent on the postharvest storage time and preservation process of dried figs. CONCLUSION: Each of the studied fig cultivars and liquors showed a unique aroma profile, and the obtained results were used for the preparation of the first dried fig liqueur for potential commercial use with the highest content of ß-damascenone. © 2017 Society of Chemical Industry.

Greek Pinus essential oils: larvicidal activity and repellency against Aedes albopictus (Diptera: Culicidae).

The needle volatiles metabolites of seven Pinus spp.: Pinus nigra (3 samples), Pinus stankewiczii, Pinus brutia, Pinus halepensis, Pinus canariensis, Pinus pinaster and Pinus strobus from Greece were determined by gas chromatography and gas chromatography-mass spectrometry. P. nigra and P. canariensis essential oils were dominated by α-pinene (24.9-28.9 % and 15 %, respectively) and germacrene D (20.3-31.9 % and 55.8 %, respectively), whereas P. brutia and P. strobus by α-pinene (20.6 % and 31.4 %, respectively) and ß-pinene (31.7 % and 33.6 %, respectively). P. halepensis and P. pinaster oils were characterized by ß-caryophyllene (28.5 % and 22.5 %, respectively). Finally, ß-pinene (31.4 %), germacrene D (23.3 %) and α-pinene (17.5 %) were the most abundant compounds in the needle oil of P. stankewiczii. Additionally the larvicidal and repellent properties of their essential oils were evaluated against Aedes albopictus, a mosquito of great ecological and medical importance. The results of bioassays revealed that repellent abilities of the tested essential oils were more potent than their larvicidal activities. The essential oils of P. brutia, P. halepensis and P. stankewiczii presented considerable larvicidal activity (LC50 values 67.04 mgL(-1) and 70.21 mgL(-1), respectively), while the others were weak to inactive against larvae. The essential oils of P. halepensis, P. brutia, and P. stankewiczii presented a high repellent activity, even at the dose of 0.2 µL cm(-2), while in the dose of 0.4 µL cm(-2), almost all the tested EOs displayed protection against the mosquito.

Molecular detection of Usutu virus in pools of Culex pipiens mosquitoes in Greece.

Usutu virus (USUV) is a mosquito-borne flavivirus originating from Africa, that belongs to the Japanese encephalitis virus (JEV) complex. In nature, USUV involves Culex spp. mosquitoes acting as vectors and birds as amplifying hosts. The virus has recently spread in Europe and is considered an emerging human pathogen. This is the first research study performed in Greece revealing the presence and circulation of USUV in Culex spp. mosquito populations. Out of the 1,500 mosquito pools tested with real-time RT-PCR, four (Roesch et al., 2019) were positive for USUV. All four pools were collected from the region of Central Macedonia, Northern Greece.

Mitigating the Threat of Invasive Mosquito Species Expansion: A Comprehensive Entomological Surveillance Study on Kastellorizo, a Remote Greek Island.

The expansion of the tiger mosquito, a vector that can transmit diseases such as dengue, chikungunya, and Zika virus, poses a growing threat to global health. This study focuses on the entomological surveillance of Kastellorizo, a remote Greek island affected by its expansion. This research employs a multifaceted approach, combining KAP survey (knowledge, attitude, practices), mosquito collection using adult traps and human landing catches, and morphological and molecular identification methods. Results from questionnaires reveal community awareness and preparedness gaps, emphasizing the need for targeted education. Mosquito collections confirm the presence of the Aedes albopictus, Aedes cretinus, and Culex pipiens mosquitoes, highlighting the importance of surveillance. This study underscores the significance of community engagement in entomological efforts and proposes a citizen science initiative for sustained monitoring. Overall, this research provides essential insights for developing effective mosquito control programs in remote island settings, thereby emphasizing the importance of adopting a One Health approach to mitigate the spread of vector-borne diseases.

Overwintering West Nile virus in active Culex pipiens mosquito populations in Greece.

The flavivirus West Nile Virus (WNV), which is transmitted by mosquitoes, poses a significant threat to both humans and animals, and its outbreaks often challenge public health in Europe and other continents. In recent years, there is an increasing trend of WNV incidence rates across several European countries. However, whether there is a year-round circulation or seasonal introduction has yet to be elucidated. Real-time polymerase chain reaction (PCR) identified WNV-positive Culex pipiens mosquitos in 6 out of 146 pools examined in winter 2022 that correspond to three out of the 24 study areas, located in two coastal regions units in Attica, Greece. Spatial dispersion of the six positive pools in the same region suggests a clustered circulation of WNV during the winter of 2022. This is the first study that documents the identification of WNV in Cx. pipiens populations, captured in adult traps during winter period. Our findings underscore the need to extend entomological surveillance programs to include the winter period, specifically in temperate climates and historically affected areas by WNV.

