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Schematic of FlyBox. Illustration of FlyBox schematic (upper panel) and the front view of FlyBox (lower left panel) were shown. The example of recording image used for mosquito behavior monitoring is provided in lower right panel.
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Anopheles gambiae and Aedes aegypti are perhaps the best studied mosquito species and important carriers of human malaria and arbovirus, respectively. Mosquitoes have daily rhythms in behaviors and show a wide range of activity patterns. Although Anopheles is known to be principally nocturnal and Aedes principally diurnal, details of mosquito activ...
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... monitored the locomotor activity patterns of An. gambiae and Ae. aegypti using FlyBox ( Fig. 1 and see Supplementary Video), a relatively new behavior monitoring system first developed for Drosophila 21 . We evaluated the ability of the FlyBox basic version (without optogenetic LEDs; see below) to assay mosquito activity by comparing two important tropical disease vectors, An. gambiae and Ae. aegypti. The mean locomotor activity ...
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... drawbacks are: i) 24-well plates detect fewer mosquitoes than the standard 32 glass behavior tubes of the DAM system; ii) The limited space within each well of the 24-well plate is non-physiological and almost certainly restricts the natural (flight) activity of the insect (Fig. 1 and see Supplementary Video) albeit less severely than the DAM tubes. Indeed, video (see Supplementary Video) shows that the mosquitoes are jumping and attempting to fly. However, this drawback underscores the extent to which the circadian clock must regulate the temporal patterns such that that they still resemble previous data in the ...
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... insectary under the conditions described, but it can be used anywhere as long as it is connected to a computer to control its parameters and record data. FlyBox contains three main features: an entrainment light (5000 K daylight white LED to fully mimic daylight), an infrared camera to record locomotor activity and a plate platform (Fig. 1). The FlyBox can also be configured to include optional red and green LEDs for optogenetics, which were not used in this study. A 24-well plate sufficiently large for mosquitoes replaced the 96-well plate used for fruit flies 21 . The plate platform, made of white acrylic transparent to infrared light, is positioned in the center of the ...
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... observation periods. After covering the 24-well plate with a transparent plastic film and poking five holes over each well to promote air circulation, it was placed on the platform inside the FlyBox. 2. Configuring and running FlyBox. The fully loaded 24-well plate can be seen in real time with the FlyBox camera, which is connected to a computer (Fig. 1). Locomotor activity starts recording when the software (WebCamImageSave) is opened, and an image is captured every 10 seconds. All recordings were in a 12 h :12 h LD cycle or in DD conditions. Time of day in LD is reported in 24 hr Zeitgeber Time (ZT); ZT12 is defined as the time of lights off under the LD cycle, and ZT0 defined as ...
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This study reports the results of a molecular screening for Wolbachia (Wb) infection in Aedes albopictus (Skuse) populations recently established in the Yucatan Peninsula, Mexico. To do so, collections of free-flying adults with BG traps and emerged adults from eggs after ovitrap field collections were performed in three suburban localities of the...
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Citations
... aegypti. Similar results of diurnal behavior were also observed in earlier studies where measurements were done using the Rockefeller and West African strains (Taylor and Jones, 1969;Lima-Camara et al., 2014;Rivas et al., 2018;Araujo et al., 2020;Teles-de-Freitas et al., 2020). Our current study is one of several studies where strain differences did not impact chronotype. ...
Aedes aegypti is an important mosquito vector of human disease with a wide distribution across the globe. Climatic conditions and ecological pressure drive differences in the biology of several populations of this mosquito, including blood-feeding behavior and vector competence. However, no study has compared activity and/or sleep among different populations/lineages of Ae. aegypti . Having recently established sleep-like states in three mosquito species with observable differences in timing and amount of sleep among species, we investigated differences in activity and sleep levels among 17 Ae. aegypti lines drawn from both its native range in Africa and its invasive range across the global tropics. Activity monitoring indicates that all the lines show consistent diurnal activity, but significant differences in activity level, sleep amount, number of sleep bouts, and bout duration were observed among the lines. Variations in specific activity and sleep parameters were explained by differences in host preference, ancestry, and human population density for the lineages collected in Africa. This study provides evidence that the diurnal sleep and activity profiles for Ae. aegypti are consistent, but there are significant population differences for Ae. aegypti sleep and activity levels and interactions with humans may significantly impact mosquito activity and sleep.
