Female Aedes Aegypti Research Articles

Phosphoproteomics Analyses of Aedes aegypti fat body reveals blood meal-induced Signaling and Metabolic Pathways.

The mosquito fat body is the principal source of yolk protein precursors (YPP) during mosquito egg development in female Aedes aegypti. To better understand the metabolic and signaling pathways involved in mosquito reproduction, we investigated changes in the mosquito fat body phosphoproteome at multiple time points after a blood meal. Using LC/MS, we identified 3,570 phosphorylated proteins containing 14,551 individual phosphorylation sites. We observed protein phosphorylation changes in cellular pathways required for vitellogenesis, as well as proteins involved in primary cellular functions. Specifically, after a blood meal, proteins involved in ribosome synthesis, transcription, translation, and autophagy showed dynamic changes in their phosphorylation patterns. Our results provide new insight into blood meal-induced fat body dynamics and reveal potential proteins that can be targeted for interference with mosquito reproduction. Considering the devastating impact of mosquitoes on human health, worldwide, new approaches to control mosquitoes are urgently needed.

The V410L kdr allele in the VGSC confers higher levels of field resistance to permethrin in urban mosquito populations of Aedes aegypti (L.).

Females of Aedes aegypti transmit emerging arboviruses including Zika, dengue, yellow fever, and chikungunya. Control of these adult mosquitoes heavily relies on synthetic insecticides, including pyrethroids. However, insecticide resistance development in populations poses a significant challenge to vector control, particularly from knockdown resistance (kdr) mutations in the voltage-gated sodium channel (VGSC), the target of pyrethroids. This study investigated the field efficacy of Permanone, a pyrethroid-based insecticide, against Ae. aegypti by assessing the impact of three common kdr mutations (V410L, V1016I, F1534C) on mosquito survival under a real operational mosquito control scenario, by quantifying the pesticide delivered in the field. Field cage tests (FCTs) were conducted while conducting a realistic mosquito control application. Female mosquitoes from six operational areas from Harris County, TX, USA were exposed to Permanone delivered with a handheld sprayer. Permanone deposited near the cages was estimated from aluminum boats placed in the field during FCTs using gas chromatography-mass spectrometry (GC-MS). Mortality rates were recorded, and individual mosquitoes were genotyped for kdr mutations. A probit regression model was used to analyze the factors influencing mosquito survivorship. As the distance from the application source route increased, the amount of Permanone deposited decreased, resulting in higher survivorship frequency of Ae. aegypti females with the triple-resistant kdr genotype (LL/II/CC). The L allele at the 410-site significantly contributed to an increased resistance level when co-occurring with other kdr mutations. This study linked the survival probabilities of mosquitoes with different kdr genotypes, and the amount of pesticide they received in the field. Pesticide quantification, control efficacy results and genotyping allowed us to empirically determine the impact of genotypic resistance on vector control in the field. © 2024 Society of Chemical Industry.

Bacillus thuringiensis israelensis VCRC B650 culture filtrate useful for mosquito oviposition attractant and larvicidal action

Oviposition attractants hold the potential attention for monitoring and controlling mosquitoes by enticing them to deposit eggs at specific locations. In this study, the bacterial culture filtrate of Bacillus thuringiensis var. israelensis VCRC B650, previously isolated from clay soil at an agricultural site in U.T of Puducherry, was investigated for the first time. This larvicidal bacterium was assessed for oviposition attractancy against dengue and filarial vectors, namely, gravid females of Aedes aegypti and Culex quinquefasciatus, respectively. The investigation revealed that, at 1:1 dilution of bacterial culture filtrate and water, Cx. quinquefasciatus exhibited significant attraction, with an oviposition activity index (OAI) value of +0.92, surpassing the standard threshold value of +0.30. The OAI of Bti culture filtrate at a 1:1 dilution was also compared with a standard oviposition attractant, p-cresol, at a concentration of 10ppm. It was observed that the Bti culture filtrate demonstrated a significant oviposition attractant effect, with 80 per cent more egg rafts laid, compared to p-cresol at 20 per cent. GC-MS analysis showed that there were 25 compounds present in the Bti culture supernatant, and out of those, only one compound, namely, benzaldehyde, was effective in showing oviposition attraction.