Sterile Insect Technique (SIT) field trial targeting the suppression of Aedes albopictus in Greece.

The sterile insect technique (SIT) involves releasing large numbers of sterile males to outcompete wild males in mating with females, leading to a decline in pest populations. In the current study, we conducted a suppression trial in Greece against the invasive dengue vector mosquito Aedes albopictus (Skuse) through the weekly release of sterile males for 22 weeks from June to September 2019. Our approach included the long-distance transport of sterile mosquitoes, and their release at a density of 2,547 ± 159 sterile males per hectare per week as part of an area-wide integrated pest management strategy (AW-IPM). The repeated releases of sterile males resulted in a gradual reduction in egg density, reaching 78% from mid-June to early September. This reduction remained between 70% and 78% for four weeks after the end of the releases. Additionally, in the SIT intervention area, the ovitrap index, representing the percentage of traps containing eggs, remained lower throughout the trial than in the control area. This trial represents a significant advance in the field of mosquito control, as it explores the viability and efficacy of producing and transporting sterile males from a distant facility to the release area. Our results provide valuable insights for future SIT programmes targeting Ae. Albopictus, and the methodology we employed can serve as a starting point for developing more refined and effective release protocols, including the transportation of sterile males over long distances from production units to intervention areas.

Title: Essai sur le terrain de la Technique de l'Insecte Stérile (TIS) ciblant la suppression d'Aedes albopictus en Grèce. Abstract: La technique de l'insecte stérile (TIS) consiste à libérer un grand nombre de mâles stériles pour supplanter les mâles sauvages lors de l'accouplement avec les femelles, entraînant ainsi un déclin des populations de nuisibles. Dans la présente étude, nous avons mené un essai de suppression en Grèce contre le moustique vecteur invasif de la dengue, Aedes albopictus (Skuse), par le biais de la libération hebdomadaire de mâles stériles pendant 22 semaines de juin à septembre 2019. Notre approche comprenait le transport sur de longues distances de moustiques stériles, et leur lâcher à une densité de 2 547 ± 159 mâles stériles par hectare et par semaine dans le cadre d'une stratégie de lutte intégrée contre les nuisibles à l'échelle de la zone (AW-IPM). Les lâchers répétés de mâles stériles ont entraîné une réduction progressive de la densité des œufs, atteignant 78 % de la mi-juin au début septembre. Cette réduction est restée entre 70 % et 78 % pendant quatre semaines après la fin des lâchers. De plus, dans la zone d'intervention de la TIS, l'indice d'oviposition, représentant le pourcentage de pièges contenant des œufs, est resté plus faible que dans la zone témoin tout au long de l'essai. Cet essai représente une avancée significative dans le domaine de la lutte contre les moustiques, car il explore la viabilité et l'efficacité de la production et du transport de mâles stériles depuis une installation éloignée vers la zone de lâcher. Nos résultats fournissent des informations précieuses pour les futurs programmes de TIS ciblant Ae. albopictus et la méthodologie que nous avons utilisée pourra servir de point de départ pour développer des protocoles de libération plus raffinés et plus efficaces, y compris le transport de mâles stériles sur de longues distances depuis les unités de production jusqu'aux zones d'intervention.

Essential oil composition, adult repellency and larvicidal activity of eight Cupressaceae species from Greece against Aedes albopictus (Diptera: Culicidae).

The present study evaluated leaf essential oils from eight Cupresaceae species; Cupressus arizonica, Cupressus benthamii, Cupressus macrocarpa, Cupressus sempervirens, Cupressus torulosa, Chamaecyparis lawsoniana, Juniperus phoenicea, and Tetraclinis articulata for their larvicidal and repellent properties against Aedes albopictus, a mosquito of great ecological and medical importance. Based on the LC(50) values, C. benthamii essential oil was the most active (LC(50) = 37.5 mg/L) while the other tested Cupressaceae essential oils provided rather moderate toxicity against larvae (LC(50) = 47.9 to 70.6 mg/L). Under the used laboratory conditions, three of the essential oils (C. benthamii, C. lawsoniana, and C. macrocarpa) provided sufficient protection against mosquito adults, equivalent to the standard repellent "Deet" in the 0.2 mg/cm(2) dose, while C. macrocarpa assigned as the superior repellent oil in the 0.08 mg/cm(2) dose. Chemical analysis of the essential oils using gas chromatography and gas chromatography-mass spectrometry revealed the presence of 125 components.

