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1.
Pestic Biochem Physiol ; 174: 104823, 2021 May.
Article in English | MEDLINE | ID: mdl-33838716

ABSTRACT

Conventional and volatile pyrethroids are widely used to control the vectors of dengue arboviral diseases, Aedes albopictus in China. The development of resistance to conventional pyrethroids has become an increasing problem, potentially affecting the use of volatile pyrethroid. The Ae. albopictus dimefluthrin-resistant (R) strain by selecting the field population with dimefluthrin were investigated the multiple and cross-resistance levels between conventional and volatile pyrethroids and analyzed both target-site and metabolic resistant mechanisms to dimefluthrin compared with three volatile pyrethroids metofluthrin, meperfluthrin and esbiothrin and type II pyrethroid deltamethrin. The R strain displayed moderate to low resistance to selected pyrethroids (dimefluthrin, metofluthrin, meperfluthrin, esbiothrin and deltamethrin) associated with metabolic enzymes, but less distinctly to selected pyrethroids (dimefluthrin and metofluthrin) associated with a high frequency of sodium channel gene mutation (F1534S). Profiles of the multiple and cross-resistance of the R strain to other three volatile pyrethroids and type II pyrethroid deltamethrin were detected. Both synergistic and enzyme activity studies indicated that multifunctional oxidase (MFO) played an important role in this resistance.


Subject(s)
Aedes , Insecticides , Pyrethrins , Aedes/genetics , Animals , China , Insecticide Resistance/genetics , Insecticides/pharmacology , Mosquito Vectors , Pyrethrins/pharmacology
2.
BMC Genomics ; 22(1): 253, 2021 Apr 09.
Article in English | MEDLINE | ID: mdl-33836668

ABSTRACT

BACKGROUND: Aedes aegypti mosquito, the principal global vector of arboviral diseases, lays eggs and undergoes larval and pupal development to become adult mosquitoes in fresh water (FW). It has recently been observed to develop in coastal brackish water (BW) habitats of up to 50% sea water, and such salinity tolerance shown to be an inheritable trait. Genomics of salinity tolerance in Ae. aegypti has not been previously studied, but it is of fundamental biological interest and important for controlling arboviral diseases in the context of rising sea levels increasing coastal ground water salinity. RESULTS: BW- and FW-Ae. aegypti were compared by RNA-seq analysis on the gut, anal papillae and rest of the carcass in fourth instar larvae (L4), proteomics of cuticles shed when L4 metamorphose into pupae, and transmission electron microscopy of cuticles in L4 and adults. Genes for specific cuticle proteins, signalling proteins, moulting hormone-related proteins, membrane transporters, enzymes involved in cuticle metabolism, and cytochrome P450 showed different mRNA levels in BW and FW L4 tissues. The salinity-tolerant Ae. aegypti were also characterized by altered L4 cuticle proteomics and changes in cuticle ultrastructure of L4 and adults. CONCLUSIONS: The findings provide new information on molecular and ultrastructural changes associated with salinity adaptation in FW mosquitoes. Changes in cuticles of larvae and adults of salinity-tolerant Ae. aegypti are expected to reduce the efficacy of insecticides used for controlling arboviral diseases. Expansion of coastal BW habitats and their neglect for control measures facilitates the spread of salinity-tolerant Ae. aegypti and genes for salinity tolerance. The transmission of arboviral diseases can therefore be amplified in multiple ways by salinity-tolerant Ae. aegypti and requires appropriate mitigating measures. The findings in Ae. aegypti have attendant implications for the development of salinity tolerance in other fresh water mosquito vectors and the diseases they transmit.


Subject(s)
Aedes , Aedes/genetics , Animals , Larva , Proteomics , Salinity , Sea Level Rise , Transcriptome
3.
Ecol Lett ; 24(4): 698-707, 2021 Apr.
Article in English | MEDLINE | ID: mdl-33554374

ABSTRACT

Recurring seasonal changes can lead to the evolution of phenological cues. For example, many arthropods undergo photoperiodic diapause, a programmed developmental arrest induced by short autumnal day length. The selective mechanisms that determine the timing of autumnal diapause initiation have not been empirically identified. We quantified latitudinal clines in genetically determined diapause timing of an invasive mosquito, Aedes albopictus, on two continents. We show that variation in diapause timing within and between continents is explained by a novel application of a growing degree day (GDD) model that delineates a location-specific deadline after which it is not possible to complete an additional full life cycle. GDD models are widely used to predict spring phenology by modelling growth and development as physiological responses to ambient temperatures. Our results show that the energy accumulation dynamics represented by GDD models have also led to the evolution of an anticipatory life-history cue in autumn.