Low humidity enhances Zika virus infection and dissemination in Aedes aegypti mosquitoes.

As climate change alters Earth's biomes, it is expected the transmission dynamics of mosquito-borne viruses will change. While the effects of temperature changes on mosquito-virus interactions and the spread of the pathogens have been elucidated over the last decade, the impact of relative humidity changes is still relatively unknown. To overcome this knowledge gap, we exposed Aedes aegypti females to various humidity conditions. We measured different components of vectorial capacity such as survival, blood-feeding rates, and changes in infection and dissemination of Zika virus. Survival decreased as the humidity level decreased, while infection rates increased as the humidity level decreased. Alternatively, blood feeding rates and disseminated infection rates peaked at the intermediate 50% relative humidity treatment but were the same in the 30% and 80% relative humidity treatments. These results provide empirical evidence that Ae. aegypti exposure to low humidity can enhance Zika virus infection in the mosquito, which has important implications in predicting how climate change will impact mosquito-borne viruses.IMPORTANCEViruses transmitted by mosquitoes to humans are a major public health burden and are expected to increase under climate change. While we know that temperature is an important driver of variation in arbovirus replication in the mosquito, very little is known about how other relevant climate variables such as humidity will influence the interaction between mosquitoes and the viruses they transmit. Given the variability in humidity across environments, and the predicted changes in humidity under climate change, it is imperative that we also study the impact that it has on mosquito infection and transmission of arboviruses.

Hybridization between Aedes aegypti and Aedes mascarensis mosquitoes leads to disruption of male sex determination

Understanding the sex determination pathway and its disruptions in mosquitoes is critical for the effective control of disease vectors through genetic manipulations based on sex separation. When male hybrids of Aedes aegypti females and Ae. mascarensis males are backcrossed to Ae. aegypti females, a portion of the backcross progeny manifests as males with abnormal sexual differentiation. We discovered a significant correlation between pupal abnormalities and the feminization of subsequent adults exemplified by the relative abundance of ovarian and testicular tissues. All intersex individuals were genetic males as they expressed a male determining factor, Nix. Further, our analysis of the sex-specific splicing of doublesex and fruitless transcripts demonstrated the presence of both male and female splice variants indicating that sex determination is disrupted. A comparative transcriptomic analysis revealed similar expression levels of most female-associated genes in reproductive organs and carcasses between intersexual males and normal females. Moreover, intersexes had largely normal gene expression in testes but significant gene downregulation in male accessory glands when compared with normal males. We conclude that evolving hybrid incompatibilities between Ae. aegypti and Ae. mascarensis involve disruption of sex determination and are accompanied by changes in gene expression associated with sexual differentiation.

Immuno-Haematologic Aspects of Dengue Infection: Biologic Insights and Clinical Implications.

Dengue infection is caused by the dengue virus (DENV) and is transmitted to humans by infected female Aedes aegypti and Aedes albopictus mosquitoes. There are nearly 100 million new dengue cases yearly in more than 120 countries, with a five-fold increase in incidence over the past four decades. While many patients experience a mild illness, a subset suffer from severe disease, which can be fatal. Dysregulated immune responses are central to the pathogenesis of dengue, and haematologic manifestations are a prominent feature of severe disease. While thrombocytopaenia and coagulopathy are major causes of bleeding in severe dengue, leucocyte abnormalities are emerging as important markers of prognosis. In this review, we provide our perspective on the clinical aspects and pathophysiology of haematologic manifestations in dengue. We also discuss the key gaps in our current practice and areas to be addressed by future research.

Bloodmeals fuel dengue virus replication in the female mosquito Aedes aegypti.

Through evolution, the mosquito-borne viruses have adapted to the blood-feeding behaviors of their opportunist hosts to fulfill a complete lifecycle in humans and mosquitos. Disruption in the mosquitos' ability to host these viruses offers strategies to prevent diseases caused by them. With the advent of genomic tools, we discovered that dengue virus (DENV) benefited from the female mosquitos' bloodmeals for metabolic and energetic supplies for replication. Chemical or genetic disruption in these supplies reduced DENV replication in the female mosquitos. Our discovery can be exploited to produce genetically modified mosquitos, in which DENV infection leads to disruption in the supplies and thereby reduces replication and transmission. Our discovery might be extrapolated to prevent mosquito-borne virus transmission and the diseases they cause.

