Light and dark cycles modify the expression of clock genes in the ovaries of Aedes aegypti in a noncircadian manner.

Circadian oscillators (i.e., circadian clocks) are essential to producing the circadian rhythms observed in virtually all multicellular organisms. In arthropods, many rhythmic behaviors are generated by oscillations of the central pacemaker, specific groups of neurons of the protocerebrum in which the circadian oscillator molecular machinery is expressed and works; however, oscillators located in other tissues (i.e., peripheral clocks) could also contribute to certain rhythms, but are not well known in non-model organisms. Here, we investigated whether eight clock genes that likely constitute the Aedes aegypti clock are expressed in a circadian manner in the previtellogenic ovaries of this mosquito. Also, we asked if insemination by conspecific males would alter the expression profiles of these clock genes. We observed that the clock genes do not have a rhythmic expression profile in the ovaries of virgin (VF) or inseminated (IF) females, except for period, which showed a rhythmic expression profile in ovaries of IF kept in light and dark (LD) cycles, but not in constant darkness (DD). The mean expression of seven clock genes was affected by the insemination status (VF or IF) or the light condition (LD 12:12 or DD), among which five were affected solely by the light condition, one solely by the insemination status, and one by both factors. Our results suggest that a functional circadian clock is absent in the ovaries of A. aegypti. Still, their differential mean expression promoted by light conditions or insemination suggests roles other than circadian rhythms in this mosquito's ovaries.

Experiências em divulgação científica e sensibilização da população: importância do controle mecânico do vetor Aedes aegypti / Experiences in scientific outreach and public awareness: the importance of mechanical control of the Aedes aegypti vector / Experiencias en divulgación científica y sensibilización de la población: importancia del control mecánico del vector Aedes aegypti

Aedes aegypti é o principal vetor dos agentes etiológicos de dengue, zika e chikungunya, doenças para as quais não existem vacinas totalmente eficazes. Alternativas de controle visando mitigar essas arboviroses são primordiais. Entre essas, o controle mecânico aborda práticas de eliminação e/ou limpeza de criadouros do vetor. Neste relato, apresentamos e avaliamos criticamente ações realizadas pelo grupo, ocorridas entre 2016 e 2019, nas quais divulgamos informação científica clara através do diálogo com a população. Os métodos utilizados foram: 1) palestras em escolas (público infantojuvenil) utilizando slides, fotos e vídeos; 2) oficinas (público misto), estande com material in vivo do ciclo de vida do Aedes, jogos e desenhos. Analisamos dez palestras em escolas do ensino fundamental e médio e vinte oficinas realizadas em diferentes regiões do Brasil. Concluímos que tais ações e suas análises críticas devem ser realizadas continuamente para que sejam bem-sucedidas

Aedes aegypti is the main vector of dengue, zika, and chikungunya etiological agents, diseases for which no effective vaccines are available. Control alternatives aimed at mitigating these arboviruses are essential. Among such, mechanical control addresses practices of elimination and/or cleaning of vector breeding sites. Here, we presented and critically evaluated actions carried out by ourselves. These actions took place between 2016 and 2019, where we disseminated clear scientific information through dialogue with the population. The following methods were employed: 1) lectures in schools (children and youth audiences) using slides, photos, and videos; 2) workshops (mixed audience), stand with in vivo material from the Aedeslife cycle, games, and drawings. Ten lectures in elementary and high schools and twenty workshops held in different regions of Brazil were analyzed. It was concluded that such actions and their critical analyzes must be carried out continuously to be successful

Aedes aegypti es el principal vector de los agentes etiológicos del dengue, zika y chikungunya, enfermedades para las que no existen vacunas totalmente eficaces. Las alternativas de control para mitigar estas arbovirosis son fundamentales. El control mecánico, representa una de estas alternativas, aborda prácticas de eliminación y/o limpieza de criaderos del vector. En este informe presentamos y evaluamos de manera crítica las acciones realizadas por el grupo entre los años 2016 y 2019. Presentamos información científica clara a través del diálogo con la población mediante los siguientes métodos: 1) conferencias en escuelas (público infantil) utilizando diapositivas, fotos y videos; 2) Talleres (público mixto), stand con material in vivo del ciclo de vida del Aedes, juegos y dibujos. Analizamos diez conferencias en escuelas (primarias y secundarias) y veinte talleres realizados en diferentes regiones de Brasil. Concluimos que tales acciones y el análisis crítico de las mismas deben llevarse a cabo de manera continua para que resulten exitosas.