Evaluating the Sublethal Effects of Origanum vulgare Essential Oil and Carvacrol on the Biological Characteristics of Culex pipiens biotype molestus (Diptera: Culicidae).

Culex pipiens is a mosquito species complex spread worldwide that poses a serious threat to human health as the primary vector of West Nile virus. Its control is mainly based on larvicidal applications with synthetic insecticides on mosquito breeding sites. However, the excessive use of synthetic larvicides may provoke mosquito resistance issues and negative side effects to the aquatic environment and human health. Plant-derived essential oils, including those from the Lamiaceae family, can be eco-friendly alternative larvicidal agents causing acute larval toxicity and/or growth inhibitory effects on the developmental stages of mosquitoes through different modes of action. In the current laboratory study, we evaluated the sublethal effects of carvacrol-rich oregano essential oil and pure carvacrol on Cx. pipiens biotype molestus, the autogenous member of the Cx. pipiens species complex, after the exposure of 3rd-4th instar larvae to LC50 concentrations. The short-term (24 h) larvicidal treatment with the sublethal concentrations of both tested materials exhibited an acute lethal effect on the exposed larvae as well as significant delayed mortality for surviving larvae and pupae. Larvicidal treatment with carvacrol reduced the longevity of the emerged males. In addition, the morphological abnormalities that were observed at the larval and pupal stage along with failed adult emergence indicate the potential growth inhibitory properties of the tested bioinsecticides. Our findings suggest that carvacrol and carvacrol-rich oregano oil are effective plant-based larvicides at doses lower than the acute lethal ones, thus promoting an environmentally friendly and more affordable perspective for their use against the WNV vector Cx. pipiens biotype molestus.

Two invasions at once: update on the introduction of the invasive species Aedes aegypti and Aedes albopictus in Cyprus - a call for action in Europe.

Aedes aegypti, the yellow fever mosquito and Aedes albopictus, the tiger mosquito, continue to expand their geographical distribution, reshaping the European epidemiological risks for mosquito-borne diseases. The reintroduction of Aedes aegypti near the airport and port in Larnaka and the detection of Aedes albopictus near the marina and old port of the Limassol area in Cyprus are reported herein. The measures taken to investigate these events included (i) communication to health authorities, (ii) expert on-site visits and verification of findings, (iii) enhanced active surveillance, and (iv) development of an Emergency Action Plan followed by a Contingency Plan. These emergency action plans were developed to delimitate the infested areas and to prevent the spreading of the mosquito populations into new areas. The general principles are presented along with their rationale to serve as guidelines for other geographical regions targeting suppression/eradication with a sterile insect technique component. In parallel, this manuscript serves as a call for action at the European level to impede the further spread of these species and support the activities being undertaken in Cyprus to combat the incursions of Aedes invasive species.

Title: Deux invasions à la fois : le point sur l'introduction des espèces envahissantes Aedes aegypti et Aedes albopictus à Chypre ­ un appel à l'action en Europe. Abstract: Aedes aegypti, le moustique de la fièvre jaune, et Aedes albopictus, le moustique tigre, continuent à étendre leurs aires de distribution, redessinant les risques épidémiologiques des maladies transmises par les moustiques en Europe. La réintroduction d'Aedes aegypti près de l'aéroport et du port de Larnaka, et la détection d'Aedes albopictus près de la marina et du vieux port de la zone de Limassol à Chypre sont rapportées dans cette étude. Les mesures prises pour investiguer ces évènements ont inclus : (i) la communication avec les autorités sanitaires, (ii) des visites d'experts sur site et la vérification des résultats, (iii) une surveillance active augmentée et (iv) le développement d'un Plan d'Actions d'Urgence suivi d'un Plan de Contingence. Des plans d'action d'urgence ont été développés pour délimiter les zones infestées et prévenir l'expansion des populations de moustiques à d'autres zones. Les principes généraux sont présentés, en même temps que leur justification, pour guider d'autres zones géographiques visant la suppression / éradication avec une composante de technique de l'insecte stérile. En parallèle, cet article sert d'appel, au niveau européen, à prévenir la diffusion toujours plus importante de ces espèces et pour appuyer les activités à mener à Chypre pour combattre les incursions des espèces invasives d'Aedes.