Subject(s)
Aedes , Introduced Species , Aedes/genetics , Animals , Climate , Photoperiod , Seasons
4.
Mem Inst Oswaldo Cruz ; 115: e200313, 2021.
Article in English | MEDLINE | ID: mdl-33533870

ABSTRACT

BACKGROUND: Aedes aegypti is the sole vector of urban arboviruses in French Guiana. Overtime, the species has been responsible for the transmission of viruses during yellow fever, dengue, chikungunya and Zika outbreaks. Decades of vector control have produced resistant populations to deltamethrin, the sole molecule available to control adult mosquitoes in this French Territory. OBJECTIVES: Our surveillance aimed to provide public health authorities with data on insecticide resistance in Ae. aegypti populations and other species of interest in French Guiana. Monitoring resistance to the insecticide used for vector control and to other molecule is a key component to develop an insecticide resistance management plan. METHODS: In 2009, we started to monitor resistance phenotypes to deltamethrin and target-site mechanisms in Ae. aegypti populations across the territory using the WHO impregnated paper test and allelic discrimination assay. FINDINGS: Eight years surveillance revealed well-installed resistance and the dramatic increase of alleles on the sodium voltage-gated gene, known to confer resistance to pyrethroids (PY). In addition, we observed that populations were resistant to malathion (organophosphorous, OP) and alpha-cypermethrin (PY). Some resistance was also detected to molecules from the carbamate family. Finally, those populations somehow recovered susceptibility against fenitrothion (OP). In addition, other species distributed in urban areas revealed to be also resistant to pyrethroids. CONCLUSION: The resistance level can jeopardize the efficiency of chemical adult control in absence of other alternatives and conducts to strongly rely on larval control measures to reduce mosquito burden. Vector control strategies need to evolve to maintain or regain efficacy during epidemics.


Subject(s)
Aedes/drug effects , Insect Vectors/genetics , Insecticide Resistance/genetics , Insecticides/pharmacology , Mosquito Vectors/drug effects , Pyrethrins/pharmacology , Aedes/genetics , Aedes/virology , Animals , French Guiana , Insect Vectors/drug effects , Mosquito Control/methods , Mosquito Vectors/virology , Spatio-Temporal Analysis
5.
Nat Commun ; 12(1): 942, 2021 02 11.
Article in English | MEDLINE | ID: mdl-33574256

ABSTRACT

The mosquito microbiota impacts the physiology of its host and is essential for normal larval development, thereby influencing transmission of vector-borne pathogens. Germ-free mosquitoes generated with current methods show larval stunting and developmental deficits. Therefore, functional studies of the mosquito microbiota have so far mostly been limited to antibiotic treatments of emerging adults. In this study, we introduce a method to produce germ-free Aedes aegypti mosquitoes. It is based on reversible colonisation with bacteria genetically modified to allow complete decolonisation at any developmental stage. We show that, unlike germ-free mosquitoes previously produced using sterile diets, reversibly colonised mosquitoes show no developmental retardation and reach the same size as control adults. This allows us to uncouple the study of the microbiota in larvae and adults. In adults, we detect no impact of bacterial colonisation on mosquito fecundity or longevity. In larvae, data from our transcriptome analysis and diet supplementation experiments following decolonisation suggest that bacteria support larval development by contributing to folate biosynthesis and by enhancing energy storage. Our study establishes a tool to study the microbiota in insects and deepens our knowledge on the metabolic contribution of bacteria to mosquito development.


Subject(s)
Host Microbial Interactions/physiology , Microbiota/physiology , Mosquito Vectors/microbiology , Aedes/genetics , Aedes/growth & development , Aedes/microbiology , Animals , Bacteria/classification , Bacteria/genetics , Bacteria/growth & development , Folic Acid , Food, Fortified , Gastrointestinal Tract/microbiology , Gene Expression Regulation , Germ-Free Life , Larva/genetics , Larva/growth & development , Larva/microbiology , Lipid Metabolism , Mosquito Vectors/growth & development , RNA, Ribosomal, 16S
6.
Parasit Vectors ; 14(1): 12, 2021 Jan 06.
Article in English | MEDLINE | ID: mdl-33407824