... Ae. aegypti has diurnal locomotor activity under natural conditions (Jones, 1981;Lima-Camara et al., 2011;Araujo et al., 2020), with bimodal rhythms peaking at sunrise and sunset (Araujo et al., 2020;Lima-Camara, 2010) genetically determined for its circadian rhythms (Araripe et al., 2018;Johnson et al., 2003;Roenneberg et al., 2003;Taylor and Jones, 1969). Under laboratory conditions, sunrise and sunset are artificially generated for light and dark phases, with locomotor activity peaks observed in the transition of these phases (Araujo et al., 2020;Lima-Camara, 2010;Jones, 1981). ...
... Ae. aegypti has diurnal locomotor activity under natural conditions (Jones, 1981;Lima-Camara et al., 2011;Araujo et al., 2020), with bimodal rhythms peaking at sunrise and sunset (Araujo et al., 2020;Lima-Camara, 2010) genetically determined for its circadian rhythms (Araripe et al., 2018;Johnson et al., 2003;Roenneberg et al., 2003;Taylor and Jones, 1969). Under laboratory conditions, sunrise and sunset are artificially generated for light and dark phases, with locomotor activity peaks observed in the transition of these phases (Araujo et al., 2020;Lima-Camara, 2010;Jones, 1981). ...
... Ae. aegypti has diurnal locomotor activity under natural conditions (Jones, 1981;Lima-Camara et al., 2011;Araujo et al., 2020), with bimodal rhythms peaking at sunrise and sunset (Araujo et al., 2020;Lima-Camara, 2010) genetically determined for its circadian rhythms (Araripe et al., 2018;Johnson et al., 2003;Roenneberg et al., 2003;Taylor and Jones, 1969). Under laboratory conditions, sunrise and sunset are artificially generated for light and dark phases, with locomotor activity peaks observed in the transition of these phases (Araujo et al., 2020;Lima-Camara, 2010;Jones, 1981). Ae. aegypti resistant to insecticides has increased locomotor activity (Nakazato et al., 2021). ...
Aedes aegypti are vector insects of arboviruses such as dengue, Zika, and chikungunya. All available vector control methods have limited efficacy, highlighting the urgent need to find alternative ones. Evidence shows that arachnids like ticks are sources of biologically active compounds. Moreover, chemical modulation of the locomotor and immune systems of vector insects can be used to control arbovirus transmission. The present study evaluated the effectiveness of crude saliva of female Amblyomma cajennense sensu stricto (s.s.) ticks in reducing locomotor activity and inducing an immune response in Ae. aegypti females. Additionally, the study evaluated the protein constitution of tick saliva. For this purpose, the crude saliva obtained from several semi-engorged A. cajennense females was used. A volume of 0.2 nL of crude tick saliva was administered to mosquitoes by direct intrathoracic microinjection. The effect of the tick's saliva on the locomotor activity of the mosquito was observed using Flybox, a video-automated monitoring system, and the hemolymph hemocyte levels were quantified by reading slides under a light microscope. The protein concentration of the crude tick saliva was 1.27 μg/μL, and its electrophoretic profile indicates the presence of proteins with a molecular weight ranging between ∼17 and 95 kDa. Microplusins, ixodegrins, cystatin, actins, beta-actin, calponin, albumin, alpha-globulins, and hemoglobin were the main proteins identified by proteomics in the saliva of A. cajennense. The microinjected saliva had low toxicity for Ae. aegypti females and significantly reduced their locomotor activity, especially in the transition between the light and dark phases. The crude tick saliva did not change the period and rhythmicity of the circadian cycle. The tick saliva significantly increased the number of hemocytes two days after injection and reduced it after five days. These results suggest that further evaluation of the biological properties of tick saliva proteins against Ae. aegypti would be of interest.