Winter survival of adults of two geographically distant populations of Aedes albopictus in a microclimatic environment of Athens, Greece.

Overwintering capacity is a major step towards establishment of invasive mosquitoes from the tropics in temperate zone areas and one of the main elements determining next seasons' population size that regulates disease transmission of competent invasive vector species. The Asian tiger mosquito, Aedes albopictus (Skuse) (Diptera: Culiciidae) is an aggressive invasive species that has greatly expanded its geographical boundaries over the last few decades. The species' ability to induce photoperiodic-based egg diapause allows its overwintering in temperate regions, which favors its establishment in higher latitudes. In warmer temperate areas winter survival can be accomplished in the adult stage as well especially in human-made shelters. Aedes albopictus is already showing signs of adaptation to colder climates which may result in disease transmission in new areas. Although the Asian tiger mosquito has invaded Greece since 2003-4, little is known regarding its overwintering capacity in the country, especially as far as adults are regarded. We studied the survival of Ae. albopictus adults during winter in a protected shelter in Athens, the capital city of Greece. The study involved two geographically isolated populations originating from Chania (Crete, most southern part of Greece), and Palaio Faliro (Athens, Central Greece). We exposed different cohorts of F3 adults of the above two populations that emerged from field collected eggs to "winter condition" from November 2018 to beginning of January 2019. in a protected microclimatic environment. Adult mortality was recorded systematically until the death of the last individual in the cohort. Results demonstrated evidence of winter survival of adults for both populations. Longevity of both females and males of the Palaio Faliro population was longer than that of the Chania population for almost all installation dates. Survival curves, regardless of the date of installation, were steeper for females of the Chania population compared to those from Palaio Faliro. In conclusion, overwinter survival of both populations in the adult stage highlights the need for future studies, considering local and microclimatic condition that vary significantly between regions. Winter climatic conditions play vital role in adult survival of this highly important public health species concerning distribution limits and knowledge on winter survival of Ae. albopictus transmitted viruses.

Identification of volatile compounds from fruits aroma and citrus essential oils and their effect on oviposition of Ceratitis capitata (Diptera: Tephritidae).

Ceratitis capitata (Wiedemann), also known as the Mediterranean fruit fly, is one of the most serious pests for several fresh fruit commodities causing extensive fruit losses worldwide. The response of C. capitata adults to fruit and nonfruit volatiles has been extensively studied. However, the linkage between fruit volatiles and the female ovipositional choice has not been fully elucidated. The present study focused on identifying the volatile organic compounds emitted by detached intact fresh fruits (oranges, lemons, bergamots, and apples) and citrus essential oils and evaluate their effect on Mediterranean fruit fly oviposition. There were more than 130 and 45 volatiles compounds detected in fruits odors and citrus essential oils, respectively. The volatile profile of fruits was dominated either from terpenes and terpenoids or from esters of butanoic, hexanoic, and octanoic acids while limonene was by far the most abundant compound in all citrus essential oils. Oviposition of C. capitata was strongly affected by volatiles emanated from both intact fruit and the citrus essential oils. Regarding the volatiles of the intact fruits, the odor of sweet orange elicited strong oviposition responses to females, while bergamot had the least stimulatory effect on oviposition. Bergamot oil also elicited the least oviposition stimulation compared to sweet orange and lemon essential oils. Our discussion elaborates on the role of fruit volatiles on host finding behavior and fruit susceptibility to C. capitata infestation and includes possible practical implication of the above findings.

Field Evaluation of Commonly Used Adult Mosquito Traps in Greece.

Background: Entomological monitoring activities are a major part of mosquito and mosquito-borne diseases surveillance. Several trapping methods are implemented worldwide, aiming to gather data on species composition and their abundance in various study areas. Methods: Several methodological modifications, such as trapping systems baited with attractants or carbon dioxide, have been proposed to increase trap efficiency. The aim of this study was to test different trap types, commonly used in Greece to collect mosquitoes, with the addition of the Biogents Sentinel lure. Moreover, traps were placed in two distinct land types and two different heights above the ground to compare their efficacy. West Nile Virus is endemic in Greece, so we also aimed to detect viral presence and circulation in selected mosquito pools. Results: Adult mosquitoes of Aedes albopictus, Culex pipiens s.l., and Culiseta longiareolata were collected in both study areas. The trap type had a significant impact on the total collections, while the trap position and interaction between trap and position did not significantly affect mosquito catches. WNV was detected in Cx. pipiens s.l. pools examined from the two study areas. Conclusion: This study emphasizes the role of trapping methods as a key component for monitoring and surveillance of adult mosquito populations, reflecting that several trap types present with different mosquito species capture and catch rates.

