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International audience; Background: Identification of the source of mosquito blood meals is an important component for disease control and surveillance. Recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling has emerged as an effective tool for mosquito blood meal identification, using the abdomens of freshly engorged mosquitoes. In the field, mosquito abdomens are crushed on Whatman filter papers to determine the host feeding patterns by identifying the origin of their blood meals. The aim of this study was to test whether crushing engorged mosquito abdomens on Whatman filter papers was compatible with MALDI-TOF MS for mosquito blood meal identification. Both laboratory reared and field collected mosquitoes were tested.Material and methods: Sixty Anopheles gambiae Giles were experimentally engorged on the blood of six distinct vertebrate hosts (human, sheep, rabbit, dog, chicken and rat). The engorged mosquito abdomens were crushed on Whatman filter papers for MALDI-TOF MS analysis. 150 Whatman filter papers, with mosquitoes engorged on cow and goat blood, were preserved. A total of 77 engorged mosquito abdomens collected in the Comoros Islands and crushed on Whatman filter papers were tested with MALDI-TOF MS. Results The MS profiles generated from mosquito engorged abdomens crushed on Whatman filter papers exhibited high reproducibility according to the original host blood. The blood meal host was correctly identified from mosquito abdomens crushed on Whatman filter papers by MALDI-TOF MS. The MS spectra obtained after storage were stable regardless of the room temperature and whether or not they were frozen. The MS profiles were reproducible for up to three months. For the Comoros samples, 70/77 quality MS spectra were obtained and matched with human blood spectra. This was confirmed by molecular tools.Conclusion: The results demonstrated that MALDI-TOF MS could identify mosquito blood meals from Whatman filter papers collected in the field during entomological surveys. The application of MALDI-TOF MS has proved to be rapid and successful, making it a new and efficient tool for mosquito-borne disease surveillance.

In over a year, the COVID-19 pandemic caused 2.69 million deaths and 122 million infections. Social isolation and distancing measures have been the only prevention available for months. Scientific research has done a great deal of work, developing in a few months safe and effective vaccines against COVID-19. In the European Union, nowadays, four vaccines have been authorized for use: Pfizer-BioNTech, Moderna, ChAdOx1 (AstraZeneca/Oxford), Janssen (Johnson & Johnson), and three others are currently under rolling review. Vaccine allocation policy is crucial to optimize the advantage of treatment preferring people with the highest risk of contagion. These days the priority in the vaccination program is of particular importance since it has become clear that the number of vaccines is not sufficient for the entire Italian population in the short term. Cardiovascular diseases are frequently associated with severe COVID-19 infections, leading to the worst prognosis. The elderly population suffering from cardiovascular diseases is, therefore, to be considered a particularly vulnerable population. However, age cannot be considered the only discriminating factor because in the young-adult population suffering from severe forms of heart disease, the prognosis, if affected by COVID-19, is particularly ominous and these patients should have priority access to the vaccination program. The aim of this position paper is to establish a consensus on a priority in the vaccination of COVID-19 among subjects suffering from different cardiovascular diseases.

This article focuses on some representations of the origin of AIDS and Ebola in Burkina Faso, against a new background of Covid-19 which began in early 2020 in connection with two animals: the spider and the bat. These are also, if not first and foremost, heroes of oral literature (from tales to myths) from this region of West Africa. It is up to anthropologists to explore the meandering symbolism and imagination of these liminal animals that move back and forth between the worlds inhabited by humans and the "bush" worlds of non-humans. Here arises a mythological anamnesis. These "trickster" animals challenge categories and understanding of both virologists and anthropologists.Cet article porte sur quelques représentations de l'origine du sida et d'Ebola en pays lobi burkinabè, avec la Covid-19 en nouvel arrière-plan depuis le début de l'année 2020, en lien avec deux animaux : l'araignée et la chauve-souris. Ce sont aussi, voire d'abord, des héros de la littérature orale (des contes aux mythes) de cette région d'Afrique de l'Ouest. Des anthropologues ont exploré les méandres des symboliques et des imaginaires de ces animaux liminaires qui vont et viennent entre les mondes habités par les humains et les univers de « brousse » des non-humains. Une anamnèse mythologique est mise à jour. Ces animaux rusés se jouent de nos catégories et de notre entendement, virologues et anthropologues ici confondus.

