The effects of female age on blood-feeding, insemination, sperm storage, and fertility in the dengue vector mosquito Aedes aegypti (Diptera: Culicidae).

Mating induces behavioral and physiological changes in female insects-collectively referred to as the female post-mating response (PMR)-that facilitate the production of progeny. PMRs are elicited by transfer of male-derived seminal components during mating, but are altered by other factors, including adult age. Increased female age is often accompanied by declines in fertility. However, mating shortly after emergence also impacts fertility in the insect model Drosophila melanogaster. Here, we determined the age post-emergence when females of the vector mosquito Aedes aegypti can be inseminated and blood-feed. We next examined fecundity, fertility, and the storage of sperm in the female reproductive tract in "young" (30-41 hours-old) and "old" (2- and 3-week-old) females, finding that blood-feeding began at 14 hours, and mating at ∼24 hours post-emergence. Although young females consumed smaller blood quantities and stored fewer sperm, they were similarly fertile to 4-day-old controls. Old females, however, suffered significant declines in fecundity by 2 weeks of age. Our results show that female Ae. aegypti start to become sexually receptive 1 day after their emergence, but can ingest blood much sooner, suggesting that mating is not a prerequisite to blood-feeding, and that females can ingest an arbovirus infected blood-meal shortly after emergence.

wMel Wolbachia alters female post-mating behaviors and physiology in the dengue vector mosquito Aedes aegypti.

Globally invasive Aedes aegypti disseminate numerous arboviruses that impact human health. One promising method to control Ae. aegypti populations is transinfection with Wolbachia pipientis, which naturally infects ~40-52% of insects but not Ae. aegypti. Transinfection of Ae. aegypti with the wMel Wolbachia strain induces cytoplasmic incompatibility (CI), allows infected individuals to invade native populations, and inhibits transmission of medically relevant arboviruses by females. Female insects undergo post-mating physiological and behavioral changes-referred to as the female post-mating response (PMR)-required for optimal fertility. PMRs are typically elicited by male seminal fluid proteins (SFPs) transferred with sperm during mating but can be modified by other factors, including microbiome composition. Wolbachia has modest effects on Ae. aegypti fertility, but its influence on other PMRs is unknown. Here, we show that Wolbachia influences female fecundity, fertility, and re-mating incidence and significantly extends the longevity of virgin females. Using proteomic methods to examine the seminal proteome of infected males, we found that Wolbachia moderately affects SFP composition. However, we identified 125 paternally transferred Wolbachia proteins, but the CI factor proteins (Cifs) were not among them. Our findings indicate that Wolbachia infection of Ae. aegypti alters female PMRs, potentially influencing control programs that utilize Wolbachia-infected individuals.

Characterization of the reproductive tract bacterial microbiota of virgin, mated, and blood-fed Aedes aegypti and Aedes albopictus females.

BACKGROUND: Aedes aegypti and Ae. albopictus are vectors of numerous arboviruses that adversely affect human health. In mosquito vectors of disease, the bacterial microbiota influence several physiological processes, including fertility and vector competence, making manipulation of the bacterial community a promising method to control mosquito vectors. In this study, we describe the reproductive tract tissue microbiota of lab-reared virgin Ae. aegypti and Ae. albopictus males, and virgin, mated, and mated + blood-fed females of each species, comparing the bacterial composition found there to the well-described gut microbiota. METHODS: We performed metabarcoding of the 16S rRNA isolated from the gut, upper reproductive tract (URT; testes or ovaries), and lower reproductive tract (LRT; males: seminal vesicles and accessory glands; females: oviduct, spermathecae, and bursa) for each species, and evaluated the influence of host species, tissue, nutritional status, and reproductive status on microbiota composition. Finally, based on the identified taxonomic profiles of the tissues assessed, bacterial metabolic pathway abundance was predicted. RESULTS: The community structure of the reproductive tract is unique compared to the gut. Asaia is the most prevalent OTU in the LRTs of both Ae. aegypti and Ae. albopictus. In the URT, we observed differences between species, with Wolbachia OTUs being dominant in the Ae. albopictus URT, while Enterobacter and Serratia were dominant in Ae. aegypti URT. Host species and tissue were the best predictors of the community composition compared to reproductive status (i.e., virgin or mated) and nutritional status (i.e., sugar or blood-fed). The predicted functional profile shows changes in the abundance of specific microbial pathways that are associated with mating and blood-feeding, like energy production in mated tissues and siderophore synthesis in blood-fed female tissues. CONCLUSIONS: Aedes aegypti and Ae. albopictus have distinct differences in the composition of microbiota found in the reproductive tract. The distribution of the bacterial taxonomic groups indicates that some bacteria have tissue-specific tropism for reproductive tract tissue, such as Asaia and Wolbachia. No significant differences in the taxonomic composition were observed in the reproductive tract between virgin, mated, and mated + blood-fed females, but changes in the abundance of specific metabolic pathways were found in the predicted microbial functional profiles in mated and blood-fed females.