ABSTRACT

BACKGROUND: Aedes albopictus is an indigenous primary vector for dengue and Zika viruses in China. Compared with its insecticide resistance, biology and vector competence, little is known about its genetic variation, which corresponds to environmental variations. Thus, the present study examines how Ae. albopictus varies among different climatic regions in China and deciphers its potential dispersal patterns. METHODS: The genetic variation and population structure of 17 Ae. albopictus populations collected from three climatic regions of China were investigated with 11 microsatellite loci and the mitochondrial coxI gene. RESULTS: Of 44 isolated microsatellite markers, 11 pairs were chosen for genotyping analysis and had an average PIC value of 0.713, representing high polymorphism. The number of alleles was high in each population, with the ne value increasing from the temperate region (3.876) to the tropical region (4.144). Twenty-five coxI haplotypes were detected, and the highest diversity was observed in the tropical region. The mean Ho value (ca. 0.557) of all the regions was significantly lower than the mean He value (ca. 0.684), with nearly all populations significantly departing from HWE and displaying significant population expansion (p value < 0.05). Two genetically isolated groups and three haplotype clades were evaluated via STRUCTURE and haplotype phylogenetic analyses, and the tropical populations were significantly isolated from those in the other regions. Most genetic variation in Ae. albopictus was detected within populations and individuals at 31.40 and 63.04%, respectively, via the AMOVA test, and a relatively significant positive correlation was observed among only the temperate populations via IBD analysis (R2 = 0.6614, p = 0.048). Recent dispersions were observed among different Ae. albopictus populations, and four major migration trends with high gene flow (Nm > 0.4) were reconstructed between the tropical region and the other two regions. Environmental factors, especially temperature and rainfall, may be the leading causes of genetic diversity in different climatic regions. CONCLUSIONS: Continuous dispersion contributes to the genetic communication of Ae. albopictus populations across different climatic regions, and environmental factors, especially temperature and rainfall, may be the leading causes of genetic variation.


Subject(s)
Aedes/genetics , Animal Distribution , Genetics, Population , Aedes/virology , Animals , China/epidemiology , Climate , Dengue/transmission , Electron Transport Complex IV/genetics , Genes, Insect , Genetic Variation , Microsatellite Repeats/genetics , Mosquito Vectors/genetics , Mosquito Vectors/virology , Phylogeny , Polymorphism, Genetic , Zika Virus Infection/transmission
7.
PLoS One ; 16(1): e0243992, 2021.
Article in English | MEDLINE | ID: mdl-33428654

ABSTRACT

Insecticide resistance is a worldwide threat for vector control around the world, and Aedes aegypti, the main vector of several arboviruses, is a particular concern. To better understand the mechanisms of resistance, four isofemale strains originally from French Guiana were isolated and analysed using combined approaches. The activity of detoxification enzymes involved in insecticide resistance was assayed, and mutations located at positions 1016 and 1534 of the sodium voltage-gated channel gene, which have been associated with pyrethroid resistance in Aedes aegypti populations in Latin America, were monitored. Resistance to other insecticide families (organophosphates and carbamates) was evaluated. A large-scale proteomic analysis was performed to identify proteins involved in insecticide resistance. Our results revealed a metabolic resistance and resistance associated with a mutation of the sodium voltage-gated channel gene at position 1016. Metabolic resistance was mediated through an increase of esterase activity in most strains but also through the shifts in the abundance of several cytochrome P450 (CYP450s). Overall, resistance to deltamethrin was linked in the isofemale strains to resistance to other class of insecticides, suggesting that cross- and multiple resistance occur through selection of mechanisms of metabolic resistance. These results give some insights into resistance to deltamethrin and into multiple resistance phenomena in populations of Ae. aegypti.


Subject(s)
Aedes/metabolism , Cytochrome P-450 Enzyme System/genetics , Insect Proteins/genetics , Insecticide Resistance/genetics , Voltage-Gated Sodium Channels/genetics , Aedes/drug effects , Aedes/genetics , Animals , Esterases/metabolism , Female , French Guiana , Gene Knockdown Techniques , Genotype , Inactivation, Metabolic/genetics , Insect Proteins/antagonists & inhibitors , Insect Proteins/metabolism , Insecticides/pharmacology , Intestinal Mucosa/metabolism , Nitriles/pharmacology , Oligonucleotides/metabolism , Polymorphism, Single Nucleotide , Proteome/analysis , Proteomics , Pyrethrins/pharmacology , Voltage-Gated Sodium Channels/chemistry , Voltage-Gated Sodium Channels/metabolism
8.
BMC Genomics ; 22(1): 71, 2021 Jan 21.
Article in English | MEDLINE | ID: mdl-33478394

ABSTRACT

BACKGROUND: In the light of dengue being the fastest growing transmissible disease, there is a dire need to identify the mechanisms regulating the behaviour of the main vector Aedes aegypti. Disease transmission requires the female mosquito to acquire the pathogen from a blood meal during one gonotrophic cycle, and to pass it on in the next, and the capacity of the vector to maintain the disease relies on a sustained mosquito population. RESULTS: Using a comprehensive transcriptomic approach, we provide insight into the regulation of the odour-mediated host- and oviposition-seeking behaviours throughout the first gonotrophic cycle. We provide clear evidence that the age and state of the female affects antennal transcription differentially. Notably, the temporal- and state-dependent patterns of differential transcript abundance of chemosensory and neuromodulatory genes extends across families, and appears to be linked to concerted differential modulation by subsets of transcription factors. CONCLUSIONS: By identifying these regulatory pathways, we provide a substrate for future studies targeting subsets of genes across disparate families involved in generating key vector behaviours, with the goal to develop novel vector control tools.