International audience; Mosquito control is implemented when arboviruses are detected in patients or in field-collected mosquitoes. However, mass screening of mosquitoes is usually laborious and expensive, requiring specialized expertise and equipment. Detection of virus in mosquito saliva using honey-impregnated filter papers seems to be a promising method as it is non-destructive and allows monitoring the viral excretion dynamics over time from the same mosquito. Here we test the use of filter papers to detect chikungunya virus in mosquito saliva in laboratory conditions, before proposing this method in large-scale mosquito surveillance programs. We found that 0.9 cm 2 cards impregnated with a 50% honey solution could replace the forced salivation technique as they offered a viral RNA detection until 7 days after oral infection of Aedes aegypti and Aedes albopictus mosquitoes with CHIKV.

Background In 2005, Bangladesh, India and Nepal agreed to eliminate visceral leishmaniasis (VL) as a public health problem. The approach to this was through improved case detection and treatment, and controlling transmission by the sand fly vector Phlebotomus argentipes, with indoor residual spraying (IRS) of insecticide. Initially, India applied DDT with stirrup pumps for IRS, however, this did not reduce transmission. After 2015 onwards, the pyrethroid alpha-cypermethrin was applied with compression pumps, and entomological surveillance was initiated in 2016. Methods Eight sentinel sites were established in the Indian states of Bihar, Jharkhand and West Bengal. IRS coverage was monitored by household survey, quality of insecticide application was measured by HPLC, presence and abundance of the VL vector was monitored by CDC light traps, insecticide resistance was measured with WHO diagnostic assays and case incidence was determined from the VL case register KAMIS. Results Complete treatment of houses with IRS increased across all sites from 57% in 2016 to 70% of houses in 2019, rising to >80% if partial house IRS coverage is included (except West Bengal). The quality of insecticide application has improved compared to previous studies, average doses of insecticide on filters papers ranged from 1.52 times the target dose of 25mg/m2 alpha-cypermethrin in 2019 to 1.67 times in 2018. Resistance to DDT has continued to increase, but the vector was not resistant to carbamates, organophosphates or pyrethroids. The annual and seasonal abundance of P. argentipes declined between 2016 to 2019 with an overall infection rate of 0.03%. This was associated with a decline in VL incidence for the blocks represented by the sentinel sites from 1.16 per 10,000 population in 2016 to 0.51 per 10,000 in 2019. Conclusion Through effective case detection and management reducing the infection reservoirs for P. argentipes in the human population combined with IRS keeping P. argentipes abundance and infectivity low has reduced VL transmission. This combination of effective case management and vector control has now brought India within reach of the VL elimination targets., Author summary Visceral Leishmaniasis (VL), also known as kala-azar, is a major parasitic disease in South Asia (Indian subcontinent), with 85% of the disease incidence in India. Historically VL had been controlled and almost eliminated with Indoor Residual Spraying (IRS) using dichlorodiphenyltrichloroethane (DDT). However, reinitiating this approach in 2015 failed due to high insecticide resistance in the sand fly vector and poor IRS quality, meaning that VL elimination targets were not met. To improve this the National Vector Borne Disease Control Programme changed to an effective insecticide, alpha-cypermethrin and altered the mode of application to compression pumps. Sentinel sites were established to monitor the entomological indicators, these showed the positive impact of these changes from 2016 to 2019. During this period the overall incidence of disease has decreased, and India is now on track to reach it’s target incidence for VL of less than 1/1000 people at the sub-district (block) level.

Background Reduced susceptibility of mosquito vectors to currently used insecticides hampers control interventions. Recently pyriproxyfen, an insect growth regulator has been demonstrated to effectively reduce the reproductive potential in vector mosquitoes. Methods Pyriproxyfen (PPF), in different concentrations (0.75%, 0.075%, 0.0075%, and 0.00075%) was applied on papers and Indian wild type Aedes aegypti female mosquitoes (N ≥ 20 for each treatment) were exposed onto it as per WHO guidelines, to study the reproductive disruption. PPF concentration on treated papers was quantitatively cross-determined using HPLC method. Reduction in fecundity, fertility and adult emergence in exposed female Ae. aegypti was determined. Abnormal development in ovary and eggs of exposed females was studied microscopically after different time intervals. Results Eggs laid, eggs hatched, pupae formed and adults emerged per female exposed in both before blood meal and after blood meal groups declined significantly from lowest to highest concentration of PPF (F ≥ 5.2; p < 0.02). Adult emergence inhibition in females exposed to PPF before and after blood meal groups ranged from 58.8% [OR = 0.18 (95% CI = 0.09–0.36)] to 79.2% [OR = 0.04 (95% CI = 0.02–0.10)] and 64.4% [OR = 0.12 (95% CI = 0.05–0.28)] to 77.1% [OR = 0.05 (95% CI = 0.02–0.14)] respectively in different concentrations. The probit model used suggested that FI50 (50% fertility inhibition) and EI50 (50% emergence inhibition) were 0.002% (p = 0.82) and 0.0001% (p = 0.99) for females exposed before blood meal, while 0.01% (p = 0.63) and, Author summary Development of resistance against insecticides has challenged mosquito control programmes globally and prompted the research of alternative options that can complement insecticides. An insect growth regulator, pyriproxyfen (PPF) usage against mosquitoes can effectively reduce the vector population. PPF mainly inhibits the metamorphosis of mosquito larvae into pupae and prevent the adult emergence, therefore it is generally applied in mosquito breeding habitats. PPF has been shown to exhibit delayed residual effect that may impair the reproductive capacity by affecting the survival, fecundity and fertility of adult mosquito exposed through tarsal contact. Presently, the effectiveness of different concentrations of PPF intended to be delivered through contact have been evaluated against dengue vector Ae. aegypti. Results suggested that very low PPF concentration treated surfaces drastically reduce the fecundity, fertility and adult emergence in mosquitoes. Study suggests that control interventions based on PPF-treated surfaces could provide an additional route to target mosquito vector control by overall population density reduction.

