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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: The chemical control of the mosquito Aedes aegypti, the major vector of dengue, is being seriously threatened due to the development of pyrethroid resistance. Substitutions in the 1016 and 1534 sites of the voltage gated sodium channel (AaNaV), commonly known as kdr mutations, confer the mosquito with knockdown resistance. Our aim was to evaluate the allelic composition of natural populations of Brazilian Ae. aegypti at both kdr sites. METHODS: The AaNaVIIIS6 region was cloned and sequenced from three Brazilian populations. Additionally, individual mosquitoes from 30 populations throughout the country were genotyped for 1016 and 1534 sites, based in allele-specific PCR. For individual genotypes both sites were considered as a single locus. RESULTS: The 350 bp sequence spanning the IIIS6 region of the AaNa V gene revealed the occurrence of the kdr mutation Phe1534Cys in Brazil. Concerning the individual genotyping, beyond the susceptible wild-type (NaVS), two kdr alleles were identified: substitutions restricted to the 1534 position (NaVᴿ¹) or simultaneous substitutions in both 1016 and 1534 sites (NaVᴿ²). A clear regional distribution pattern of these alleles was observed. The NaVᴿ¹kdr allele occurred in all localities, while NaVᴿ²was more frequent in the Central and Southeastern localities. Locations that were sampled multiple times in the course of a decade revealed an increase in frequency of the kdr mutations, mainly the double mutant allele NaVᴿ². Recent samples also indicate that NaVᴿ²is spreading towards the Northern region. CONCLUSIONS: We have found that in addition to the previously reported Val1016Ile kdr mutation, the Phe1534Cys mutation also occurs in Brazil. Allelic composition at both sites was important to elucidate the actual distribution of kdr mutations throughout the country. Studies to determine gene flow and the fitness costs of these kdr alleles are underway and will be important to better understand the dynamics of Ae. aegypti pyrethroid resistance.

Background The study of behavioral and physiological traits in mosquitoes has been mainly focused on females since males are not hematophagous and thus do not transfer the parasites that cause diseases in human populations. However, the performance of male mosquitoes is key for the expansion of populations and the perpetuation of mosquito species. Pre-copulatory communication between males and females is the initial and essential step for the success of copulation and studying the male facet of this interaction provides fertile ground for the improvement of vector control strategies. Like in most animals, reproduction, feeding, and oviposition are closely associated with locomotor activity in mosquitoes. Rhythmic cycles of locomotor activity have been previously described in Aedes aegypti , and in females, they are known to be altered by blood-feeding and arbovirus infection. In previous work, we found that males in the presence of females significantly change their locomotor activity profiles, with a shift in the phase of the activity peak. Here, we investigated whether this shift is associated with changes in the expression level of three central circadian clock genes. Methods Real-time PCR reactions were performed for the gene period , cycle , and cryptochrome 2 in samples of heads, antennae, and abdominal tips of solitary males and males in the presence of females. Assays with antennae-ablated males were also performed, asking whether this is an essential organ mediating the communication and the variation in activity profiles. Results The gene period showed a conserved expression pattern in all tissues and conditions, while the other two genes varied according to the male condition. A remarking pattern was observed in cry2 , where the difference between the amplitude of expression at the beginning of photophase and the expression peak in the scotophase was greater when males were in the presence of females. Antennae ablation in males did not have a significant effect on the expression profiles, suggesting that female recognition may involve other senses besides hearing and olfaction. Conclusion Our results suggest that the expression of gene cryptochrome 2 varies in association with the interaction between males and females. Graphical abstract

DNA barcoding has been an effective tool for species identification in several animal groups. Here, we used DNA barcoding to discriminate between 47 morphologically distinct species of Brazilian sand flies. DNA barcodes correctly identified approximately 90% of the sampled taxa (42 morphologically distinct species) using clustering based on neighbor-joining distance, of which four species showed comparatively higher maximum values of divergence (range 4.23-19.04%), indicating cryptic diversity. The DNA barcodes also corroborated the resurrection of two species within the shannoni complex and provided an efficient tool to differentiate between morphologically indistinguishable females of closely related species. Taken together, our results validate the effectiveness of DNA barcoding for species identification and the discovery of cryptic diversity in sand flies from Brazil.