Subject(s)
Aedes , Dengue , Aedes/genetics , Animals , Dengue/genetics , Female , Humans , Insect Vectors/genetics , Mosquito Vectors/genetics , Transcriptome
9.
BMC Genet ; 21(Suppl 2): 142, 2020 12 18.
Article in English | MEDLINE | ID: mdl-33339503

ABSTRACT

BACKGROUND: Aedes aegypti is the primary vector of arthropod-borne viruses and one of the most widespread and invasive mosquito species. Due to the lack of efficient specific drugs or vaccination strategies, vector population control methods, such as the sterile insect technique, are receiving renewed interest. However, availability of a reliable genetic sexing strategy is crucial, since there is almost zero tolerance for accidentally released females. Development of genetic sexing strains through classical genetics is hindered by genetic recombination that is not suppressed in males as is the case in many Diptera. Isolation of naturally-occurring or irradiation-induced inversions can enhance the genetic stability of genetic sexing strains developed through genetically linking desirable phenotypes with the male determining region. RESULTS: For the induction and isolation of inversions through irradiation, 200 male pupae of the 'BRA' wild type strain were irradiated at 30 Gy and 100 isomale lines were set up by crossing with homozygous 'red-eye' (re) mutant females. Recombination between re and the M locus and the white (w) gene (causing a recessive white eye phenotype when mutated) and the M locus was tested in 45 and 32 lines, respectively. One inversion (Inv35) reduced recombination between both re and the M locus, and wand the M locus, consistent with the presence of a rather extended inversion between the two morphological mutations, that includes the M locus. Another inversion (Inv5) reduced recombination only between w and the M locus. In search of naturally-occurring, recombination-suppressing inversions, homozygous females from the red eye and the white eye strains were crossed with seventeen and fourteen wild type strains collected worldwide, representing either recently colonized or long-established laboratory populations. Despite evidence of varying frequencies of recombination, no combination led to the elimination or substantial reduction of recombination. CONCLUSION: Inducing inversions through irradiation is a feasible strategy to isolate recombination suppressors either on the M or the m chromosome for Aedes aegypti. Such inversions can be incorporated in genetic sexing strains developed through classical genetics to enhance their genetic stability and support SIT or other approaches that aim to population suppression through male-delivered sterility.


Subject(s)
Aedes/genetics , Aedes/radiation effects , Infertility/genetics , Recombination, Genetic/radiation effects , Animals , Female , Gamma Rays , Genes, Insect , Genetic Markers , Insect Control , Male , Mosquito Vectors/genetics , Mosquito Vectors/radiation effects
10.
PLoS Negl Trop Dis ; 14(12): e0008971, 2020 12.
Article in English | MEDLINE | ID: mdl-33338046

ABSTRACT

Aedes aegypti is a vector of dengue, chikungunya, and Zika viruses. Current vector control strategies such as community engagement, source reduction, and insecticides have not been sufficient to prevent viral outbreaks. Thus, interest in novel strategies involving genetic engineering is growing. Female mosquitoes rely on flight to mate with males and obtain a bloodmeal from a host. We hypothesized that knockout of genes specifically expressed in female mosquitoes associated with the indirect flight muscles would result in a flightless female mosquito. Using CRISPR-Cas9 we generated loss-of-function mutations in several genes hypothesized to control flight in mosquitoes, including actin (AeAct-4) and myosin (myo-fem) genes expressed specifically in the female flight muscle. Genetic knockout of these genes resulted in 100% flightless females, with homozygous males able to fly, mate, and produce offspring, albeit at a reduced rate when compared to wild type males. Interestingly, we found that while AeAct-4 was haplosufficient, with most heterozygous individuals capable of flight, this was not the case for myo-fem, where about half of individuals carrying only one intact copy could not fly. These findings lay the groundwork for developing novel mechanisms of controlling Ae. aegypti populations, and our results suggest that this mechanism could be applicable to other vector species of mosquito.


Subject(s)
Aedes/genetics , CRISPR-Cas Systems , Insecticides/pharmacology , Mosquito Control , Mosquito Vectors/genetics , Zika Virus Infection/prevention & control , Zika Virus/physiology , Aedes/physiology , Aedes/virology , Animals , Female , Flight, Animal , Gene Knockout Techniques , Humans , Male , Mosquito Vectors/physiology , Mosquito Vectors/virology , Phenotype , Zika Virus Infection/transmission , Zika Virus Infection/virology
11.
PLoS One ; 15(10): e0228695, 2020.
Article in English | MEDLINE | ID: mdl-33022007