Introduction Leishmaniasis is a vector-borne infection endemic in 98 countries. Its cutaneous form (CL) causes skin lesions on exposed parts of the body and may lead to scarring. The numbers of imported cases of CL are increasing in Europe but the incidence of CL importation in Poland is unknown. Material and methods A list of all patients diagnosed with CL at the Department in the years 2005-2017 was obtained. The study presents their data including age, sex, areas visited, purpose of travel, time from the onset of symptoms to correct diagnosis, appearance of lesions, results of impression smears and PCR, and superinfection, if detected, as well as treatment methods and their outcomes. Results Altogether, 14 cases of cutaneous leishmaniasis were identified. The study demonstrates an increase in the number of cases of imported CL at our center over the last 5 years. The time to correct diagnosis was long in spite of the fact that the lesions had usually an appearance typical of CL. Conclusions Intensified education of physicians and travelers, as well as improvement in the access to travel medicine services and to the diagnosis and treatment methods appropriate for CL, are needed in our country. In our opinion, surveillance of leishmaniasis should be introduced in Poland.

Since the 1980s, dengue incidence has increased 30-fold. However, in 2017, there was a noticeable reduction in reported dengue incidence cases within the Americas, including severe and fatal cases. Understanding the mechanism underlying dengue's incidence and decline in the Americas is vital for public health planning. We aimed to provide plausible explanations for the decline in 2017.An expert panel of representatives from scientific and academic institutions, Ministry of Health officials from Latin America and PAHO/WHO staff met in October 2017 to propose hypotheses. The meeting employed six moderated plenary discussions in which participants reviewed epidemiological evidence, suggested explanatory hypotheses, offered their expert opinions on each and developed a consensus.The expert group established that in 2017, there was a generalised decreased incidence, severity and number of deaths due to dengue in the Americas, accompanied by a reduction in reported cases of both Zika and chikungunya virus infections, with no change in distribution among age groups affected. This decline was determined to be unlikely due to changes in epidemiological surveillance systems, as similar designs of surveillance systems exist across the region. Although sudden surveillance disruption is possible at a country or regional level, it is unlikely to occur in all countries simultaneously. Retrospective modelling with epidemiological, immunological and entomological information is needed. Host or immunological factors may have influenced the decline in dengue cases at the population level through immunity; however, herd protection requires additional evidence. Uncertainty remains regarding the effect on the outcome of sequential infections of different dengue virus (DENV) types and Zika virus (ZIKV), and vice versa. Future studies were recommended that examine the epidemiological effect of prior DENV infection on Zika incidence and severity, the epidemiological effect of prior Zika virus infection on dengue incidence and severity, immune correlates based on new-generation ELISA assays, and impact of prior DENV/other arbovirus infection on ZIKV immune response in relation to number of infections and the duration of antibodies in relation to interval of protection. Follow-up studies should also investigate whether increased vector control intensification activities contributed to the decline in transmission of one or more of these arboviruses. Additionally, proposed studies should focus on the potential role of vector competence when simultaneously exposed to various arboviruses, and on entomological surveillance and its impact on circulating vector species, with a goal of applying specific measures that mitigate seasonal occurrence or outbreaks.Multifactorial events may have accounted for the decline in dengue seen in 2017. Differing elements might explain the reduction in dengue including elements of immunity, increased vector control, and even vector and\or viruses changes or adaptations. Most of the results of this expert consensus group meeting are hypothetical and based on limited evidence. Further studies are needed.Depuis les années 1980, l'incidence de