MATHEMATICAL MODEL OF REPELLENT EFFECT IN DENGUE TRANSMISSION

Dengue is a disease caused by the dengue virus, transmitted through the bite of an infected female Aedes aegypti. Dengue virus is a member of the genus Flavivirus, family Flaviviridae. Indonesia is one of the countries with the most dengue cases in Southeast Asia. Therefore, dengue transmission must be controlled to reduce the increase in dengue cases. One of the controls is by using repellents. Repellent is one of the human protection strategies to avoid mosquito bites used by spraying or smearing. This study models dengue transmission by reviewing the effect and control of repellent. A mathematical model of repellent effect and control in dengue transmission uses a SIR compartment model. The SIR model is modified by involving mosquitoes and the human population. Repellent is used in both susceptible humans, infected humans, and recovered humans. Numerical and analytical simulations are conducted to analyze the behavior of each compartment of the mosquito and human populations in dengue transmission. Analytical results show that the factors affecting the spread of infection are the transmission rate of the dengue virus and the loss of human-repellent protection. The transmission rate of dengue virus in the interval increases the infected human by 2.73%, while the rate of loss of human repellent protection in the interval increases the infected human by 0.03%. Optimal control is used to minimize the number of infected humans who do not use repellent. The results of numerical simulations on the optimal control problem show that an increase in the proportion of healthy humans who have campaign effect and use repellent regularly in the range of 14.67% can reduce infected individuals by 0.647%.

Oviposition site selection and subsequent offspring performance of Aedes aegypti in short- and long-term detritus accumulation conditions

The “oviposition preference-offspring performance” hypothesis proposes that females lay their eggs in habitats that maximize the fitness of their offspring. The aim of this study was to assess the oviposition site selection by Aedes aegypti females and the success of their larvae in habitats with different detritus accumulation times, under conditions representative of the natural spatial variability of detritus quality and quantity in the Metropolitan Area of Buenos Aires, Argentina. Two experiments were performed, one assessing oviposition site selection and the other analyzing developmental success. In both experiments, two levels of detritus accumulation time were compared, one with short-time detritus accumulation (2 weeks), and the other with long-time detritus accumulation (8 weeks). Naturally fallen detritus was used in both experiments, collected in ten sites across the Metropolitan Area of Buenos Aires. In the oviposition experiment, two contiguous ovitraps corresponding to each accumulation time were placed at each of the ten sites and the number of eggs received for each accumulation time was compared. In the development experiment, always 19 larvae were raised in containers of both accumulation times and overall performance was compared using an integrated index that considers survival, development time and female size. A large variability in the amount of detritus collected at the different sites was observed. The number of eggs was significantly higher in the long-time than in the short-time detritus accumulation containers, and approximately proportional to the amount of detritus in each ovitrap. The performance was not affected by the detritus accumulation time, but a better performance was detected in containers that received a higher amount of organic detritus, regardless of the accumulation time. Leaves were on average the most abundant type of detritus, with an average of 53 % of the total detritus collected. The amount of leaves added 2 weeks before hatching showed a positive effect on larval performance. Our results do not support the “oviposition preference-offspring performance” hypothesis, since Ae. aegypti females laid eggs in containers where larvae did not show a better performance. Moreover, at larval densities related to the number of eggs actually laid in each of the accumulation times, it is expected that the performance would be even worse in the most selected containers, due to the density-dependent effects of crowding. Since the results obtained reflect the natural heterogeneity of the environmental conditions in the region studied, they might be a fairly good indicator of what occurs in natural larval habitats.

Ammonia transport in the excretory system of mosquito larvae (Aedes aegypti): Rh protein expression and the transcriptome of the rectum