The impact of mating and sugar feeding on blood-feeding physiology and behavior in the arbovirus vector mosquito Aedes aegypti.

BACKGROUND: Aedes aegypti mosquitoes are globally distributed vectors of viruses that impact the health of hundreds of millions of people annually. Mating and blood feeding represent fundamental aspects of mosquito life history that carry important implications for vectorial capacity and for control strategies. Females transmit pathogens to vertebrate hosts and obtain essential nutrients for eggs during blood feeding. Further, because host-seeking Ae. aegypti females mate with males swarming near hosts, biological crosstalk between these behaviors could be important. Although mating influences nutritional intake in other insects, prior studies examining mating effects on mosquito blood feeding have yielded conflicting results. METHODOLOGY/PRINCIPAL FINDINGS: To resolve these discrepancies, we examined blood-feeding physiology and behavior in virgin and mated females and in virgins injected with male accessory gland extracts (MAG), which induce post-mating changes in female behavior. We controlled adult nutritional status prior to blood feeding by using water- and sugar-fed controls. Our data show that neither mating nor injection with MAG affect Ae. aegypti blood intake, digestion, or feeding avidity for an initial blood meal. However, sugar feeding, a common supplement in laboratory settings but relatively rare in nature, significantly affected all aspects of feeding and may have contributed to conflicting results among previous studies. Further, mating, MAG injection, and sugar intake induced declines in subsequent feedings after an initial blood meal, correlating with egg production and laying. Taking our evaluation to the field, virgin and mated mosquitoes collected in Colombia were equally likely to contain blood at the time of collection. CONCLUSIONS/SIGNIFICANCE: Mating, MAG, and sugar feeding impact a mosquito's estimated ability to transmit pathogens through both direct and indirect effects on multiple aspects of mosquito biology. Our results highlight the need to consider natural mosquito ecology, including diet, when assessing their physiology and behavior in the laboratory.

Male Age Influences Re-mating Incidence and Sperm Use in Females of the Dengue Vector Aedes aegypti.

Diseases transmitted by female Aedes aegypti mosquitoes are public health issues in countries in the tropics and sub-tropics. As in other insects, A. aegypti females undergo behavioral and physiological changes upon mating that principally act to facilitate the production of progeny. The primary effectors of A. aegypti female post-mating responses are male-derived seminal proteins that are transferred to females during mating. Increased male age reduces ejaculate function in numerous taxa and alters seminal protein composition in Drosophila melanogaster, but the impacts of male age on female A. aegypti post-mating responses are unknown. Here, we used "old" (21-22 days old) and "young" (4-5 days old) A. aegypti males to assess the influence of male age on oviposition, fertility, and re-mating incidence in their mates. We also examined how age influenced paternity share in females initially mated to young or old males that subsequently re-mated with a transgenic male that transferred RFP-labeled sperm and whose progeny inherited a larval-expressed GFP marker. We found that increased male age had no effect on female fecundity or fertility but significantly impacted their ability to prevent re-mating in their mates-more than half (54.5%) of the females mated to an old male re-mated, compared to 24% of females initially mated to a young male. Polyandrous A. aegypti females displayed first male precedence regardless of the age of their initial mate. However, young males were better able to compete with rival male sperm, siring significantly more progeny (77%) compared to old males (64%). Young males had significantly more sperm in their seminal vesicles than old males at the time of mating, although males of both age groups transferred similar numbers of sperm to their mates. Our results suggest that male senescence differentially impacts the induction of some post-mating changes in A. aegypti females. As the effect of age may be further exacerbated in the field, age-related declines in male ability to induce sexual refractoriness have implications for A. aegypti population control programs that release adults into the environment.

Identification and Removal of Potential Contaminants in 16S rRNA Gene Sequence Data Sets from Low-Microbial-Biomass Samples: an Example from Mosquito Tissues.