The influence of different sources of blood meals on the physiology of Aedes aegypti harboring Wolbachia wMel: mouse blood as an alternative for mosquito rearing.

BACKGROUND: Aedes aegypti control programs have failed to restrain mosquito population expansion and, consequently, the spread of diseases such as dengue, Zika, and Chikungunya. Wolbachia infection of mosquitoes is a new and promising complementary tool for the control of arbovirus transmission. The use of Wolbachia-infected mosquitoes, mass reared using human blood, is currently being tested in several countries. However, the use of human blood for mass rearing mosquitoes, and thus expansion of this strategy, is problematic. With the aim of overcoming this problem, we tested the effect of different types of blood source on the fitness parameters of female Ae. aegypti and the Wolbachia titer over generations to be able to guarantee the suitability of an alternative source to human blood for mass rearing Wolbachia-infected mosquitoes. METHODS: We investigated and compared essential parameters of the vector capacity of laboratory strains of Ae. aegypti with and without Wolbachia that fed on blood of different types of host (human, guinea pig, and mouse). The parameters analyzed were fecundity, fertility, pupation dynamics, and adult survival. Also, we tested whether it is possible to maintain mosquitoes with Wolbachia on mouse blood over generations without losing the bacterium titer. RESULTS: The average number of eggs per female, egg viability and pupation dynamics in the Wolbachia-infected mosquito (wMelBr) strain were similar, regardless of the blood source. The F1 progenies of females that fed on mouse blood or human blood were analyzed. The longevity of males was lower than that of females. F1 female survival differed depending on the presence of Wolbachia in the mother. In subsequent generations analyzed up until F35, the relative Wolbachia density was even higher when mosquitoes fed on mouse blood in comparison to human blood. CONCLUSIONS: Taken together, our results provide no evidence that the different types of blood influenced the fitness of the Wolbachia-infected mosquitoes. The presence of the bacterium in the colonies of Wolbachia-infected Ae. aegypti after 35 generations under the conditions evaluated indicates that they can be maintained on mouse blood. Based on these results, we show that it is possible to use mouse blood to feed female mosquitoes when using human blood for this purpose is problematic.

Unlike Zika, Chikungunya virus interferes in the viability of Aedes aegypti eggs, regardless of females' age.

Chikungunya and Zika are arboviruses transmitted by the mosquito Aedes aegypti. Mosquito fecundity and egg viability are important parameters of vectorial capacity. Here we aim to understand, comparatively, the effects of Chikungunya virus (CHIKV) and Zika virus (ZIKV) infections on the fecundity and fertility of young and old Aedes aegypti females. Using artificial infection blood feeding experiments we observed that both CHIKV and ZIKV do not alter the number of eggs laid when compared to uninfected females, although the egg fertility significantly decreases in both young and old CHIKV-infected females. There is an upward trend of null females (infertile females) from 2.1% in young to 6.8% in old ZIKV-infected females. Together, our data revealed that CHIKV and ZIKV affects differently Ae. aegypti physiology, that may be related to different viral spread in nature.

Embryonic development and egg viability of wMel-infected Aedes aegypti.

BACKGROUND: Aedes aegypti is a major disease vector in urban habitats, involved in the transmission of dengue, chikungunya and Zika. Despite innumerous attempts to contain disease outbreaks, there are neither efficient vaccines nor definite vector control methods nowadays. In recent years, an innovative strategy to control arboviruses, which exploits the endosymbiotic bacterium Wolbachia pipientis, emerged with great expectations. The success of the method depends on many aspects, including Wolbachia's cytoplasmic incompatibility and pathogen interference phenotypes, as well as its effect on host fitness. In this work, we investigated the influence the Wolbachia strain wMel exerts on embryo development and egg viability and speculate on its field release use. METHODS: Wild-type (Br or Rockefeller) and Wolbachia-harboring specimens (wMelBr) were blood-fed and submitted to synchronous egg laying for embryo development assays. Samples were analyzed for morphological markers, developmental endpoint and egg resistance to desiccation (ERD). Quiescent egg viability over time was also assessed. RESULTS: wMelBr samples completed embryogenesis 2-3 hours later than wild-type. This delay was also observed through the onset of both morphological and physiological markers, respectively by the moments of germband extension and ERD acquisition. Following the end of embryonic development, wMelBr eggs were slightly less resistant to desiccation and showed reduced viability levels, which rapidly decayed after 40 days into quiescence, from approximately 75% to virtually 0% in less than a month. CONCLUSIONS: Our data revealed that the wMel strain of Wolbachia slightly delays embryogenesis and also affects egg quality, both through reduced viability and desiccation resistance. These findings suggest that, although embryonic fitness is somehow compromised by wMel infection, an efficient host reproductive manipulation through cytoplasmic incompatibility seems sufficient to overcome these effects in nature and promote bacterial invasion, as shown by successful ongoing field implementation.