la dengue a été multipliée par 30. Cependant, en 2017, il y a eu une réduction notable du nombre de cas d'incidence de dengue rapportés dans les Amériques. Nous voulions fournir des explications plausibles à la baisse en 2017. MÉTHODES: Un groupe d'experts constitué de représentants d'institutions scientifiques et académiques, d'officiels des Ministères de la Santé d'Amérique Latine et de membres du personnel de l’OPS/OMS s'est réuni en octobre 2017 pour proposer et évaluer des hypothèses. RÉSULTATS: En 2017, il y a eu une baisse généralisée de l'incidence, de la sévérité et du nombre de décès dus à la dengue dans les Amériques, accompagnée d'une réduction des cas rapportés d'infections par le virus Zika et par le virus du chikungunya, sans modification dans la répartition entre les groupes d’âge affectés. Il a été déterminé que ce déclin était peu probablement dû aux changements dans les systèmes de surveillance épidémiologique, étant donné que des systèmes de surveillance similaires existaient dans toute la région. Bien que des perturbations soudaines dans la surveillance soient possibles au niveau national ou régional, il est peu probable que cela se produise simultanément dans tous les pays. Une modélisation rétrospective avec des informations épidémiologiques, immunologiques et entomologiques est nécessaire. Des facteurs liés à l'hôte ou immunologiques peuvent avoir influencé le déclin des cas de dengue au niveau de la population par le biais de l'immunité; cependant, l’évidence d'une protection conférée par l'effet du troupeau nécessite des données supplémentaires. Une incertitude subsiste quant à l'effet sur le résultat des infections séquentielles de différents types du virus de la dengue (DENV) et du virus Zika (ZIKV), et vice-versa. Les études à venir devraient examiner (1) l'effet épidémiologique d'une infection antérieure par le DENV sur l'incidence et la sévérité du virus Zika, (2) l'effet épidémiologique d'une infection antérieure par le virus Zika sur l'incidence et la sévérité de la dengue, (3) les corrélats immunitaires basés sur des tests ELISA de nouvelle génération, (4) l’ impact d'une infection antérieure à DENV/autres arbovirus sur la réponse immunitaire au ZIKV en fonction du nombre d'infections et de la durée des anticorps en fonction de l'intervalle de protection, (5) si des activités d'intensification de la lutte antivectorielle ont contribué à la diminution de la transmission d'un ou plusieurs de ces arbovirus, (6) le rôle potentiel de la compétence vectorielle lorsqu'ils sont exposés simultanément à différents arbovirus, (7) la surveillance entomologique et son impact sur la circulation d'espèces de vecteurs, dans le but d'appliquer des mesures spécifiques qui réduisent l'occurrence saisonnière d’épidémies.Des événements multifactoriels pourraient expliquer le déclin observé de la dengue en 2017. La plupart des résultats de cette réunion du groupe de consensus d'experts sont hypothétiques, reposent sur des données limitées et requièrent des investigations supplémentaires.

Owned, free-roaming domestic cats are abundant in the Chilean countryside, having high probability of contact with wildlife and potentially participating as reservoirs of zoonotic pathogens. In the present study, 131 cats from two remote study areas (Valdivia and Chiloe Island) in southern Chile were analyzed for infection/exposure to eight pathogens. Serum samples from 112 cats were tested for antigens against feline leukemia virus (FeLV antigen-ELISA) and antibodies against feline immunodeficiency virus (FIV-ELISA) and canine distemper virus (CDV-serum neutralization), yielded occurrence of 8.9, 1.7 and 0.8% respectively. The presence of DNA of five vector-borne pathogens, piroplasmids, Ehrlichia spp., Anaplasma spp., Rickettsia spp. and Bartonella spp. was investigated in thirty cats. Overall observed occurrence was 6.6% (2/30) for both Anaplasma platys, and B. henselae, and 3.3% (1/30) for both Bartonella sp. and Theileria equi. Observed occurrence for all vector-borne pathogens in Valdivia area was significantly higher than in Chiloe Island (5/15 vs 0/15; P=0.04). Our results represent the first description of exposure to CDV and DNA detection of T. equi and A. platys in domestic cats in Chile. The results highlight the importance of performing pathogen screening in owned, free-roaming rural cats to evaluate their potential role as reservoirs of infection and vectors for disease transmission to wildlife.