2D finger-printing and molecular docking studies identified potent mosquito repellents targeting odorant binding protein 1.

Personal protection measures against the mosquitoes like the use of repellents constitute valuable tools in the effort to prevent the transmission of vector-borne diseases. Therefore, the discovery of novel repellent molecules which will be effective at lower concentrations and provide a longer duration of protection remains an urgent need. Mosquito Odorant-Binding Proteins (OBPs) involved in the initial steps of the olfactory signal transduction cascade have been recognized not only as passive carriers of odors and pheromones but also as the first molecular filter to discriminate semiochemicals, hence serving as molecular targets for the design of novel pest control agents. Among the three-dimensional structures of mosquito OBPs solved in the last decades, the OBP1 complexes with known repellents have been widely used as reference structures in docking analysis and molecular dynamics simulation studies for the structure-based discovery of new molecules with repellent activity. Herein, ten compounds known to be active against mosquitoes and/or displaying a binding affinity for Anopheles gambiae AgamOBP1 were used as queries in an in silico screening of over 96 million chemical samples in order to detect molecules with structural similarity. Further filtering of the acquired hits on the basis of toxicity, vapor pressure, and commercial availability resulted in 120 unique molecules that were subjected to molecular docking studies against OBP1. For seventeen potential OBP1-binders, the free energy of binding (FEB) and mode of interaction with the protein were further estimated by molecular docking simulations leading to the selection of eight molecules exhibiting the highest similarity with their parental compounds and favorable energy values. The in vitro determination of their binding affinity to AgamOBP1 and the evaluation of their repellent activity against female Aedes albopictus mosquitoes revealed that our combined ligand similarity screening and OBP1 structure-based molecular docking successfully detected three molecules with enhanced repellent properties. A novel DEET-like repellent with lower volatility (8.55 × 10-4 mmHg) but a higher binding affinity for OBP1 than DEET (1.35 × 10-3 mmHg). A highly active repellent molecule that is predicted to bind to the secondary Icaridin (sIC)-binding site of OBP1 with higher affinity than to the DEET-site and, therefore, represents a new scaffold to be exploited for the discovery of binders targeting multiple OBP sites. Finally, a third potent repellent exhibiting a high degree of volatility was found to be a strong DEET-site binder of OBP1 that could be used in slow-release formulations.

Secondary vectors of Zika Virus, a systematic review of laboratory vector competence studies.

BACKGROUND: After the unprecedented Zika virus (ZIKV) outbreak in the western hemisphere from 2015-2018, Aedes aegypti and Ae. albopictus are now well established primary and secondary ZIKV vectors, respectively. Consensus about identification and importance of other secondary ZIKV vectors remain. This systematic review aims to provide a list of vector species capable of transmitting ZIKV by reviewing evidence from laboratory vector competence (VC) studies and to identify key knowledge gaps and issues within the ZIKV VC literature. METHODS: A search was performed until 15th March 2022 on the Cochrane Library, Lilacs, PubMed, Web of Science, WHOLIS and Google Scholar. The search strings included three general categories: 1) "ZIKA"; 2) "vector"; 3) "competence", "transmission", "isolation", or "feeding behavior" and their combinations. Inclusion and exclusion criteria has been predefined and quality of included articles was assessed by STROBE and STROME-ID criteria. FINDINGS: From 8,986 articles retrieved, 2,349 non-duplicates were screened by title and abstracts,103 evaluated using the full text, and 45 included in this analysis. Main findings are 1) secondary vectors of interest include Ae. japonicus, Ae. detritus, and Ae. vexans at higher temperature 2) Culex quinquefasciatus was not found to be a competent vector of ZIKV, 3) considerable heterogeneity in VC, depending on the local mosquito strain and virus used in testing was observed. Critical issues or gaps identified included 1) inconsistent definitions of VC parameters across the literature; 2) equivalency of using different mosquito body parts to evaluate VC parameters for infection (mosquito bodies versus midguts), dissemination (heads, legs or wings versus salivary glands), and transmission (detection or virus amplification in saliva, FTA cards, transmission to neonatal mice); 3) articles that fail to use infectious virus assays to confirm the presence of live virus; 4) need for more studies using murine models with immunocompromised mice to infect mosquitoes. CONCLUSION: Recent, large collaborative multi-country projects to conduct large scale evaluations of specific mosquito species represent the most appropriate approach to establish VC of mosquito species.