... The Fly Box activity monitor was constructed using a modified design of da Silva Araujo et al. [66]. An infrared LED floodlight (CMVision, CM-IR130-198 Illuminator) was used to illuminate the mosquito plate from below, and placed in a separate, ventilated compartment to minimize the amount of heat generated in the mosquito housing. ...
... Image files were processed for analysis according to da Silva Araujo et al. [66]. All image files were loaded into PySolo-Video [67]. ...
The Northern House mosquito, Culex pipiens, is an important disease vector, and females are capable of surviving the winter in a state of overwintering diapause. This species’ diapause response has been extensively studied, and recent evidence suggests that the circadian clock is involved in measuring seasonal changes in daylength to initiate the diapause response. However, differences in the circadian activity of diapausing and non-diapausing Cx. pipiens have not been thoroughly investigated. Additionally, recent findings indicate that artificial light at night (ALAN) can disrupt mosquito diapause, potentially prolonging the mosquito biting season. We compared the circadian locomotor activity of mosquitoes reared in diapause-averting, long-day conditions and diapause-inducing, short-day conditions with and without ALAN to elucidate the interplay between circadian activity, diapause, and light pollution. We also uncovered metabolic differences between mosquitoes reared under diapausing and non-diapausing photoperiods with and without ALAN by measuring the concentration of protein, fructose, glycogen, water-soluble carbohydrates, and lipids. We found that ALAN exposure altered several diapause-associated phenotypes including slightly, but not significantly, increasing activity levels in short day-reared mosquitoes; and preventing some short day-reared mosquitoes from accumulating lipids. ALAN also significantly reduced glycogen and water-soluble carbohydrate levels in long day-reared mosquitoes. Based on our findings, light pollution may decrease insect fitness by perturbing metabolism, and may also impact several phenotypes associated with insect diapause, potentially extending the mosquito biting season and preventing insects in urban environments from overwintering successfully.
... 78 Adult female Ae. aegypti are primarily diurnal 79,80 and readily enter human habitations to seek a blood meal or rest 72 ; however, they are weak fliers and don't often fly more than a few hundred meters from breeding sites unless inadvertently transported by humans. 81 Taxonomy and identification. ...
Several invasive mosquitoes have become established in Canada, including important pathogen vectors such as Aedes albopictus , Ae. japonicus , and Culex pipiens . Some species have been present for decades, while others are recent arrivals. Several species present new health concerns and may result in autochthonous seasonal outbreaks of pathogens, particularly in southern Canada, that were previously restricted to imported cases. This review provides an overview of current knowledge of the biological, medical, and veterinary perspectives of these invasive species and highlights the need for increased monitoring efforts and information sharing.
... LesAedes sont principalement actifs pendant la journée, avec deux pics d'activité, un au petit matin et un autre au crépuscule. Quant aux Culex, l'heure à laquelle piquent les femelles débute à la tombée du jour et dure jusqu'à l'aube(Araujo, Guo, and Rosbash., 2020). donc la combinaison de la compétence vectorielle et de ces facteurs qui offrent au vecteur sa capacité à transmettre le virus aux hôtes, donc sa capacité vectorielle.Les vecteurs Aedes sont ainsi majoritairement compétents pour les virus du chikungunyaAuerswald et al. 2021; Eynde et al. 2022). ...