The role of the mosquito excretory organs (Malpighian tubules, MT and hindgut, HG) in ammonia transport as well as expression and function of the Rhesus (Rh protein) ammonia transporters within these organs was examined in Aedes aegypti larvae and adult females. Immunohistological examination revealed that the Rh proteins are co-localized with V-type H+-ATPase (VA) to the apical membranes of MT and HG epithelia of both larvae and adult females. Of the two Rh transporter genes present in A. aegypti, AeRh50-1 and AeRh50-2, we show using quantitative real-time PCR (qPCR) and an RNA in-situ hybridization (ISH) assay that AeRh50-1 is the predominant Rh protein expressed in the excretory organs of larvae and adult females. Further assessment of AeRh50-1 function in larvae and adults using RNAi (i.e. dsRNA-mediated knockdown) revealed significantly decreased [NH4+] (mmoll-1) levels in the secreted fluid of larval MT which does not affect overall NH4+ transport rates, as well as significantly decreased NH4+ flux rates across the HG (haemolymph to lumen) of adult females. We also used RNA sequencing to identify the expression of ion transporters and enzymes within the rectum of larvae, of which limited information currently exists for this important osmoregulatory organ. Of the ammonia transporters in A. aegypti, AeRh50-1 transcript is most abundant in the rectum thus validating our immunohistochemical and RNA ISH findings. In addition to enriched VA transcript (subunits A and d1) in the rectum, we also identified high Na+-K+-ATPase transcript (α subunit) expression which becomes significantly elevated in response to HEA, and we also found enriched carbonic anhydrase 9, inwardly rectifying K+ channel Kir2a, and Na+-coupled cation-chloride (Cl-) co-transporter CCC2 transcripts. Finally, the modulation in excretory organ function and/or Rh protein expression was examined in relation to high ammonia challenge, specifically high environmental ammonia (HEA) rearing of larvae. NH4+ flux measurements using the scanning-ion selective electrode (SIET) technique revealed no significant differences in NH4+ transport across organs comprising the alimentary canal of larvae reared in HEA vs freshwater. Further, significantly increased VA activity, but not NKA, was observed in the MT of HEA-reared larvae. Relatively high Rh protein immunostaining persists within the hindgut epithelium, as well as the ovary, of females at 24-48h post blood meal corresponding with previously demonstrated peak levels of ammonia formation. These data provide new insight into the role of the excretory organs in ammonia transport physiology and the contribution of Rh proteins in mediating ammonia movement across the epithelia of the MT and HG, and the first comprehensive examination of ion transporter and channel expression in the mosquito rectum.

Cell fusing agent virus rarely transmits vertically in artificially infected laboratory-colonized Aedes aegypti mosquitoes

BackgroundVertical transmission (VT) of arboviruses (arthropod-borne viruses) can serve as an essential link in the transmission cycle during adverse environmental conditions. The extent of VT among mosquito-borne arboviruses can vary significantly among different virus families and even among different viruses within the same genus. For example, orthobunyaviruses exhibit a higher VT rate than orthoflaviviruses and alphaviruses. Mosquitoes are also the natural hosts of a large number of insect-specific viruses (ISV) that belong to several virus families, including Bunyaviridae, Flaviviridae, and Togaviridae. Cell fusing agent virus (CFAV), an insect-specific orthoflavivirus, displays higher VT rates than other dual-host orthoflaviviruses, such as Zika and dengue viruses. High VT rates require establishment of stabilized infections in the germinal tissues of female vectors. To delve deeper into understanding the mechanisms governing these differences in VT rates and the establishment of stabilized infections, the ovary infection patterns and VT of Zika virus (ZIKV) and CFAV were compared.MethodsLaboratory colonized Aedes aegypti females were infected with either ZIKV or CFAV by intrathoracic injection. Ovary infection patterns were monitored by in situ hybridization using virus-specific probes, and VT was determined by detecting the presence of the virus among the progeny, using a reverse-transcription quantitative polymerase chain reaction (PCR) assay.ResultsBoth ZIKV and CFAV infect mosquito ovaries after intrathoracic injection. Infections then become widespread following a non-infectious blood meal. VT rates of ZIKV are similar to previously reported results (3.33%). CFAV, on the contrary transmits vertically very rarely. VT was not observed in the first gonotrophic cycle following intrathoracic injection, and only rarely in the second gonotrophic cycle. VT of CFAV is mosquito population independent, since similar results were obtained with Aedes aegypti collected from two different geographic locations.ConclusionsAlthough CFAV infects mosquito ovaries, the occurrence of VT remains infrequent in artificially infected Ae. aegypti, despite the observation of high VT rates in field-collected mosquitoes. These results suggest that infections of insect-specific viruses are stabilized in mosquitoes by some as yet unidentified mechanisms.Graphical