ABSTRACT

Aedes aegypti is the main vector of dengue, chikungunya, and Zika viruses, which are of great public health importance in Colombia. Aedes control strategies in Colombia rely heavily on the use of organophosphate and pyrethroid insecticides, providing constant selection pressure and the emergence of resistant populations. In recent years, insecticide use has increased due to the increased incidence of dengue and recent introductions of chikungunya and Zika. In the present study, pyrethroid resistance was studied across six populations of Ae. aegypti from the Caribbean coast of Colombia. Susceptibility to λ-cyhalothrin, deltamethrin, and permethrin was assessed, and resistance intensity was determined. Activity levels of enzymes associated with resistance were measured, and the frequencies of three kdr alleles (V1016I, F1534C, V410L) were calculated. Results showed variations in pyrethroid susceptibility across Ae. aegypti populations and altered enzyme activity levels were detected. The kdr alleles were detected in all populations, with high variations in frequencies: V1016I (frequency ranging from 0.15-0.70), F1534C (range 0.94-1.00), and V410L (range 0.05-0.72). In assays of phenotyped individuals, associations were observed between the presence of V1016I, F1534C, and V410L alleles and resistance to the evaluated pyrethroids, as well as between the VI1016/CC1534/VL410 tri-locus genotype and λ-cyhalothrin and permethrin resistance. The results of the present study contribute to the knowledge of the mechanisms underlying the resistance to key pyrethroids used to control Ae. aegypti along the Caribbean coast of Colombia.


Subject(s)
Aedes/genetics , Insecticide Resistance , Mutation , Pyrethrins/pharmacology , Vascular Endothelial Growth Factor Receptor-2/genetics , Aedes/drug effects , Animals , Colombia , Gene Frequency , Insect Proteins/genetics , Male , Nitriles/pharmacology , Permethrin/pharmacology , Phenotype
12.
Pestic Biochem Physiol ; 170: 104666, 2020 Nov.
Article in English | MEDLINE | ID: mdl-32980073

ABSTRACT

Despite the substantial progress achieved in the characterization of cytochrome P450 (CYP) -based resistance mechanisms in mosquitoes, a number of questions remain unanswered. These include: (i) the regulation and physiology of resistance conferring CYPs; (ii) the actual contribution of CYPs in resistance alone or in combination with other detoxification partners or other resistance mechanisms; (iii) the association between overexpression levels and allelic variation, with the catalytic activity and the intensity of resistance and (iv) the true value of molecular diagnostics targeting CYP markers, for driving decision making in the frame of Insecticide Resistance Management applications. Furthermore, the translation of CYP - based insecticide resistance research in mosquitoes into practical applications, is being developed, but it is not fully exploited, as yet. Examples include the production of high throughput platforms for screening the liability (stability) or inhibition potential of novel insecticidal leads and synergists (add-ons), as well as the exploration of the negative cross resistance concept (i.e. detoxification of certain insecticides, but activation of others pro-insecticides). The goal of this review is to critically summarise the current knowledge and the gaps of the CYP-based metabolic insecticide resistance in Anopheles and Aedes mosquito vectors. The progress and limitations of the protein and the reverse/forward genetic approaches, the understanding and importance of molecular and physiological aspects, as well as the current and future exploitation routes of CYP research are discussed.


Subject(s)
Aedes/drug effects , Aedes/genetics , Anopheles/drug effects , Anopheles/genetics , Insecticides/pharmacology , Pyrethrins , Animals , Cytochrome P-450 Enzyme System/genetics , Insecticide Resistance/drug effects , Insecticide Resistance/genetics , Mosquito Vectors/genetics
13.
PLoS Negl Trop Dis ; 14(9): e0008531, 2020 09.
Article in English | MEDLINE | ID: mdl-32911504

ABSTRACT

Pathogens may manipulate their human and mosquito hosts to enhance disease transmission. Dengue, caused by four viral serotypes, is the fastest-growing transmissible disease globally resulting in 50-100 million infections annually. Transmission of the disease relies on the interaction between humans and the vector Aedes aegypti and is largely dependent on the odor-mediated host seeking of female mosquitoes. In this study, we use activity monitors to demonstrate that dengue virus-1 affects the locomotion and odor-mediated behavior of Ae. aegypti, reflecting the progression of infection within the mosquito. Mosquitoes 4-6 days post-infection increase locomotion, but do not alter their odor-driven host-seeking response. In contrast, females 14-16 days post-infection are less active, yet more sensitive to human odors as assessed by behavioral and electrophysiological assays. Such an increase in physiological and behavioral sensitivity is reflected by the antennal-specific increase in abundance of neural signaling transcripts in 14 days post-infection females, as determined by transcriptome analysis. This suggests that the sensitivity of the mosquito peripheral olfactory system is altered by the dengue virus by enhancing the overall neural responsiveness of the antenna, rather than the selective regulation of chemosensory-related genes. Our study reveals that dengue virus-1 enhances vector-related behaviors in the early stages post-infection that aid in avoiding predation and increasing spatial exploration. On the other hand, at the later stages of infection, the virus enhances the host-seeking capacity of the vector, thereby increasing the risk of virus transmission. A potential mechanism is discussed.