The bacterial microbiota of the mosquito influences numerous physiological processes of the host. As low-microbial-biomass ecosystems, mosquito tissues are prone to contamination from the laboratory environment and from reagents commonly used to isolate DNA from tissue samples. In this report, we analyzed nine 16S rRNA data sets, including new data obtained by us, to gain insight into the impact of potential contaminating sequences on the composition, diversity, and structure of the mosquito tissue microbial community. Using a clustering-free approach based on the relative abundance of amplicon sequence variants (ASVs) in tissue samples and negative controls, we identified candidate contaminating sequences that sometimes differed from, but were consistent with, results found using established methodologies. Some putative contaminating sequences belong to bacterial taxa previously identified as contaminants that are commonly found in metagenomic studies but that have also been identified as part of the mosquito core microbiota, with putative physiological relevance for the host. Using different relative abundance cutoffs, we show that contaminating sequences have a significant impact on tissue microbiota diversity and structure analysis. IMPORTANCE The study of tissue-associated microbiota from mosquitoes (primarily from the gut) has grown significantly in the last several years. Mosquito tissue samples represent a challenge for researchers given their low microbial biomass and similar taxonomic composition commonly found in the laboratory environment and in molecular reagents. Using new and published data sets that identified mosquito tissue microbiota from gut and reproductive tract tissues (and their respective negative controls), we developed a simple method to identify contamination microbiota. This approach uses an initial taxonomic identification without operational taxonomic unit (OTU) clustering and evaluates the relative abundance of control sample sequences, allowing the identification and removal of purported contaminating sequences in data sets obtained from low-microbial-biomass samples. While it was exemplified with the analysis of tissue microbiota from mosquitos, it can be extended to other data sets dealing with similar technical artifacts.

Spatial and temporal population dynamics of male and female Aedes albopictus at a local scale in Medellín, Colombia.

BACKGROUND: Diseases transmitted by invasive Aedes aegypti and Aedes albopictus mosquitoes are public health issues in the tropics and subtropics. Understanding the ecology of mosquito vectors is essential for the development of effective disease mitigation programs and will allow for accurate predictions of vector occurrence and abundance. Studies that examine mosquito population dynamics are typically focused on female presence or total adult captures without discriminating the temporal and spatial distribution of both sexes. METHODS: We collected immature and adult mosquitoes bimonthly for 2 years (2018-2019) in the Medellín Botanical Garden. Collection sites differed in proximity to buildings and nearby vegetation, and were classified by their overhead vegetation cover. We used linear mixed models (LMMs) and Spatial Analysis by Distance Indices (SADIE) to assess the spatial distribution of Ae. aegypti and Ae. albopictus. Using our Ae. albopictus captures exclusively, we assessed (1) the spatial and temporal distribution of males and females using SADIE and a generalized linear mixed model (GLMM), (2) the relationship between climatic variables/vegetation coverage and adult captures using GLMMs and LMMs, and (3) the correlation of male and female size in relation to climatic variables and vegetation coverage using LMMs. RESULTS: Spatial analysis showed that Ae. aegypti and Ae. albopictus were distributed at different locations within the surveilled area. However, Ae. albopictus was the predominant species in the park during the study period. Adult Ae. albopictus captures were positively correlated with precipitation and relative humidity, and inversely correlated with temperature and wind speed. Moreover, we observed a spatial misalignment of Ae. albopictus males and females-the majority of males were located in the high vegetation coverage sites, while females were more evenly distributed. We observed significant associations of the size of our adult Ae. albopictus captures with precipitation, temperature, and wind speed for both sexes and found that overhead vegetation cover influenced male size, but observed no effect on female size. CONCLUSIONS: Our work elucidates the differential dynamics of Ae. albopictus males and females, which is pivotal to develop accurate surveillance and the successful establishment of vector control programs based on the disruption of insect reproduction.

Mating and blood-feeding induce transcriptome changes in the spermathecae of the yellow fever mosquito Aedes aegypti.

Aedes aegypti mosquitoes are the primary vectors of numerous viruses that impact human health. As manipulation of reproduction has been proposed to suppress mosquito populations, elucidation of biological processes that enable males and females to successfully reproduce is necessary. One essential process is female sperm storage in specialized structures called spermathecae. Aedes aegypti females typically mate once, requiring them to maintain sperm viably to fertilize eggs they lay over their lifetime. Spermathecal gene products are required for Drosophila sperm storage and sperm viability, and a spermathecal-derived heme peroxidase is required for long-term Anopheles gambiae fertility. Products of the Ae. aegypti spermathecae, and their response to mating, are largely unknown. Further, although female blood-feeding is essential for anautogenous mosquito reproduction, the transcriptional response to blood-ingestion remains undefined in any reproductive tissue. We conducted an RNAseq analysis of spermathecae from unfed virgins, mated only, and mated and blood-fed females at 6, 24, and 72 h post-mating and identified significant differentially expressed genes in each group at each timepoint. A blood-meal following mating induced a greater transcriptional response in the spermathecae than mating alone. This study provides the first view of elicited mRNA changes in the spermathecae by a blood-meal in mated females.