Novel chemistry for vector control is urgently needed to counter insecticide resistance in mosquitoes. Here a new meta-diamide insecticide, broflanilide (TENEBENALTM), was evaluated in East African experimental huts in Moshi, northern Tanzania. Two consecutive experimental hut trials with broflanilide 50WP were conducted; the first evaluating the efficacy of three concentrations, 50 mg/m2, 100 mg/m2, and 200 mg/m2 using a prototype formulation, and the second trial evaluating an improved formulation. The IRS treatments were applied on both mud and concrete surfaces and efficacy was monitored over time. The mortality, blood-feeding inhibition and exiting behaviour of free-flying wild mosquitoes was compared between treatment arms. Additionally, cone assays with pyrethroid-susceptible and resistant mosquito strains were conducted in the huts to determine residual efficacy. The first trial showed a dosage-mortality response of the prototype formulation and 3–8 months of residual activity, with longer activity on concrete than mud. The second trial with an improved formulation showed prolonged residual efficacy of the 100 mg/m2 concentration to 5–6 months on mud, and mosquito mortality on the concrete surface ranged between 94–100% for the full duration of the trial. In both trials, results with free-flying, wild Anopheles arabiensis echoed the mortality trend shown in cone assays, with the highest dose inducing the highest mortality and the improved formulation showing increased mortality rates. No blood-feeding inhibition or insecticide-induced exiting effects were observed with broflanilide. Broflanilide 50WP was effective against both susceptible and pyrethroid-resistant mosquito strains, demonstrating an absence of cross resistance between broflanilide and pyrethroids. The improved formulation, which has now been branded VECTRONTM T500, resulted in a prolonged residual efficacy. These results indicate the potential of this insecticide as an addition to the arsenal of IRS products needed to maintain both control of malaria and resistance management of malaria-transmitting mosquitoes.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causing agent of Coronavirus Disease-2019 (COVID-19), is likely to be originated from bat and transmitted through intermediate hosts. However, the immediate source species of SARS-CoV-2 have not yet been confirmed. Here, we used diversity analysis of the angiotensin I converting enzyme 2 (ACE2) that serves as cellular receptor for SARS-CoV-2 and transmembrane protease serine 2 (TMPRSS2), which has been proved to be utilized by SARS-CoV-2 for spike protein priming. We also simulated the structure of receptor-binding domain of SARS-CoV-2 spike protein (SARS-CoV-2S RBD) with the ACE2s to investigate their binding affinity to determine the potential intermediate animal hosts that could spread the SARS-CoV-2 to humans in South Asia. We identified cow, buffalo, goat and sheep, which are predominant species in the household farming system in South Asia that can potentially be infected by SARS-CoV-2. All the bird species studied along with rat and mouse were considered less potential to interact with SARS-CoV-2. The interaction interfaces of SARS-CoV-2S RBD and ACE2 protein complex suggests pangolin as a potential intermediate host in SARS-CoV-2. Our results provide a valuable resource for the identification of potential hosts for SARS-CoV-2 in South Asia and henceforth reduce the opportunity for a future outbreak of COVID-19., Highlights • Host range of SARS-CoV-2 was predicted based on ACE2 and TMPRSS2 protein sequences of the hosts. • Cow, buffalo, goat and sheep were identified as highly potential for SARS-CoV-2 infection in South Asia. • Pangolin was suggested as the intermediate host of SARS-CoV-2.

Globally, the economic losses due to hard ticks infestation and the control of the associated diseases have been calculated at USD $13.9–18.7 billion per year. The economic impact is related to its direct damage to the skins, blood loss, anemia, severe immunological reactions and indirect losses that related to the effects of hemoparasites, cost of treatment for clinical cases and expenses incurred in the control of ticks. The current study evaluated the acaricidal activities of fennel Foeniculum vulgare essential oil and its main components; trans-anethole and fenchone; against R. annulatus. GC–MS analysis revealed that this oil contained 16 components representing 99.9% of the total identified compounds with E-anethole being the predominant component(64.29%), followed by fenchone (9.94%). The fennel oil and trans-anethole showed significant acaricidal activities. The LC50 of the fennel oil was attained at concentrations of 12.96% for adult ticks and 1.75% for tick larvae meanwhile the LC50 of trans-anethole was reached at concentrations of 2.36% for adult tick and 0.56% for tick larvae. On the contrary, fenchone showed no any significant adulticidal activities and its LC50 attained at a concentration of 9.11% for tick larvae. Regarding repellence activities, trans-anethole achieved 100% repellency at the concentration of 10% while fennel showed 86% repellency at the same concentration. Fenchone showed no repellency effect. Treatment of larvae with fennel, trans-anethole, and fenchone LC50 concentrations significantly inhibited the acetylcholinesterase activity. Meanwhile, glutathione s-transferase activity was significantly decreased in fennel treated larvae but no significant effect was found in the larvae of trans-anethole and fenchone groups. These results indicate that the acaricide effect of fennel oil may attributed to its high content of trans-anethole. This was supported by potent adulticidal, larvicidal, and repellency effects of trans-anethole against Rhipeciphalus annulatus tick and therefore it could be included in the list of acaricide of plant origin.

