Molecular diversity of genes related to biological rhythms (period and timeless) and insecticide resistance (Na V and ace-1) in Anopheles darlingi.

BACKGROUND: Malaria is a public health concern in the Amazonian Region, where Anopheles darlingi is the main vector of Plasmodium spp. Several studies hypothesised the existence of cryptic species in An. darlingi, considering variations in behaviour, morphological and genetic aspects. Determining their overall genetic background for vector competence, insecticide resistance, and other elements is essential to better guide strategies for malaria control. OBJECTIVES: This study aimed to evaluate the molecular diversity in genes related to behaviour and insecticide resistance, estimating genetic differentiation in An. darlingi populations from Amazonian localities in Brazil and Pacific Colombian region. METHODS: We amplified, cloned and sequenced fragments of genes related to behaviour: timeless (tim) and period (per), and to insecticide resistance: voltage-gated sodium channel (Na V ) and acetylcholinesterase (ace-1) from 516 An. darlingi DNA samples from Manaus, Unini River, Jaú River and Porto Velho - Brazil, and Chocó - Colombia. We discriminated single nucleotide polymorphisms (SNPs), determined haplotypes and evaluate the phylogenetic relationship among the populations. FINDINGS: The genes per, tim and ace-1 were more polymorphic than Na V . The classical kdr and ace-1 R mutations were not observed. Phylogenetic analyses suggested a significant differentiation between An. darlingi populations from Brazil and Colombia, except for the Na V gene. There was a geographic differentiation within Brazilian populations considering per and ace-1. CONCLUSIONS: Our results add genetic data to the discussion about polymorphisms at population levels in An. darlingi. The search for insecticide resistance-related mechanisms should be extended to more populations, especially from localities with a vector control failure scenario.

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.

Male accessory gland substances from Aedes albopictus affect the locomotor activity of Aedes aegypti females.

Dengue is one of the world's most important mosquito-borne diseases and is usually transmitted by one of two vector species: Aedes aegypti or Aedes albopictus . These two diurnal mosquitoes are frequently found coexisting in similar habitats, enabling interactions between adults, such as cross-mating. The objective of this study was to assess cross-mating between Ae. aegypti females and Ae. albopictus males under artificial conditions and evaluate the locomotor activity of Ae. aegypti virgin females injected with male accessory gland (MAG) homogenates to infer the physiological and behavioural responses to interspecific mating. After seven days of exposure, 3.3-16% of Ae. aegypti females mated with Ae. albopictus males. Virgin Ae. aegypti females injected with conspecific and heterospecific MAGs showed a general decrease in locomotor activity compared to controls and were refractory to mating with conspecific males. The reduction in diurnal locomotor activity induced by injections of conspecific or heterospecific MAGs is consistent with regulation of female reproductive activities by male substances, which are capable of sterilising female Ae. aegypti through satyrisation by Ae. albopictus.

Circadian clock of Aedes aegypti: effects of blood-feeding, insemination and RNA interference.

Mosquitoes are the culprits of some of the most important vector borne diseases. A species' potential as a vector is directly dependent on their pattern of behaviour, which is known to change according to the female's physiological status such as whether the female is virgin/mated and unfed/blood-fed. However, the molecular mechanism triggered by and/or responsible for such modulations in behaviour is poorly understood. Clock genes are known to be responsible for the control of circadian behaviour in several species. Here we investigate the impact mating and blood-feeding have upon the expression of these genes in the mosquito Aedes aegypti. We show that blood intake, but not insemination, is responsible for the down-regulation of clock genes. Using RNA interference, we observe a slight reduction in the evening activity peak in the fourth day after dstim injection. These data suggest that, as in Drosophila, clock gene expression, circadian behaviour and environmental light regimens are interconnected in Ae. aegypti.

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.

Correction: Frequency of co-seropositivities for certain pathogens and their relationship with clinical and histopathological changes and parasite load in dogs infected with Leishmania infantum.

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

Males of Aedes aegypti show different clock gene expression profiles in the presence of conspecific females.

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.

Potential impact of a presumed increase in the biting activity of dengue-virus-infected Aedes aegypti (Diptera: Culicidae) females on virus transmission dynamics.

Recently, we showed that infection with dengue virus increases the locomotor activity of Aedes aegypti females. We speculate that the observed increased locomotor activity could potentially increase the chances of finding a suitable host and, as a consequence, the relative biting rate of infected mosquitoes. We used a mathematical model to investigate the impact of the increased locomotor activity by assuming that this activity translated into an increased biting rate for infected mosquitoes. The results show that the increased biting rate resulted in dengue outbreaks with greater numbers of primary and secondary infections and with more severe biennial epidemics.

Spatiotemporal transcription of the P element and the 412 retrotransposon during embryogenesis of Drosophila melanogaster and D. willistoni.

Transposable elements (TEs) are mobile nucleotide sequences which, through changing position in host genomes, partake in important evolutionary processes. The expression patterns of two TEs, P element transposon and 412 retrotransposon, were investigated during Drosophila melanogaster and D. willistoni embryogenesis, by means of embryo hybridization using riboprobes. Spatiotemporal transcription patterns for both TEs were similar to those of developmental genes. Although the two species shared the same P element transcription pattern, this was not so with 412 retrotransposon. These findings suggest that the regulatory sequences involved in the initial development of Drosophila spp are located in the transposable element sequences, and differences, such as in this case of the 412 retrotransposon, lead to losses or changes in their transcription patterns.

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.

Frequency of co-seropositivities for certain pathogens and their relationship with clinical and histopathological changes and parasite load in dogs infected with Leishmania infantum.