Subject(s)
Aedes/virology , Dengue , Host-Seeking Behavior , Aedes/genetics , Aedes/metabolism , Aedes/physiology , Animals , Arthropod Antennae/physiology , Behavior, Animal , Dengue Virus/physiology , Female , Gene Expression Profiling , Humans , Locomotion , Mosquito Vectors/physiology , Mosquito Vectors/virology
14.
PLoS Negl Trop Dis ; 14(9): e0008657, 2020 09.
Article in English | MEDLINE | ID: mdl-32997656

ABSTRACT

Aedes albopictus, along with Ae. aegypti, are key arbovirus vectors that have been expanding their geographic range over the last decades. In 2017, Ae. albopictus was detected for the first time at two distinct locations in Portugal. In order to understand how the Ae. albopictus populations recently introduced in Portugal are genetically related and which is their likely route of invasion, we performed an integrative cytochrome C oxidase I gene (COI)- and mitogenome-based phylogeographic analysis of mosquitoes samples collected in Portugal in 2017 and 2018 in the context of the global Ae. albopictus diversity. COI-based analysis (31 partial sequences obtained from 83 mosquitoes) revealed five haplotypes (1 to 5), with haplotype 1 (which is widely distributed in temperate areas worldwide) being detected in both locations. Haplotypes 2 and 3 were exclusively found in Southern region (Algarve), while haplotype 4 and 5 were only detected in the North of Portugal (Penafiel, Oporto region). Subsequent high discriminatory analyses based on Ae. albopictus mitogenome (17 novel sequences) not only confirmed a high degree of genetic variability within and between populations at both geographic locations (compatible with the Ae. albopictus mosquito populations circulating in Europe), but also revealed two mitogenome mutational signatures not previously reported at worldwide level. While our results generally sustain the occurrence of multiple introduction events, fine mitogenome sequence inspection further indicates a possible Ae. albopictus migration within the country, from the Northern introduction locality to the Southern region. In summary, the observed scenario of high Ae. albopictus genetic diversity in Portugal, together with the detection of mosquitoes in successive years since 2017 in Algarve and Penafiel, points that both Ae. albopictus populations seem to be already locally established, as its presence has been reported for three consecutive years, raising the public health awareness for future mosquito-borne diseases outbreaks.


Subject(s)
Aedes/genetics , Genetic Variation , Genome, Mitochondrial/genetics , Mosquito Vectors/genetics , Aedes/classification , Aedes/virology , Animals , Arboviruses , Electron Transport Complex IV/genetics , Female , Haplotypes , High-Throughput Nucleotide Sequencing , Mosquito Vectors/virology , Phylogeography , Portugal , Sequence Analysis, DNA
15.
PLoS One ; 15(8): e0234892, 2020.
Article in English | MEDLINE | ID: mdl-32817668

ABSTRACT

The mosquito Aedes aegypti vectors the arboviral diseases yellow fever, dengue, Zika and chikungunya. Larvae are usually found developing in freshwater; however, more recently they have been increasingly found in brackish water, potential habitats which are traditionally ignored by mosquito control programs. Aedes aegypti larvae are osmo-regulators maintaining their hemolymph osmolarity in a range of ~ 250 to 300 mOsmol l-1. In freshwater, the larvae must excrete excess water while conserving ions while in brackish water, they must alleviate an accumulation of salts. The compensatory physiological mechanisms must involve the transport of ions and water but little is known about the water transport mechanisms in the osmoregulatory organs of these larvae. Water traverses cellular membranes predominantly through transmembrane proteins named aquaporins (AQPs) and Aedes aegypti possesses 6 AQP homologues (AaAQP1 to 6). The objective of this study was to determine if larvae that develop in freshwater or brackish water have differential aquaporin expression in osmoregulatory organs, which could inform us about the relative importance and function of aquaporins to mosquito survival under these different osmotic conditions. We found that AaAQP transcript abundance was similar in organs of freshwater and brackish water mosquito larvae. Furthermore, in the Malpighian tubules and hindgut AaAQP protein abundance was unaffected by the rearing conditions, but in the gastric caeca the protein level of one aquaporin, AaAQP1 was elevated in brackish water. We found that AaAQP1 was expressed apically while AaAQP4 and AaAQP5 were found to be apical and/or basal in the epithelia of osmoregulatory organs. Overall, the results suggest that aquaporin expression in the osmoregulatory organs is mostly consistent between larvae that are developing in freshwater and brackish water. This suggests that aquaporins may not have major roles in adapting to longterm survival in brackish water or that aquaporin function may be regulated by other mechanisms like post-translational modifications.


