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.

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. 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. 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. 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.

Background: Aedes aegypticontrol programs have failed to restrain mosquito population expansion and, consequently, the spread of diseases such as dengue, Zika, and Chikungunya.Wolbachiainfection of mosquitoes is a new and promising complementary tool for the control of arbovirus transmission. The use ofWolbachia-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 femaleAe. aegyptiand theWolbachiatiter over generations to be able to guarantee the suitability of an alternative source to human blood for mass rearingWolbachia-infected mosquitoes.Methods: We investigated and compared essential parameters of the vector capacity of laboratory strains ofAe. aegyptiwith and withoutWolbachiathat 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 withWolbachiaon mouse blood over generations without losing the bacterium titer.Results: The average number of eggs per female, egg viability and pupation dynamics in theWolbachia-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 ofWolbachiain the mother. In subsequent generations analyzed up until F35, the relativeWolbachiadensity 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 theWolbachia-infected mosquitoes. The presence of the bacterium in the colonies ofWolbachia-infectedAe. aegyptiafter 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.Graphical

Aedes aegypti é o principal vetor de dengue, Zika e chikungunya, doenças para as quais não existem vacinas totalmente eficazes. Alternativas de controle visando mitigar essas arboviroses são primordiais. Dentre 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, onde 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), stand com material in vivo do ciclo de vida do Aedes, jogos e desenhos. Analisamos dez palestras em escolas do ensino fundamental e médio e 20 oficinas realizadas em diferentes regiões do Brasil. Concluímos que tais ações e suas análises críticas devem ser realizadas continuamente para que sejam bem-sucedidas.

Mosquito vectors lay their eggs in the aquatic milieu. During early embryogenesis water passes freely through the transparent eggshell, 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 secretes the serosal cuticle, localized right below the endochorion, which greatly reduces water flow and allows the egg to survive 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 melanization enhances 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 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.

An amendment to this paper has been published and can be accessed via the original article.