In canine leishmaniosis caused by the protozoan Leishmania infantum, little is known about how co-infections with or co-seropositivities for other pathogens can influence aggravation of this disease. Therefore, the objectives of this study were to evaluate the frequency of co-infections with or co-seropositivities for certain pathogens in dogs seropositive for L. infantum and their relationship with clinical signs, histological changes and L. infantum load. Sixty-six L. infantum-seropositive dogs were submitted to clinical examination, collection of blood and bone marrow, culling, and necropsy. Antibodies against Anaplasma spp., Borrelia burgdorferi sensu lato, Ehrlichia spp. and Toxoplasma gondii and Dirofilaria immitis antigens were investigated in serum. Samples from different tissues were submitted to histopathology and immunohistochemistry for the detection of Leishmania spp. and T. gondii. Quantitative real-time PCR was used to assess the L. infantum load in spleen samples. For detection of Coxiella burnetii, conventional PCR and nested PCR were performed using bone marrow samples. All 66 dogs tested positive for L. infantum by qPCR and/or culture. Fifty dogs (76%) were co-seropositive for at least one pathogen: T. gondii (59%), Ehrlichia spp., (41%), and Anaplasma spp. (18%). Clinical signs were observed in 15 (94%) dogs monoinfected with L. infantum and in 45 (90%) dogs co-seropositive for certain pathogens. The L. infantum load in spleen and skin did not differ significantly between monoinfected and co-seropositive dogs. The number of inflammatory cells was higher in the spleen, lung and mammary gland of co-seropositive dogs and in the mitral valve of monoinfected dogs. These results suggest that dogs infected with L. infantum and co-seropositive for certain pathogens are common in the region studied. However, co-seropositivities for certain pathogens did not aggravate clinical signs or L. infantum load, although they were associated with a more intense inflammatory reaction in some organs.

The Summer Is Coming: nocte and timeless Genes Are Influenced by Temperature Cycles and May Affect Aedes aegypti Locomotor Activity.

Mosquitoes exhibit activity rhythms, crucial for the transmission of pathogens, under the control of a circadian clock. Aedes aegypti is one of the world's leading vectors. For decades, several studies have linked the rise in ambient temperature with the increase in their activity. Here, we identify candidate genes whose expression is influenced by temperature cycles and may affect Aedes locomotor activity. We observed that timeless completely lost its rhythmic expression in light/dark, with out-of-phase temperature cycles, and by RNAi mediated knockdown of nocte, an important gene for Drosophila circadian synchronization by temperature cycles. Thus, timeless and nocte are important genes for synchronization by temperature cycles in Aedes aegypti. To reinforce our findings, we simulated in the laboratory the gradual temperature fluctuations that were as close as possible to daily temperature variations in Brazil. We observed that the activity and the expression of the molecular circadian clock of Ae. aegypti differs significantly from that of mosquitoes subjected to constant or rectangular abrupt changes in temperature. We suggest that for understanding the circadian behavior of Aedes with possible implications for intervention strategies, the seminatural paradigm needs to replace the traditional laboratory study.

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.

Frequency, active infection and load of Leishmania infantum and associated histological alterations in the genital tract of male and female dogs.

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.

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.

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.

Locomotor activity in males of Aedes aegypti can shift in response to females' presence.

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.

In tune with nature: Wolbachia does not prevent pre-copula acoustic communication in Aedes aegypti.

BACKGROUND: Mosquito-borne diseases are rapidly spreading to vast territories, putting at risk most of the world's population. A key player in this scenario is Aedes aegypti, a hematophagous species which hosts and transmits viruses causing dengue and other serious illnesses. Since vector control strategies relying only on insecticides have proven unsustainable, an alternative method involving the release of Wolbachia-harboring individuals has emerged. Its successful implementation vastly depends on how fit the released individuals are in the natural habitat, being able to mate with wild populations and to spread Wolbachia to subsequent generations. In mosquitoes, an important aspect of reproductive fitness is the acoustic communication between males and females, which translates to interactions between harmonic frequencies in close proximity flight. This study aimed to characterize the flight tone produced by individuals harboring Wolbachia, also evaluating their ability to establish stable acoustic interactions. METHODS: Wild-type (WT) and Wolbachia-harboring specimens (wMelBr) were thorax-tethered to blunt copper wires and placed at close proximity to sensitive microphones. Wing-beat frequencies (WBFs) were characterized at fundamental and harmonic levels, for both single individuals and couples. Harmonic interactions in homogeneous and heterogeneous couples of WT and wMelBr variants were identified, categorized and quantified accordingly. RESULTS: In tethered 'solo' flights, individuals harboring Wolbachia developed WBFs, differing slightly, in a sex-dependent way, from those of the WT strain. To test the ability to form harmonic 'duets', tethered couples of wMelBr and WT individuals were shuffled in different sex pairs and had their flight tones analyzed. All couple types, with WT and/or wMelBr individuals, were able to interact acoustically in the frequency range of 1300-1500 Hz, which translates to the convergence between male's second harmonic and female's third. No significant differences were found in the proportions of interacting couples between the pair types. Surprisingly, spectrograms also revealed the convergence between alternative harmonic frequencies, inside and outside the species putative hearing threshold. CONCLUSIONS: Wolbachia infection leads to small sex-dependent changes on the flight tones of Ae. aegypti, but it does not seem to prevent the stereotyped harmonic interaction between males and females. Therefore, when released in the natural habitat to breed with native individuals, Wolbachia-harboring individuals shall be fit enough to meet the criteria of acoustically-related mating behavior and promote bacteria dispersion effectively.