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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.

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.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.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.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.

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.

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.

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.

Mosquito vectors lay their white eggs in the aquatic milieu. During early embryogenesis water passes freely through the transparent eggshell, which at this moment is composed of exochorion and endochorion. Within two hours the endochorion darkens via melanization but even so eggs shrink and perish if removed from moisture. However, during mid-embryogenesis, cells of the extraembryonic serosa secrete the serosal cuticle, localized right below the endochorion, becoming the third and innermost eggshell layer. Serosal cuticle formation greatly reduces water flow and allows egg survival outside the water. The degree of egg resistance to desiccation (ERD) at late embryogenesis varies among different species: Aedes aegypti, Anopheles aquasalis and Culex quinquefasciatus eggs can survive in a dry environment for ≥ 72, 24 and 5 hours, respectively. In some adult insects, darker-body individuals show greater resistance to desiccation than lighter ones. We asked if egg melanization enhances mosquito serosal cuticle-dependent ERD. Species with higher ERD at late embryogenesis exhibit more melanized eggshells. The melanization-ERD hypothesis was confirmed employing two Anopheles quadrimaculatus strains, the wild type and the mutant GORO, with a dark-brown and a golden eggshell, respectively. In all cases, serosal cuticle formation is fundamental for the establishment of an efficient ERD but egg viability outside the water is much higher in mosquitoes with darker eggshells than in those with lighter ones. The finding that pigmentation influences egg water balance is relevant to understand the evolutionary history of insect egg coloration. Since eggshell and adult cuticle pigmentation ensure insect survivorship in some cases, they should be considered regarding species fitness and novel approaches for vector or pest insects control., Author summary Mosquitoes transmit various causative agents of diseases and the blockage of vector life cycle is an effective way to hamper disease transmission. The egg is the least known life stage and understanding it can contribute with novel strategies for mosquito control. Mosquitoes lay eggs in water collections, some of which are temporary. At early embryogenesis eggs are prone to lose water, leading to dehydration and death. During embryogenesis the serosal cuticle is produced, it wraps the embryo and contributes to the egg protection, allowing it to survive outside the water. Curiously, this resistance varies among mosquitoes: Aedes, Anopheles and Culex eggs can survive outside the water for long, intermediate and short periods, respectively. Here, we show that these differences are related to the degree of eggshell melanization (melanin is a dark pigment): darker eggs resists more against water loss. We confirmed that melanin increases survival outside the water employing a mosquito mutant that does not melanize properly. The protection conferred by melanin is dependent on the formation of the serosal cuticle. Our results contribute to the study of the evolution of egg coloration in insects and we identified one of the reasons why Aedes aegypti eggs survive for several months outside water.

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.

Population control of the dengue vector mosquito, Aedes aegypti, is difficult due to many reasons, one being the development of resistance to neurotoxic insecticides employed. The biosynthesis of chitin, a major constituent of insect cuticle, is a novel target for population control. Novaluron is a benzoylphenylurea (BPU) that acts as a chitin synthesis inhibitor, already used against mosquitoes. However, information regarding BPU effects on immature mosquito stages and physiological parameters related with mosquito larval development are scarce. A set of physiological parameters were recorded in control developing larvae and novaluron was administered continuously to Ae. aegypti larvae, since early third instar. Larval instar period duration was recorded from third instar until pupation. Chitin content was measured during third and fourth instars. Fourth instars were processed histochemically at the mesothorax region, stained with hematoxylin and eosin (HE) for assessment of internal tissues, and labeled with WGA-FITC to reveal chitinized structures. In control larvae: i) there is a chitin content increase during both third and fourth instars where late third instars contain more chitin than early fourth instars; ii) thoracic organs and a continuous cuticle, closely associated with the underlying epidermis were observed; iii) chitin was continuously present throughout integument cuticle. Novaluron treatment inhibited adult emergence, induced immature mortality, altered adult sex ratio and caused delay in larval development. Moreover, novaluron: i) significantly affected chitin content during larval development; ii) induced a discontinuous and altered cuticle in some regions while epidermis was often thinner or missing; iii) rendered chitin cuticle presence discontinuous and less evident. In both control and novaluron larvae, chitin was present in the peritrophic matrix. This study showed quantitatively and qualitatively evidences of novaluron effects on Ae. aegypti larval development. To our knowledge, this is the first report describing histological alterations produced by a BPU in immature vector mosquitoes.

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.