Les arboviroses, transmises par les moustiques Aedes et Culex, continuent de menacer la santé et la vie des populations des zones où elles sont endémiques et représentent des risques d’émergence dans nouveaux territoires colonisés par ces vecteurs. L’objectif principal de nos travaux était i) d’évaluer le niveau d’exposition des populations aux piqûres des Aedes aegypti et Ae. albopictus et le risque de transmission des maladies qu’ils transmettent, en milieu urbain en Afrique de l’Ouest et au Sud de la France métropolitaine et ii) d’évaluer et valider les réponses IgG anti-EGS et anti-protéine recombinante de 30 kDa de Culex quinquefasciatus comme biomarqueur d’exposition potentiel des Hommes aux piqûres des Culex. Nous avons dosé dans les sérums des individus exposés, les réponses anticorps : i) IgG anti-Nterm-34 kDa et les IgG anti- antigènes d’arbovirus (LUMINEX) chez les enfants d’Anoumabo, de Bromakoté et de Petit-Bassam, à d’Abidjan, , ii) IgG et IgM anti-Nterm-34 kDa d’Ae. aegypti à l’’Auvergne, de Corse, d’Occitanie et de PACA, en France, et iii) IgG anti-EGS et la protéine recombinante 30 kDa chez les enfants de Dar-es-Salam, N’Gattakro, Kennedy à Bouaké.A Abidjan, les IgG anti-Nterm-34 kDa étaient significativement différentes entres les quartiers d’étude avec un fort niveau à Bromakoté. Les IgG spécifiques étaient associées aux données des pièges collants. Selon la technique LUMINEX les arbovirus circulaient dans les quartiers, avec à une détection majoritaire de DENV3. Petit-Bassam était le plus affecté. L’ensemble de ces résultats démontraient que l’exposition aux Aedes et ainsi le risque de transmission étaient variables entre quartiers au sein d’une même ville africaine.En France métropolitaine, les IgG spécifiques étaient plus élevées dans les régions colonisées que dans la région non colonisée. La réponse IgG spécifique était plus élevée en Occitanie et en PACA par rapport à la Corse. Les IgM anti-peptide salivaire, indiquait une différence significative entre les régions exposées. En Occitanie, les niveaux médians des réponses IgG anti-peptide des départements 30 et 34, étaient plus élevés que celles des départements 31 et 66. En PACA, une différence significative du niveau des IgG spécifique était détectée entre le département 04 et 13 et entre les départements 05 et 13. Les IgG des départements 13 et 83 plus élevées que ceux des autres départements. Les départements colonisés entre 2010 et 2012 présentaient un taux d’IgG anti-peptide statiquement plus élevés que celui des départements non colonisés. Les IgM entre les individus des 4 régions était pratiquement nul. Ces résultats indiquaient la pertinence du biomarqueur IgG anti-peptide salivaire pour discriminer les régions et les départements les plus exposés aux piqûres d’Ae. albopictus et ainsi d’évaluer l’hétérogénéité des risques de transmission des arboviroses en France métropolitaine.Pour les Culex, les réponses IgG anti-EGS et anti-30 kDa étaient significativement plus élevées chez les enfants vivant dans les sites de forte densité de Culex quinquefasciatus. Les IgG anti-30 kDa étaient significativement plus élevées chez les enfants ne dormant pas sous les moustiquaires imprégnées d’insecticide par rapport à ceux dormant dessous. De plus, une corrélation positive a été observée entre les réponses IgG anti-EGS et les Ac IgG anti 30 kDa au sein de la population totale.Cette étude a montré que l’utilisation des biomarqueurs d’exposition aux piqûres des Aedes pourraient un outil pertinent pour l’évaluation de l’hétérogénéité d’exposition aux vecteurs d’arbovirus dans le contexte urbain africain et entre les départements d’une même région dans le Sud de la France métropolitaine. Les réponses IgG anti-EGS et anti-protéine 30 kDa représenteraient des candidats biomarqueurs potentiels aux piqûres de Culex. Toutefois, leur validation comme de ce biomarqueur d’exposition aux Culex, des études complémentaires sont nécessaires.
... In contrast, video analysis allows measuring a number of variables in parallel and is especially suitable for measuring locomotor activities (flight/walking speed and duration, resting time, angle, etc.), permitting dynamic integration of mosquito behaviour to aid the interpretation of results (Noldus et al., 2001). Video tracking has been used to study various behaviours of adult mosquitoes (Spitzen and Takken, 2018), such as diel activity patterns (Araujo et al., 2020;Boyer et al., 2013;Lebon et al., 2018), host-seeking behaviour in response to heat/odour or repellents/insecticides (Hawkes and Gibson, 2016;Parker et al., 2017;Spitzen et al., 2013), and mating behaviour (Butail et al., 2012). ...