As a means of obtaining reproducible and accurate numbers of larvae for laboratory rearing, we tested a large-particle flow-cytometer type device called the ‘Automated Particle Counter’ (APC). The APC is a gravity-fed, self-contained unit that detects changes in light intensity caused by larvae passing the detector in a water stream and controls dispensing by stopping the flow when the desired number has been reached. We determined the accuracy (number dispensed compared to the target value) and precision (distribution of number dispensed) of dispensing at a variety of counting sensitivity thresholds and larva throughput rates (larvae per second) using < 1-day old Anopheles gambiae and Aedes aegypti larvae. All measures were made using an APC algorithm called the ‘Smoothed Z-Score’ which allows the user to define how many standard deviations (Z scores) from the baseline light intensity a particle’s absorbance must exceed to register a count. We dispensed a target number of 100 An. gambiae larvae using Z scores from 2.5–8 and observed no difference among them in the numbers dispensed for scores from 2.5–6, however, scores of 7 and 8 under-counted (over-dispensed) larvae. Using a Z score ≤ 6, we determined the effect of throughput rate on the accuracy of the device to dispense An. gambiae larvae. For rates ≤ 98 larvae per second, the accuracy of dispensing a target of 100 larvae was - 2.29% ± 0.72 (95% CI of the mean) with a mode of 99 (49 of 348 samples). When using a Z score of 3.5 and rates ≤ 100 larvae per second, the accuracy of dispensing a target of 100 Ae. aegypti was - 2.43% ± 1.26 (95% CI of the mean) with a mode of 100 (6 of 42 samples). No effect on survival was observed on the number of An. gambiae first stage larvae that reached adulthood as a function of dispensing.

Background Phlebotomine sand flies are vectors of human leishmaniases, important neglected tropical diseases. In this study, we investigated diel patterns of oviposition behavior, effects of visual cues on oviposition-site selection, and whether these affect the attraction of gravid Phlebotomus papatasi (Scopoli), the vector of old-world cutaneous leishmaniasis, to olfactory cues from oviposition sites. Methodology/principal findings To evaluate these questions, we conducted a series of experiments using attraction and oviposition assays within free-flight test chambers containing gravid females entrained under a 14:10 hrs light:dark photoperiod. By replacing sticky-screens or moist filter papers every three hours, we showed that oviposition site search occurs mainly in the latest part of the night whereas peak oviposition occurs during the early part of the night. Behavioral responses to olfactory oviposition cues are regulated by time-of-day and can be disrupted by transient exposure to a constant darkness photoperiod. Gravid females, but not any other stage, age, or sex, were attracted to dark, round oviposition jars, possibly resembling rodent burrow openings. This visual attraction disappeared in the absence of an illumination source. Egg deposition rate was not affected by jar color. Olfactory cues had the strongest effect when the visual cues were minimal. Conclusion and significance Our study showed, for the first time, that visual cues in the form of oviposition-site color, lighting level, and photoperiod are important in guiding the oviposition behavior of phlebotomine sand flies. Furthermore, such visual cues could modify the flies’ sensitivity to olfactory oviposition cues. Our results suggest that chemosensory and visual cues are complementary, with visual cues used to orient gravid females towards oviposition sites, possibly at long- to medium-ranges during crepuscular periods, while olfactory cues are used to approach the burrow in darkness and assess its suitability at close-range. Implications to sand fly control are discussed., Author summary Sand flies are vectors of human leishmaniases, an important neglected tropical disease. An alternative approach to the conventional delivery of an insecticide to the vector is to bring the vector to the insecticide using oviposition (egg-laying)-site attractants. Olfactory cues originating from organic matter have been identified as important sources of oviposition attractants. However, nothing is known regarding visual assessment of oviposition sites by sand flies. Also, little is understood about diel egg-laying patterns of sand flies. Finally, it is not known if visual cues and time-of-day may affect their sensitivity to olfactory cues. In this study, we investigated these questions in a series of lab experiments using free-flight cage arenas, with Phlebotomus papatasi (vector of cutaneous leishmaniasis). We showed that peak oviposition-site search and sensitivity to olfactory cues occurs mainly in the latest part of the night whereas peak oviposition occurs during the early part of the night. We demonstrated that only gravid females, but not any other stage or sex, were attracted to dark, round oviposition jars resembling burrow openings. Finally, we showed that sensitivity to olfactory cues is reduced in the presence of strong visual cues and in the absence of natural diel photoperiod.