EVALUATION OF THERMAL FOGGER FOR EFFECTIVENESS OF LARVICIDE-ADULTICIDE MIXTURE AGAINST AEDES AEGYPTI

A hand-held thermal fogger was evaluated to spray a mixture of adulticide (Aqualuer 20-20® containing permethrin and piperonyl butoxide) and a biological larvicide (VectoBac®12AS, containing Bacillus thuringiensis subspecies israelensis). Adulticide was applied at maximum label rate of 37 mL/ha while larvicide rates were 0.58, 1.76, 2.64, and 3.50 L/ha (Maximum label rate 2.34 L/ha). Two rows, 7.6 m apart, of bioassay cages containing susceptible 25, 5-7 days old female Aedes aegypti were used to assess effectiveness of the adulticide. Along with each cage were two 473 mL empty plastic cups at 0 and 1.3 m above ground to collect the larvicide. Two control cages and two empty cups were placed upwind in the field for five minutes and removed before the spray. The study was conducted in the evening with five separated replications of a rate. The exposed cages and cups were retrieved from the field and brought back to the laboratory for further observations and tests to determine mortality at 24 and 48 h, respectively. All the adults in all treatment cages and all tests died within 24 h of the spray application. In the lab, larval mortality was assessed by introducing Ae. aegypti larvae, where mortality decreased with increasing distance from the spray line and increased with increasing application rate. The larval mortality at all distances was strongly correlated with application rate (R2 = 0.98). The highest rate used produced 64 -95% larval mortality at all distances. The results indicate that Aqualuer 20-20 and VectoBac 12AS can be applied simultaneously as a mixture achieving complete adult control and considerable larval control.

Identification of Antiviral Phytocompounds as Potential Anti‐Dengue Agents against DENV NS2B/NS3 Protease: An Integrated Molecular Modelling and Dynamics Approach

AbstractDengue fever is one of the most dreaded viral diseases caused by the bite of female Aedes aegypti and Aedes albopictus mosquitoes infected with the Dengue virus (DENV). No DENV antiviral drugs have been approved, yet they are desperately needed. The current study focuses on finding potential therapeutics from 8 medicinal plants reported to have activity against the DENV NS2B/NS3 protease in vitro. Eighty‐two phytocompounds were selected from eight different medicinal plants like Vernonia cinerea, Tridax procumbens, Cassia angustifolia, Lawsonia inermis, Dryobalanops aromatic, Punica granatum, Zingiber zerumbet and Zizyphus‐jujuba. The top five phytocompounds (Epicatechin (EPI), Chrysoeriol (CHR), Apigenin (API), Quercetin‐3,4′‐Dimethylether (QUE), and Caffeic acid (CAF)) identified from toxicity evaluation and docking studies were further subjected to 100 ns long Molecular Dynamics (MD) simulations to study their structural stability and binding affinity through MM‐PBSA based free‐energy calculations. MD results revealed overall conformational stability with all the five phytocompounds. Furthermore, MM‐PBSA results suggest binding free energy values of −154.415 kJ/mol, −151.154 kJ/mol, −139.048 kJ/mol, −122.827 kJ/mol), and −120.622 kJ/mol for compound EPI, QUE, API, CAF, and CHR respectively by interacting with essential binding site residues. These compounds could thus be studied extensively to develop nature‐based therapeutic compounds as anti‐dengue agents for DENV2.

RNAi-Mediated Knockdown of Acidic Ribosomal Stalk Protein P1 Arrests Egg Development in Adult Female Yellow Fever Mosquitoes, Aedes aegypti.

After taking a blood meal, the fat body of the adult female yellow fever mosquito, Aedes aegypti, switches from a previtellogenic state of arrest to an active state of synthesizing large quantities of yolk protein precursors (YPPs) that are crucial for egg development. The synthesis of YPPs is regulated at both the transcriptional and translational levels. Previously, we identified the cytoplasmic protein general control nonderepressible 1 (GCN1) as a part of the translational regulatory pathway for YPP synthesis. In the current study, we used the C-terminal end of GCN1 to screen for protein-protein interactions and identified 60S acidic ribosomal protein P1 (P1). An expression analysis and RNAi-mediated knockdown of P1 was performed to further investigate the role of P1 in mosquito reproduction. We showed that in unfed (absence of a blood meal) adult A. aegypti mosquitoes, P1 was expressed ubiquitously in the mosquito organs and tissues tested. We also showed that the RNAi-mediated knockdown of P1 in unfed adult female mosquitoes resulted in a strong, transient knockdown with observable phenotypic changes in ovary length and egg deposition. Our results suggest that 60S acidic ribosomal protein P1 is necessary for mosquito reproduction and is a promising target for mosquito population control.