Zika infection decreases Aedes aegypti locomotor activity but does not influence egg production or viability.

BACKGROUND: Zika has emerged as a new public health threat after the explosive epidemic in Brazil in 2015. It is an arbovirus transmitted mainly by Aedes aegypti mosquitoes. The knowledge of physiological, behavioural and biological features in virus-infected vectors may help the understanding of arbovirus transmission dynamics and elucidate their influence in vector capacity. OBJECTIVES: We aimed to investigate the effects of Zika virus (ZIKV) infection in the behaviour of Ae. aegypti females by analysing the locomotor activity, egg production and viability. METHODOLOGY: Ae. aegypti females were orally infected with ZIKV through an artificial feeder to access egg production, egg viability and locomotor activity. For egg production and viability assays, females were kept in cages containing an artificial site for oviposition and eggs were counted. Locomotor activity assays were performed in activity monitors and an average of 5th, 6th and 7th days after infective feeding was calculated. FINDINGS: No significant difference in the number of eggs laid per females neither in their viability were found between ZIKV infected and non-infected females, regardless the tested pair of mosquito population and virus strain and the gonotrophic cycles. Locomotor activity assays were performed regardless of the locomotor activity in ZIKV infected females was observed, in both LD and DD conditions. MAIN CONCLUSIONS: The lower locomotor activity may reduce the mobility of the mosquitoes and may explain case clustering within households reported during Zika outbreaks such as in Rio de Janeiro 2015. Nevertheless, the mosquitoes infected with ZIKV are still able to disseminate and to transmit the disease, especially in places where there are many oviposition sites.

The influence of a light and dark cycle on the egg laying activity of Aedes aegypti (Linnaeus, 1762) (Diptera: Culicidae).

BACKGROUND: The epidemiological importance of the mosquito Aedes aegypti as a vector of multiple human pathogens has generated a growing number of studies on the physiology and behaviour of its blood-feeding females. The activity of oviposition is one of the critical elements contributing to the expansion of Ae. aegypti's populations. Although there is a vast literature about oviposition behaviour, significant specific knowledge about egg viability and female fertility under light and dark conditions is still lacking. OBJECTIVES: We studied, in controlled laboratory conditions, the effect that light and dark cycles have on the efficiency of oviposition by Ae. aegypti females. METHODS: Physiological assays were performed using synchronised eggs obtained from forced egg laying. The number and viability of eggs was analysed under three different light/dark regimes: LD12:12 (12 h of light and 12 h of dark), DD (constant darkness) and LL (constant light). FINDINGS AND CONCLUSIONS: Our results show that females prefer to lay their eggs in dark conditions, but maximising the number and viability of eggs requires the occurrence of a light/dark cycle. Ongoing research on this theme has the potential of contributing to the proposition of new strategies for control based on the failure of egg laying and hatching.

Zika infection decreases Aedes aegypti locomotor activity but does not influence egg production or viability

BACKGROUND Zika has emerged as a new public health threat after the explosive epidemic in Brazil in 2015. It is an arbovirus transmitted mainly by Aedes aegypti mosquitoes. The knowledge of physiological, behavioural and biological features in virus-infected vectors may help the understanding of arbovirus transmission dynamics and elucidate their influence in vector capacity. OBJECTIVES We aimed to investigate the effects of Zika virus (ZIKV) infection in the behaviour of Ae. aegypti females by analysing the locomotor activity, egg production and viability. METHODOLOGY Ae. aegypti females were orally infected with ZIKV through an artificial feeder to access egg production, egg viability and locomotor activity. For egg production and viability assays, females were kept in cages containing an artificial site for oviposition and eggs were counted. Locomotor activity assays were performed in activity monitors and an average of 5th, 6th and 7th days after infective feeding was calculated. FINDINGS No significant difference in the number of eggs laid per females neither in their viability were found between ZIKV infected and non-infected females, regardless the tested pair of mosquito population and virus strain and the gonotrophic cycles. Locomotor activity assays were performed regardless of the locomotor activity in ZIKV infected females was observed, in both LD and DD conditions. MAIN CONCLUSIONS The lower locomotor activity may reduce the mobility of the mosquitoes and may explain case clustering within households reported during Zika outbreaks such as in Rio de Janeiro 2015. Nevertheless, the mosquitoes infected with ZIKV are still able to disseminate and to transmit the disease, especially in places where there are many oviposition sites.