Subject(s)
Aedes/genetics , Aquaporins/genetics , Osmoregulation/genetics , Aedes/physiology , Animals , Aquaporins/metabolism , Arbovirus Infections , Biological Transport , Ecosystem , Fresh Water , Gene Expression/genetics , Gene Expression Regulation/genetics , Hemolymph/metabolism , Insect Proteins/metabolism , Larva/genetics , Larva/metabolism , Osmoregulation/physiology , Osmosis , Saline Waters , Salinity , Water/metabolism
16.
PLoS One ; 15(8): e0237675, 2020.
Article in English | MEDLINE | ID: mdl-32797066

ABSTRACT

RNA interference (RNAi), a technique used to investigate gene function in insects and other organisms, is attracting attention as a potential new technology for mosquito control. Saccharomyces cerevisiae (baker's yeast) was recently engineered to produce interfering RNA molecules that silence genes required for mosquito survival, but which do not correspond to genes in humans or other non-target organisms. The resulting yeast pesticides, which facilitate cost-effective production and delivery of interfering RNA to mosquito larvae that eat the yeast, effectively kill mosquitoes in laboratory and semi-field trials. In preparation for field evaluation of larvicides in Trinidad, a Caribbean island with endemic diseases resulting from pathogens transmitted by Aedes mosquitoes, adult residents living in the prospective trial site communities of Curepe, St. Augustine, and Tamana were engaged. Open community forums and paper surveys were used to assess the potential acceptability, societal desirability, and sustainability of yeast interfering RNA larvicides. These assessments revealed that Trinidadians have good working knowledge of mosquitoes and mosquito-borne illnesses. A majority of the respondents practiced some method of larval mosquito control and agreed that they would use a new larvicide if it were proven to be safe and effective. During the community engagement forums, participants were educated about mosquito biology, mosquito-borne diseases, and the new yeast larvicides. When invited to provide feedback, engagement forum attendees were strongly supportive of the new technology, raised few concerns, and provided helpful advice regarding optimal larvicide formulations, insecticide application, operational approaches for using the larvicides, and pricing. The results of these studies suggest that the participants are supportive of the potential use of yeast interfering RNA larvicides in Trinidad and that the communities assessed in this investigation represent viable field sites.


Subject(s)
Aedes/genetics , Genetic Engineering/methods , Mosquito Control/methods , RNA Interference , Saccharomyces cerevisiae/genetics , Adult , Animals , Female , Humans , Larva/genetics , Male , Middle Aged , Mosquito Vectors/genetics , Pest Control, Biological/methods , RNA, Small Interfering/genetics , Residence Characteristics , Surveys and Questionnaires , Trinidad and Tobago
17.
Science ; 369(6507): 1128-1132, 2020 08 28.
Article in English | MEDLINE | ID: mdl-32855340

ABSTRACT

Hemocytes limit the capacity of mosquitoes to transmit human pathogens. Here we profile the transcriptomes of 8506 hemocytes of Anopheles gambiae and Aedes aegypti mosquito vectors. Our data reveal the functional diversity of hemocytes, with different subtypes of granulocytes expressing distinct and evolutionarily conserved subsets of effector genes. A previously unidentified cell type in An. gambiae, which we term "megacyte," is defined by a specific transmembrane protein marker (TM7318) and high expression of lipopolysaccharide-induced tumor necrosis factor-α transcription factor 3 (LL3). Knockdown experiments indicate that LL3 mediates hemocyte differentiation during immune priming. We identify and validate two main hemocyte lineages and find evidence of proliferating granulocyte populations. This atlas of medically relevant invertebrate immune cells at single-cell resolution identifies cellular events that underpin mosquito immunity to malaria infection.


Subject(s)
Aedes/immunology , Anopheles/immunology , Hemocytes/immunology , Immunity, Cellular , Malaria/transmission , Mosquito Vectors/immunology , Aedes/genetics , Animals , Anopheles/genetics , Female , Gene Expression Profiling , Gene Knockdown Techniques , Granulocytes/immunology , Hemocytes/metabolism , Malaria/immunology , Malaria/parasitology , Mice , Mosquito Vectors/genetics , RNA-Seq , Single-Cell Analysis
18.
PLoS One ; 15(7): e0235430, 2020.
Article in English | MEDLINE | ID: mdl-32722672

ABSTRACT

Phylogeographic relationships among global collections of the mosquito Aedes aegypti were evaluated using the mitochondrial Cytochrome C Oxidase 1 (CO1) and NADH dehydrogenase subunit 4 (ND4) genes including new sequences from Sri Lanka. Phylogeographic analysis estimated that Ae. aegypti arose as a species ~614 thousand years ago (kya) in the late Pleistocene. At 545 kya an "early" East African clade arose that continued to differentiate in East Africa, and eventually gave rise to three lineages one of which is distributed throughout all tropical and subtropical regions, a second that contains Southeast Asian/Sri Lankan mosquitoes and a third that contains mostly New World mosquitoes. West African collections were not represented in this early clade. The late clade continued to differentiate throughout Africa and gave rise to a lineage that spread globally. The most recent branches of the late clade are represented by South-East Asia and India/Pakistan collections. Analysis of migration rates suggests abundant gene flow between India/Pakistan and the rest of the world with the exception of Africa.