Convolutional neural network-based real-time mosquito genus identification using wingbeat frequency: A binary and multiclass classification approach

Global rises in dengue hemorrhagic fever, especially in Asia and Latin America, underscore the necessity for enhanced public health interventions. Aedes spp. mosquitoes are the primary vectors; however, species such as Culex quinquefasciatus pose significant health risks by transmitting diseases such as filariasis, impacting millions of people worldwide. This study introduces a real-time convolutional neural network-based mosquito classification system using wingbeat frequency for identifying various mosquito species, with emphasis on Aedes sp. We proposed and assessed two models: a binary classification and a multiclass system. The binary system exhibited an outstanding accuracy of 91.76% in distinguishing between Aedes aegypti and Culex quinquefasciatus. The multiclass system accurately identified female and male Aedes aegypti and Culex quinquefasciatus with a precision of 87.16%. This innovative approach serves as a potential tool for dengue infection control and a versatile instrument for combating various mosquito-borne illnesses, enhancing vector surveillance for comprehensive disease management.

Exposure to Attractive Toxic Sugar Baits containing Piriproxyfen (ATSB-PPF) in surfaces reduces the number of eggs and larvae produced by Aedes aegypti (Diptera: Culicidae) females

Exposure to Attractive Toxic Sugar Baits containing Piriproxyfen (ATSB-PPF) in surfaces reduces the number of eggs and larvae produced by Aedes aegypti (Diptera: Culicidae) females

Arbovirus Detection of Adult Female <i>Aedes aegypti</i> for Dengue Surveillance: a Cohort Study in Bandung City, Indonesia

Dengue surveillance is an important activity to prevent dengue outbreaks. This activity becomes a significant challenge for the region with limited logistic capabilities. Developing a simple mathematical model to predict the possibility of dengue incidence provides a reliable early warning system. This study compared the correlation between vector (adult female Aedes aegypti) and arbovirus detection on a vector to dengue incidence, which generalized linear mixed models tested. The incidence of adult female Aedes aegypti and dengue fever cases were interpolated through third-power inverse distance weighting (IDW). A spatial correlation between female Aedes aegypti incidence and dengue incidence was obtained from polynomial regression. Collection sites were 16 villages in Bandung city, one of the significant dengue endemic areas in January–December 2017. A total of 8,402 mosquitoes of Aedes aegypti, Aedes albopictus, and Culex sp., with 17% belonging to Aedes aegypti as the subject of the dengue virus (DENV) infection test. Data analysis only showed a weak correlation between the numbers of adult female Aedes aegypti and dengue incidence. On the other hand, there is no correlation between positive dengue infection of vector and dengue incidence. This study highlights the importance of constant arbovirus surveillance and integrated surveillance methods on all possible dengue vectors to develop an early warning system for dengue incidence.

Field Evaluation of Fyfanon Ew Against Aedes Aegypti: A Low Odor Ultra-Low Volume Formulation of Malathion.

A low odor formulation of ultra-low volume (ULV) malathion (Fyfanon EW®) mosquito adulticide was evaluated in the field for its efficacy using caged female Aedes aegypti. Cages were placed in three rows, 30 m apart at 30, 60, and 90 m from the spray line. The product was applied at the manufacturer's suggested operational rate of 0.28 liter/min (9.5 fl oz/min) and at the maximum label rate of 0.37 liter/min (12.6 fl oz/min) with a Guardian 190ES truck mounted ULV sprayer. At 1 h post-treatment for the manufacturer's suggested operational rate, adult mortality ranged from 85% at 30 m to 46% at 90 m (overall average 65.5%). At 24 h post-treatment, mortality was significantly greater at each distance with complete control of caged mosquitoes at 30 m and >95% at 90 m. Overall, 24-h average mortality at this rate was 96.8%. At the maximum label rate, mosquito mortality at 1 h was considerably greater at all distances and ranged from about 89% to 75% with an overall average of 84.3%. At 24 h post-treatment, complete mortality was recorded at all transect distances at this higher rate. In conclusion, our results showed that at 24 h, Fyfanon EW® was considered very effective when applied by ULV truck mounted ground equipment for area-wide control of mosquitoes.