Male sexual history influences female fertility and re-mating incidence in the mosquito vector Aedes aegypti (Diptera: Culicidae).

Aedes aegypti is the primary vector of several arboviruses that impact human health including the dengue, Zika, and yellow fever viruses. The potential of Ae. aegypti females to transmit viruses is enhanced by mating-induced behavioral and physiological changes that increase female host-seeking behaviors, blood-feeding frequency and longevity. The mating-induced changes are due to female receipt of male seminal fluid proteins (SFPs) during copulation. SFPs also inhibit female re-mating-re-mating incidence is significantly reduced in the initial hours after mating and nearly absent after 24 h. Males, however, are not limited in the number of females they can inseminate and are able to mate with multiple females in succession. As successive mating depletes SFPs, we examined parameters of fertility and re-mating incidence in females after mating with recently mated males. Males of two Ae. aegypti strains (Colombian and Thai) were mated five consecutive times and fecundity, resulting larvae and hatch percentage in each female of the mating sequence was assessed. In both strains, we found that males can mate three times in succession without impacting fertility in their mates. However, significant declines in fecundity, resulting larvae, and hatch percentage were observed after a third mating. Male size influenced female fecundity and fertility as mates of small males showed further reductions compared to mates of big males after mating consecutively. Seven days after the consecutive mating assays, the re-mating rate of females mated fifth in succession was significantly increased (Colombian strain: 33%; Thai strain: 48%) compared to females mated first (0% in both strains). Re-mating incidence was further increased in small, Thai strain males where 82% of fifth mated females re-mated compared to 0% of first mated females. Finally, we show that regardless of male size, mates of experimental males were similarly fertile to mates of control males when mated for a sixth time 48 h after the consecutive mating assays, showing that males recover fertility after 2 d. Our results show that male sexual history influences fertility and re-mating incidence of Ae. aegypti females.

Precopulatory acoustic interactions of the New World malaria vector Anopheles albimanus (Diptera: Culicidae).

BACKGROUND: Anopheles albimanus is a malaria vector in Central America, northern South America and the Caribbean. Although a public health threat, An. albimanus precopulatory mating behaviors are unknown. Acoustics play important roles in mosquito communication, where flight tones allow males to detect and attract potential mates. The importance of sound in precopulatory interactions has been demonstrated in Toxorhynchites brevipalpis, Aedes aegypti, Culex quinquefasciatus and Anopheles gambiae; convergence in a shared harmonic of the wing beat frequency (WBF) during courtship is thought to increase the chance of copulation. To our knowledge, An. albimanus precopulatory acoustic behaviors have not been described to date. Here, we characterized An. albimanus (i) male and female flight tones; (ii) male-female precopulatory acoustic interactions under tethered and free flight conditions; and (iii) male-male acoustic interactions during free flight. RESULTS: We found significant increases in the WBFs of both sexes in free flight compared to when tethered. We observed harmonic convergence between 79% of tethered couples. In free flight, we identified a female-specific behavior that predicts mate rejection during male mating attempts: females increase their WBFs significantly faster during mate rejection compared to a successful copulation. This behavior consistently occurred during mate rejection regardless of prior mating attempts (from the same or differing male). During group flight, males of An. albimanus displayed two distinct flying behaviors: random flight and a swarm-like, patterned flight, each associated with distinct acoustic characteristics. In the transition from random to patterned flight, males converged their WBFs and significantly decreased flight area, male-male proximity and the periodicity of their trajectories. CONCLUSIONS: We show that tethering of An. albimanus results in major acoustic differences compared to free flight. We identify a female-specific behavior that predicts mate rejection during male mating attempts in this species and show that male groups in free flight display distinct flying patterns with unique audio and visual characteristics. This study shows that An. albimanus display acoustic features identified in other mosquito species, further suggesting that acoustic interactions provide worthwhile targets for mosquito intervention strategies. Our results provide compelling evidence for swarming in this species and suggests that acoustic signaling is important for this behavior.

Molecular Responses to the Zika Virus in Mosquitoes.

The Zika virus (ZIKV), originally discovered in 1947, did not become a major concern until the virus swept across the Pacific and into the Americas in the last decade, bringing with it news of neurological complications and birth defects in ZIKV affected areas. This prompted researchers to dissect the molecular interactions between ZIKV and the mosquito vector in an attempt to better understand not only the changes that occur upon infection, but to also identify molecules that may potentially enhance or suppress a mosquito’s ability to become infected and/or transmit the virus. Here, we review what is currently known regarding ZIKV-mosquito molecular interactions, focusing on ZIKV infection of Aedes aegypti and Aedes albopictus, the primary species implicated in transmitting ZIKV during the recent outbreaks.