Subject(s)
Aedes/genetics , DNA, Mitochondrial/genetics , Mitochondria/genetics , Phylogeography , Aedes/classification , Africa , Africa, Eastern , Animals , Electron Transport Complex IV/genetics , Gene Flow , Genes, Mitochondrial/genetics , Haplotypes , India , Mitochondria/metabolism , Pakistan , Phylogeny , Sri Lanka
19.
PLoS Negl Trop Dis ; 14(7): e0008463, 2020 07.
Article in English | MEDLINE | ID: mdl-32678817

ABSTRACT

The arbovirus vectors Aedes aegypti (yellow fever mosquito) and Ae. albopictus (Asian tiger mosquito) are both common throughout the Indo-Pacific region, where 70% of global dengue transmission occurs. For Ae. aegypti all Indo-Pacific populations are invasive, having spread from an initial native range of Africa, while for Ae. albopictus the Indo-Pacific includes invasive populations and those from the native range: putatively, India to Japan to Southeast Asia. This study analyses the population genomics of 480 of these mosquitoes sampled from 27 locations in the Indo-Pacific. We investigated patterns of genome-wide genetic differentiation to compare pathways of invasion and ongoing gene flow in both species, and to compare invasive and native-range populations of Ae. albopictus. We also tested landscape genomic hypotheses that genetic differentiation would increase with geographical distance and be lower between locations with high connectivity to human transportation routes, the primary means of dispersal at these scales. We found that genetic distances were generally higher in Ae. aegypti, with Pacific populations the most highly differentiated. The most differentiated Ae. albopictus populations were in Vanuatu, Indonesia and Sri Lanka, the latter two representing potential native-range populations and potential cryptic subspeciation respectively. Genetic distances in Ae. aegypti increased with geographical distance, while in Ae. albopictus they decreased with higher connectivity to human transportation routes. Contrary to the situation in Ae. aegypti, we found evidence of long-distance Ae. albopictus colonisation events, including colonisation of Mauritius from East Asia and of Fiji from Southeast Asia. These direct genomic comparisons indicate likely differences in dispersal ecology in these species, despite their broadly sympatric distributions and similar use of human transport to disperse. Our findings will assist biosecurity operations to trace the source of invasive material and for biocontrol operations that benefit from matching genetic backgrounds of released and local populations.


Subject(s)
Aedes/genetics , Animal Distribution , Genome, Insect , Genomics , Animals , Asia
20.
Proc Natl Acad Sci U S A ; 117(30): 17702-17709, 2020 07 28.
Article in English | MEDLINE | ID: mdl-32661163

ABSTRACT

A dominant male-determining locus (M-locus) establishes the male sex (M/m) in the yellow fever mosquito, Aedes aegypti Nix, a gene in the M-locus, was shown to be a male-determining factor (M factor) as somatic knockout of Nix led to feminized males (M/m) while transient expression of Nix resulted in partially masculinized females (m/m), with male reproductive organs but retained female antennae. It was not clear whether any of the other 29 genes in the 1.3-Mb M-locus are also needed for complete sex-conversion. Here, we report the generation of multiple transgenic lines that express Nix under the control of its own promoter. Genetic and molecular analyses of these lines provided insights unattainable from previous transient experiments. We show that the Nix transgene alone, in the absence of the M-locus, was sufficient to convert females into males with all male-specific sexually dimorphic features and male-like gene expression. The converted m/m males are flightless, unable to perform the nuptial flight required for mating. However, they were able to father sex-converted progeny when presented with cold-anesthetized wild-type females. We show that myo-sex, a myosin heavy-chain gene also in the M-locus, was required for male flight as knockout of myo-sex rendered wild-type males flightless. We also show that Nix-mediated female-to-male conversion was 100% penetrant and stable over many generations. Therefore, Nix has great potential for developing mosquito control strategies to reduce vector populations by female-to-male sex conversion, or to aid in a sterile insect technique that requires releasing only non-biting males.


Subject(s)
Aedes/genetics , Flight, Animal , Genes, Insect , Genetic Association Studies , Membrane Proteins/genetics , Sex Determination Processes/genetics , Animals , Animals, Genetically Modified , CRISPR-Cas Systems , Female , Genetic Loci , Genotype , Inheritance Patterns , Male , Penetrance , Phenotype , Promoter Regions, Genetic
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