[This corrects the article DOI: 10.1371/journal.pone.0247560.].

Visceral leishmaniasis caused by the protozoan Leishmania infantum is a zoonosis. The domestic dog is the primary reservoir in urban areas. This study aimed to evaluate the frequency, active infection and load of L. infantum in the genital tract of male and female dogs seropositive for this parasite, as well as to identify histological genital alterations associated with this protozoan. We studied 45 male and 25 female L. infantum-seropositive noncastrated dogs from the same endemic area in Brazil. Tissue samples from the testis, epididymis, prostate, vulva, vagina, and uterus were examined by singleplex qPCR and parasitological tests (histopathology, immunohistochemistry, and parasitological culture). The latter were performed for the detection of active infection (parasites able to multiply and to induce lesions). Forty-four (98%) males and 25 (100%) females were positive for L. infantum in the genital tract (epididymis: 98%; vulva: 92%; vagina: 92%; testis: 91%; uterus: 84%; prostate: 66%). Active infection in the genital tract was confirmed in 69% of males and 64% of females (32% in the uterus). Parasite loads were similar in the testis, vulva, epididymis and vagina and lower in the prostate. Only the parasite load in the vagina was significantly associated with the number of clinical signs. Granulomatous inflammation predominated in all organs, except for the prostate. Only in the testis and epididymis was the inflammatory infiltrate significantly more intense among dogs with a higher parasite load in these organs. The high frequency, detection of active infection and similarity of L. infantum loads in the genital tract of infected males and females suggest the potential of venereal transmission of this parasite by both sexes and of vertical transmission by females in the area studied. Additionally, vertical transmission may be frequent since active L. infantum infection was a common observation in the uterus.

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 w Mel exerts on embryo development and egg viability and speculate on its field release use. Methods Wild-type (Br or Rockefeller) and Wolbachia -harboring specimens ( w MelBr) 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 w MelBr 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, w MelBr 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 w Mel 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 w Mel 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.

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

Background The study of physiological and behavioral traits of mosquito vectors has been of growing relevance for the proposition of alternative methods for controlling vector-borne diseases. Despite this, most studies focus on the female’s traits, including the behavior of host seeking, the physiology of disease transmission and the site-choice for oviposition. However, understanding the factors that lead to males’ reproductive success is of utmost importance, since it can help building new strategies for constraining population growth. Male behavior towards mating varies widely among species and the communication between males and females is the first aspect securing a successful encounter. Here we used an automated monitoring system to study the profile of locomotor activity of Aedes aegypti males in response to female’s presence in an adapted confinement tube. We propose a new method to quantify male response to the presence of females, which can be potentially tested as an indicator of the success of one male in recognizing a female for mating. Results Locomotor activity varies in daily cycles regulated by an endogenous clock and synchronized by external factors, such as light and temperature. Our results show the previously described startle response to light, which is displayed as a steep morning activity peak immediately when lights are on. Activity drops during the day and begins to rise again right before evening, happening about 1.5 h earlier in males than in females. Most interestingly, males’ activity shows a double peak, and the second peak is very subtle when males are alone and relatively more pronounced when females are present in the confinement tubes. The switch in the peak of activity, measured by the herein suggested Peak Matching Index (PMI), was significantly different between males with and without females. Conclusions The adapted monitoring system used here allowed us to quantify the response of individual males to nearby females in terms of the extent of the activity peak displacement. In this direction, we created the peak matching index (PMI), a new parameter that we anticipate could be interpreted as the inclination of males to respond to females’ presence, and further tested as an indicator of the potential for finding females for mating.