The influence of a light and dark cycle on the egg laying activity of Aedes aegypti (Linnaeus, 1762) (Diptera: Culicidae)

BACKGROUND The epidemiological importance of the mosquito Aedes aegypti as a vector of multiple human pathogens has generated a growing number of studies on the physiology and behaviour of its blood-feeding females. The activity of oviposition is one of the critical elements contributing to the expansion of Ae. aegypti's populations. Although there is a vast literature about oviposition behaviour, significant specific knowledge about egg viability and female fertility under light and dark conditions is still lacking. OBJECTIVES We studied, in controlled laboratory conditions, the effect that light and dark cycles have on the efficiency of oviposition by Ae. aegypti females. METHODS Physiological assays were performed using synchronised eggs obtained from forced egg laying. The number and viability of eggs was analysed under three different light/dark regimes: LD12:12 (12 h of light and 12 h of dark), DD (constant darkness) and LL (constant light). FINDINGS and CONCLUSIONS Our results show that females prefer to lay their eggs in dark conditions, but maximising the number and viability of eggs requires the occurrence of a light/dark cycle. Ongoing research on this theme has the potential of contributing to the proposition of new strategies for control based on the failure of egg laying and hatching.

Physical features and chitin content of eggs from the mosquito vectors Aedes aegypti, Anopheles aquasalis and Culex quinquefasciatus: Connection with distinct levels of resistance to desiccation.

Mosquito eggs are laid in water but freshly laid eggs are susceptible to dehydration, if their surroundings dry out at the first hours of development. During embryogenesis of different mosquito vectors the serosal cuticle, an extracellular matrix, is produced; it wraps the whole embryo and becomes part of the eggshell. This cuticle is an essential component of the egg resistance to desiccation (ERD). However, ERD is variable among species, sustaining egg viability for different periods of time. While Aedes aegypti eggs can survive for months in a dry environment (high ERD), those of Anopheles aquasalis and Culex quinquefasciatus in the same condition last, respectively, for one day (medium ERD) or a few hours (low ERD). Resistance to desiccation is determined by the rate of water loss, dehydration tolerance and total amount of water of a given organism. The ERD variability observed among mosquitoes probably derives from diverse traits. We quantified several attributes of whole eggs, potentially correlated with the rate of water loss: length, width, area, volume, area/volume ratio and weight. In addition, some eggshell aspects were also evaluated, such as absolute and relative weight, weight/area relationship (herein called surface density) and chitin content. Presence of chitin specifically in the serosal cuticle as well as aspects of endochorion external surface were also investigated. Three features could be related to differences on ERD levels: chitin content, directly related to ERD, the increase in the egg volume during embryogenesis and the eggshell surface density, which were both inversely related to ERD. Although data suggest that the amount of chitin in the eggshell is relevant for egg impermeability, the participation of other yet unidentified eggshell attributes must be considered in order to account for the differences in the ERD levels observed among Ae. aegypti, An. aquasalis and Cx. quinquefasciatus.

Serosal cuticle formation and distinct degrees of desiccation resistance in embryos of the mosquito vectors Aedes aegypti, Anopheles aquasalis and Culex quinquefasciatus.