Mosquito-borne diseases impose a high burden on human and animal health. Temperature strongly influences the physiology and life cycle of mosquitoes, but also the development and/or propagation of the pathogens they transmit. Thus, the vector capacity of mosquitoes depends strongly on temperature and their behavioural thermoregulation through microhabitat selection. Expanding on a previous study of static thermal preferences, the locomotory dynamics of temperate Aedes japonicus (reared from eggs collected in the field) and tropical Ae. aegypti (from a laboratory colony) was investigated at constant temperatures (10 °C, 25 °C, 40 °C) and in temperature gradients (10–20 °C, 20–30 °C, 30–40 °C). Blood-fed or non-blood-fed female mosquitoes were released in groups of 15 individuals into a Plexiglas box positioned on two thermoregulators connected by an aluminium plate to automatically monitor by video analysis mosquito flying, walking and resting duration, covered distances and velocity. Mosquitoes were predominantly resting, followed by walking and flying. At constant 10 °C, flights were rare and brief, and walking was slow. Most activity was observed at 25 °C for Ae. japonicus and 40 °C for Ae. aegypti. In the 30–40 °C gradient, activity of Ae. aegypti increased towards the cold end, suggesting active avoidance of very high temperatures. In the 20–30 °C gradient, edge effects were prominent, nevertheless revealing a greater proportion of mosquitoes gathered at the cooler end. Video analysis showed that this effect was not caused by a cold trap but represents true thermal preference. In the coolest gradient (10–20 °C), mosquitoes were active in all sectors without displaying a preference for either side. Overall, both the tropical and temperate mosquito species preferred cooler temperatures and actively avoided the hottest temperatures. Further studies with infected mosquitoes should provide important insights for developing models of vector-borne disease outbreaks.
... Aedes aegypti, the primary vector of numerous emerging vector-borne diseases including Yellow Fever, Dengue, Chikungunya, and more recently Zika virus [9] displays a small activity peak at the start of the light phase but is primarily active during the late afternoon hours [10]. The diurnal activity patterns of Aedes aegypti are found in flight activity, oviposition, host-seeking, and human landing/biting [11]. ...
Like other insects, Aedes aegypti displays strong daily patterns in host seeking and mating. Much of these behaviors are believed to be under the control of a circadian clock, an endogenous timekeeping mechanism relying on transcriptional/translational negative feedback loops that drive rhythmic physiology and behavior. To examine the connection between the circadian clock and various Ae. aegypti behaviors, we knocked out the core clock gene cycle using CRISPR/Cas9. We found that the rhythmic pattern and intensity of mRNA expression of seven circadian genes, including AeCyc-/-, were altered across the day/night cycle as well as in constant darkness conditions. We further show that, if expressed, the mutant CYC protein is incapable of forming a dimer with CLK to stimulate per expression and that the endogenous clock is disabled in AeCyc-/- mosquitoes. AeCyc-/- do not display the bimodal locomotor activity pattern of wild type, have a significantly reduced response to host odor, reduced egg hatching rates, delayed embryonic development, and reduced adult survival and mating success. Surprisingly, however, the propensity to blood feed in AeCyc-/- females is significantly higher than in wildtype females. Together with other recent work on the circadian clock control of key aspects of mosquito biology, our data on how cycle KO affects mosquito behavior and fitness provides a basis for further work into the pathways that connect the mosquito endogenous clock to its vector competence.
... Video technology such as this has been used to record mosquito behavior and activity in laboratory settings previously, including under light-dark and constant dark conditions (e.g. Araujo et al., 2020). We focused on the transition event (eclosion) between the pupal and adult states because it is relatively quick and discrete, and is easily noted in video footage. ...