In recent decades, the invasive Aedes albopictus vector has spread across Europe and is responsible for numerous outbreaks of autochthonous arboviral disease. The aim of this study was to identify epidemiological and sociological risk factors related to individual levels of exposure to Aedes albopictus bites. A multidisciplinary survey was conducted with volunteer blood donors living in areas either colonised or not by Aedes albopictus in mainland France. Individual levels of exposure were evaluated by measuring the IgG level specific to Aedes albopictus saliva. The most striking risk factors concerned the localisation and characteristics of the dwelling. Individuals living in areas colonised prior to 2009 or recently colonised (between 2010 and 2012) had higher anti-salivary gland extract IgG levels compared with those who were living in areas not yet colonised by Ae. albopictus. The type of dwelling did not seem to impact the level of exposure to Aedes bites. People living in apartments had a higher anti-salivary gland extract IgG level than those living in individual houses but the difference was not statistically significant. Interestingly, the presence of air conditioning or window nets was associated with a noticeable reduction in bite intensity.

BACKGROUND: Different setups and protocols have been developed for investigating insecticide effects on Anopheles (An.) mosquitoes, vectors of malaria. However, chemical uptake resulting from their tarsal contact with insecticide-treated material has seldom been investigated. To address the challenges encountered in the interpretation of bioassay data, a high throughput method for chemical analysis on malaria vectors was developed and validated for five selected insecticides including alpha-cypermethrin (aCYP), deltamethrin (DM), etofenprox (EPX), permethrin (PM), pirimiphos-methyl (PPM). METHODS: The method includes a single chemical extraction step via an ultrasound probe on mosquito samples and analysis via liquid chromatography coupled to high-resolution tandem mass spectrometry (UHPLC-MS/MS). The protocol was established for two malaria vector species, Anopheles gambiae senso stricto (s.s.) and An. stephensi, both males and females. Recovery rates ranged from 70 to 100% without any influence of sex or species. The method was efficiently applied to female An. gambiae s.s. of the KISUMU1 reference strain, after susceptibility tests using the World Health Organization´s standard protocol. RESULTS: Susceptibility tests revealed 13.4-18.4 minutes knockdown times for 50% mosquitoes during exposure to EPX and pyrethroids. The mortality rates 24 hours post-exposure to insecticides were mostly 99-100%, except in two PM and three PPM assays suggesting possible or confirmed resistance to these insecticides. The mean insecticide uptake in dead mosquitoes ranged from 23 pg (aCYP) to 1812 pg (EPX) per specimen. However, the mean uptake in survivors to PM and PPM was reduced by at least 25%, suggesting that acute doses were not achieved in these specimens during bioassays. CONCLUSIONS: The developed and validated UHPLC-MS/MS method could be used to address some limitations of bioassays or to assess the penetration of insecticides in mosquito matrix with reference to cuticle thickness and other insecticide resistance mechanisms.

Insect repellents are widely used as the first line of defense against mosquito bites and transmission of disease-causing agents. However, the cost of daily applications of even the most affordable and the gold standard of insect repellents, DEET, is still high for low-income populations where repellents are needed the most. An Indian clove-based homemade recipe has been presented as a panacea. We analyzed this homemade repellent and confirmed by behavioral measurements and odorant receptor responses that eugenol is the active ingredient in this formulation. Prepared as advertised, this homemade repellent is ineffective, whereas 5x more concentrated extracts from the brand most enriched in eugenol showed moderate repellency activity against Culex quinquefasciatus and Aedes aegypti. DEET showed higher performance when compared to the 5x concentrated formulation and is available in the same market at a lower price than the cost of the ingredients to prepare the homemade formulation.