Given their medical importance, mosquitoes have been studied as vectors of parasites since the late 1800's. However, there are still many gaps concerning some aspects of their biology, such as embryogenesis. The embryonic desiccation resistance (EDR), already described in Aedes and Anopheles gambiae mosquitoes, is a peculiar trait. Freshly laid eggs are susceptible to water loss, a condition that can impair their viability. EDR is acquired during embryogenesis through the formation of the serosal cuticle (SC), protecting eggs from desiccation. Nevertheless, conservation of both traits (SC presence and EDR acquisition) throughout mosquito evolution is unknown. Comparative physiological studies with mosquito embryos from different genera, exhibiting distinct evolutionary histories and habits is a feasible approach. In this sense, the process of EDR acquisition of Aedes aegypti, Anopheles aquasalis and Culex quinquefasciatus at 25°C was evaluated. Completion of embryogenesis occurs in Ae. aegypti, An. aquasalis and Cx. quinquefasciatus at, respectively 77.4, 51.3 and 34.3hours after egg laying, Cx. quinquefasciatus embryonic development taking less than half the time of Ae. aegypti. In all cases, EDR is acquired in correlation with SC formation. For both Ae. aegypti and An. aquasalis, EDR and SC appear at 21% of total embryonic development, corresponding to the morphological stage of complete germ band elongation/beginning of germ band retraction. Although phylogenetically closer to Ae. aegypti than to An. aquasalis, Cx. quinquefasciatus acquires both EDR and serosal cuticle later, with 35% of total development, when the embryo already progresses to the middle of germ band retraction. EDR confers distinct egg viability in these species. While Ae. aegypti eggs demonstrated high viability when left up to 72hours in a dry environment, those of An. aquasalis and Cx. quinquefasciatus supported these conditions for only 24 and 5hours, respectively. Our data suggest that serosa development is at least partially uncoupled from embryo development and that, depending upon the mosquito species, EDR bestows distinct levels of egg viability.

Embryonic development of Aedes aegypti (Diptera: Culicidae): influence of different constant temperatures.

Despite its vector importance little attention is given to Aedes aegypti embryonic development. In this study, temperature influence on time course of Ae. aegypti larvae hatching and egg viability were evaluated. The dormancy state at the end of embryogenesis could be interrupted with a proper stimulus. Temperatures tested ranged between 12-36 degrees C; the maximum temperature limit is 35 degrees C and the minimum one is below 12 degrees C. Egg viability between 16-31 degrees C was above 80%. The definition of physiological embryonic parameters at this temperature range corroborates Ae. aegypti presence on tropical and subtropical world regions.

Embryonic development of Aedes aegypti (Diptera: Culicidae): influence of different constant temperatures

Despite its vector importance little attention is given to Aedes aegypti embryonic development. In this study, temperature influence on time course of Ae. aegypti larvae hatching and egg viability were evaluated. The dormancy state at the end of embryogenesis could be interrupted with a proper stimulus. Temperatures tested ranged between 12-36°C; the maximum temperature limit is 35°C and the minimum one is below 12°C. Egg viability between 16-31°C was above 80 percent. The definition of physiological embryonic parameters at this temperature range corroborates Ae. aegypti presence on tropical and subtropical world regions.

Embryonic desiccation resistance in Aedes aegypti: presumptive role of the chitinized serosal cuticle.

BACKGROUND: One of the major problems concerning dengue transmission is that embryos of its main vector, the mosquito Aedes aegypti, resist desiccation, surviving several months under dry conditions. The serosal cuticle (SC) contributes to mosquito egg desiccation resistance, but the kinetics of SC secretion during embryogenesis is unknown. It has been argued that mosquito SC contains chitin as one of its components, however conclusive evidence is still missing. RESULTS: We observed an abrupt acquisition of desiccation resistance during Ae. aegypti embryogenesis associated with serosal cuticle secretion, occurring at complete germ band extension, between 11 and 13 hours after egglaying. After SC formation embryos are viable on dry for at least several days. The presence of chitin as one of the SC constituents was confirmed through Calcofluor and WGA labeling and chitin quantitation. The Ae. aegypti Chitin Synthase A gene (AaCHS1) possesses two alternatively spliced variants, AaCHS1a and AaCHS1b, differentially expressed during Ae. aegypti embryonic development. It was verified that at the moment of serosal cuticle formation, AaCHS1a is the sole variant specifically expressed. CONCLUSION: In addition to the peritrophic matrix and exoskeleton, these findings confirm chitin is also present in the mosquito serosal cuticle. They also point to the role of the chitinized SC in the desiccation resistance of Ae. aegypti eggs. AaCHS1a expression would be responsible for SC chitin synthesis. With this embryological approach we expect to shed new light regarding this important physiological process related to the Ae. aegypti life cycle.