Genes known to affect circadian rhythms (i.e. ‘clock genes’) also influence the photoperiodic induction of overwintering reproductive diapause in the northern house mosquito, Culex pipiens f. pipiens. This suggests that molecular changes in one or more clock genes could contribute to the inability to diapause in a second form of this mosquito, Culex pipiens f. molestus. Temperate populations of Cx. pipiens f. molestus inhabit underground locations generally devoid of predictable photoperiods. For this reason, there could be limited fitness consequences if the hypothesized molecular changes to its clock genes also eliminated this mosquito's ability to regulate circadian rhythms in response to photoperiod variation. Here, we demonstrate that in contrast to this prediction, underground derived Cx. pipiens f. molestus retain exogenously influenceable circadian rhythms. Nonetheless, our genetic analyses indicate that the gene Helicase domino (dom) has a nine-nucleotide, in-frame deletion specific to Cx. pipiens f. molestus. Previous work has shown that splice variants in this gene differentially influence circadian behavior in Drosophila melanogaster. We also find derived, non-synonymous single nucleotide polymorphisms (SNPs) in eight genes that may also affect circadian rhythms and/or diapause induction in Cx. pipiens f. molestus. Finally, four putative circadian genes were found to have no quantifiable expression during any examined life stage, suggesting potential regulatory effects. Collectively, our findings indicate that the distinct, but molecularly interconnected life-history traits of diapause induction and circadian rhythms are decoupled in Cx. pipiens f. molestus and suggest this taxon may be a valuable tool for exploring exogenously influenced phenotypes in mosquitoes more broadly.
... However, the activity cycle of the culicoid biting midges responsible for the current AHS outbreak in Thailand has not been fully investigated. In contrast, mosquitoes can be diurnal, crepuscular, and nocturnal (Clements, 1999;da Silva Araujo, Guo, & Rosbash, 2020). As a result, horses within an AHSV outbreak area are at risk for disease transmission throughout the entire day. ...
Horses were provided full-time housing in unfamiliar vector-protected facilities during the African horse sickness (AHS) outbreak in Thailand. This study aimed to investigate the impact of this housing arrangement on the equine stress response. Nine healthy horses were housed in both a traditional barn and a vector-protected barn. Equine behavior and stress response data were collected in association with the housing environment and time of day. The mean behavioral score of horses housed in the vector-protected barn was lower at night than during the day. In addition, the horses’ mean heart rate at night was lower than their heart rate during the day, irrespective of housing condition. Furthermore, although blood cortisol peaked at 6:00 AM and was lowest at 6:00 PM under both housing conditions, daily fluctuations in blood cortisol levels were correlated with changes in humidity and temperature in both environments. Finally, horses housed in the traditional barn exhibited earlier decreases in cortisol levels relative to the horses in the vector-protected barn. This result indicates that housing horses in vector-protected facilities may impose stress.
Background: Aedes albopictus (Skuse 1894) mosquitoes can transmit deadly arboviruses and are globally invasive due to their ability to survive in both tropical and temperate climates. Although adults cannot survive harsh winters, females are capable of anticipating seasonal change and producing overwintering diapause (DP) eggs that remain in a state of arrested development over the winter and hatch when favorable conditions return in the spring. Previous work has shown that shortening photoperiod (day length) alone is sufficient for DP induction. While decreasing temperatures can facilitate DP entry, temperature signals alone are not sufficient to induce DP.
Methodology/Principal Findings: To identify maternal phenotypes predictive of DP egg production, we characterized aspects of maternal physiology and behavior to identify those that correlate with DP egg production and changes in photoperiod, versus changes in temperature. Neither changes in temperature nor photoperiod impacted protein preference, blood meal consumption, or total number of eggs produced per female. Egg retention and oviposition timing were influenced by temperature, independent of DP egg production. However, females housed under short photoperiod conditions showed increased starvation resistance, despite showing similar levels of locomotor activity and internal stores of triacylglycerols, glucose, glycogen, and trehalose compared to females housed in long photoperiods.
Conclusions/Significance: These results suggest that temperature and photoperiod differentially affect maternal phenotypes and identify starvation resistance as a maternal phenotype that is influenced by photoperiod and can be used to predict DP egg status.