The arthropod melanization immune response is activated by extracellular protease cascades predominantly comprised of CLIP-domain serine proteases (CLIP-SPs) and serine protease homologs (CLIP-SPHs). In the malaria vector, Anopheles gambiae, the CLIP-SPHs SPCLIP1, CLIPA8, and CLIPA28 form the core of a hierarchical cascade downstream of mosquito complement that is required for microbial melanization. However, our understanding of the regulatory relationship of the CLIP-SPH cascade with the catalytic CLIP-SPs driving melanization is incomplete. Here, we report on the development of a novel screen to identify melanization pathway components based on the quantitation of melanotic mosquito excreta, eliminating the need for microdissections or hemolymph enzymatic assays. Using this screen, we identified CLIPC9 and subsequent functional analyses established that this protease is essential for the melanization of both Escherichia coli and the rodent malaria parasite Plasmodium berghei. Mechanistically, septic infection with E. coli promotes CLIPC9 cleavage and both full-length and cleaved CLIPC9 localize to this bacterium in a CLIPA8-dependent manner. The steady state level of CLIPC9 in the hemolymph is regulated by thioester-containing protein 1 (TEP1), suggesting it functions downstream of mosquito complement. In support, CLIPC9 cleavage is inhibited following SPCLIP1, CLIPA8, and CLIPA28 knockdown positioning it downstream of the CLIP-SPH cascade. Moreover, like CLIPA8 and CLIPA28, CLIPC9 processing is negatively regulated by serine protease inhibitor 2 (SRPN2). This report demonstrates how our novel excretion-based approach can be utilized to dissect the complex protease networks regulating mosquito melanization. Collectively, our findings establish that CLIPC9 is required for microbial melanization in An. gambiae and shed light on how the CLIP-SPH cascade regulates this potent immune response., Author summary Mosquito vector competence for Plasmodium, antifungal defense, and lifespan are all influenced by the melanization response. Despite its importance, our understanding of the proteins comprising the An. gambiae melanization cascade is incomplete. To streamline the discovery of melanization pathway components in this disease vector, we developed a screening method that is able to identify proteins with far fewer mosquitoes than other approaches. This technique facilitated our discovery that the serine protease CLIPC9 is required for the melanization of E. coli bacteria and malaria parasites. CLIPC9 activation and localization to bacteria is regulated by members of the CLIPA subfamily, highlighting that these serine protease homologs broadly regulate the melanization immune response. Traditionally viewed as ‘co-factors’ for the prophenoloxidase activating CLIPBs, this work demonstrates that CLIPAs can also control the activation of the catalytic proteases driving melanization. This work identifies a new player in the melanization hierarchy and provides the field with an efficient approach for dissecting the complex protease cascades controlling this important immune response.

Ticks are notorious vectors of disease for humans, and many species of ticks transmit multiple pathogens, sometimes in the same tick bite. Accordingly, a broad-spectrum vaccine that targets vector ticks and pathogen transmission at the tick/host interface, rather than multiple vaccines against every possible tickborne pathogen, could become an important tool for resolving an emerging public health crisis. The concept for such a tick protective vaccine comes from observations of an acquired tick resistance (ATR) that can develop in non-natural hosts of ticks following sensitization to tick salivary components. Mice are commonly used as models to study immune responses to human pathogens but normal mice are natural hosts for many species of ticks and fail to develop ATR. We evaluated HLA DR3 transgenic (tg) "humanized" mice as a potential model of ATR and assessed the possibility of using this animal model for tick protective vaccine discovery studies. Serial tick infestations with pathogen-free Ixodes scapularis ticks were used to tick-bite sensitize HLA DR3 tg mice. Sensitization resulted in a cytokine skew favoring a Th2 bias as well as partial (57%) protection to infection with Lyme disease spirochetes (Borrelia burgdorferi) following infected tick challenge when compared to tick naive counterparts. I. scapularis salivary gland homogenate (SGH) and a group of immunoinformatic-predicted T cell epitopes identified from the I. scapularis salivary transcriptome were used separately to vaccinate HLA DR3 tg mice, and these mice also were assessed for both pathogen protection and epitope recognition. Reduced pathogen transmission along with a Th2 skew resulted from SGH vaccination, while no significant protection and a possible T regulatory bias was seen in epitope-vaccinated mice. This study provides the first proof-of-concept for using HLA DR tg "humanized" mice for studying the potential tick protective effects of immunoinformatic- or otherwise-derived tick salivary components as tickborne disease vaccines.

Increased globalisation and climate change have led to concern about the increasing risk of arthropod-borne virus (arbovirus) outbreaks globally. An outbreak of equine arboviral disease in northern Europe could impact significantly on equine welfare, and result in economic losses. Early identification of arboviral disease by horse owners may help limit disease spread. In order to determine what horse owners understand about arboviral diseases of horses and their vectors, the authors undertook an open, cross-sectional online survey of UK horse owners. The questionnaire was distributed using social media and a press release and was active between May and July 2016. There were 466 respondents, of whom 327 completed the survey in full. High proportions of respondents correctly identified photographic images of biting midges (71.2 per cent) and mosquitoes (65.4 per cent), yet few were aware that they transmit equine infectious diseases (31.4 per cent and 35.9 per cent, respectively). Of the total number of respondents, only 7.4 per cent and 16.2 per cent correctly named a disease transmitted by biting midges and mosquitoes, respectively. Only 13.1 per cent and 12.5 per cent of participants identified specific clinical signs of African horse sickness (AHS) and West Nile virus (WNV), respectively. This study demonstrates that in the event of heightened disease risk educational campaigns directed towards horse owners need to be implemented, focussing on disease awareness, clinical signs and effective disease prevention strategies.