Frequent outbreaks of dengue in Nepal – causes and solutions: a narrative review

Dengue is a viral illness caused by dengue virus, a flavivirus transmitted by the bite of female Aedes aegypti and Aedes albopictus. In the year 2022, Nepal witnessed the largest ever dengue outbreak in the country. Dengue cases were reported from all seven provinces, affecting all 77 districts of the country. As of 31st December 2022, altogether 54 784 dengue cases have been identified, with Bagmati province reporting the highest. The country’s capital, Kathmandu, reported the highest burden of dengue cases (26%). The high degree of pre-monsoon rainfall because of climate change and mismanaged waste disposal system were the main cause of the 2022 dengue outbreak in Nepal. Furthermore, unplanned urbanization, Aedes vector expansion, open border policy with India, inefficient dengue surveillance system, and insufficient preventive efforts by the government are the causes of frequent dengue outbreaks. Awareness among the general public regarding vector control measures, improved surveillance of acute febrile illness, utilization of vector risk mapping techniques, and establishment of temperature-based early warning systems are utmost to halt future dengue outbreaks in Nepal.

Combined analysis of the proteome and metabolome provides insight into microRNA-1174 function in Aedes aegypti mosquitoes

BackgroundPathogenic viruses can be transmitted by female Aedes aegypti (Ae. aegypti) mosquitoes during blood-meal acquisition from vertebrates. Silencing of mosquito- and midgut-specific microRNA (miRNA) 1174 (miR-1174) impairs blood intake and increases mortality. Determining the identity of the proteins and metabolites that respond to miR-1174 depletion will increase our understanding of the molecular mechanisms of this miRNA in controlling blood-feeding and nutrient metabolism of mosquitoes.MethodsAntisense oligonucleotides (antagomirs [Ant]) Ant-1174 and Ant-Ct were injected into female Ae. aegypti mosquitoes at 12–20 h posteclosion, and depletion of miR-1174 was confirmed by reverse transcription quantitative real-time PCR (RT-qPCR). Ant-1174-injected and control mosquitoes were collected before the blood meal at 72 h post-injection for tandem mass tag-based proteomic analysis and liquid chromatography-tandom mass spectrometry non-target metabolomic analysis to identify differentially expressed proteins and metabolites, respectively. RNA interference (RNAi) using double-stranded RNA (dsRNA) injection was applied to investigate the biological roles of these differentially expressed genes. The RNAi effect was verified by RT-qPCR and western blotting assays. Triglyceride content and ATP levels were measured using the appropriate assay kits, following the manufacturers’ instructions. Statistical analyses were conducted with GraphPad7 software using the Student’s t-test.ResultsUpon depletion of mosquito- and midgut-specific miR-1174, a total of 383 differentially expressed proteins (DEPs) were identified, among which 258 were upregulated and 125 were downregulated. Functional analysis of these DEPs using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment suggested that miR-1174 plays important regulatory roles in amino acid metabolism, nucleotide metabolism, fatty acid metabolism and sugar metabolism pathways. A total of 292 differential metabolites were identified, of which 141 were upregulated and 151 were downregulated. Integrative analysis showed that the associated differential proteins and metabolites were mainly enriched in a variety of metabolic pathways, including glycolysis, citrate cycle, oxidative phosphorylation and amino acid metabolism. Specifically, the gene of one upregulated protein in miR-1174-depleted mosquitoes, purine nucleoside phosphorylase (PNP; AAEL002269), was associated with the purine, pyrimidine and niacin-nicotinamide metabolism pathways. PNP knockdown seriously inhibited blood digestion and ovary development and increased adult mortality. Mechanically, PNP depletion led to a significant downregulation of the vitellogenin gene (Vg); in addition, some important genes in the ecdysone signaling and insulin-like peptide signaling pathways related to ovary development were affected.ConclusionsThis study demonstrates differential accumulation of proteins and metabolites in miR-1174-depleted Ae. aegypti mosquitoes using proteomic and metabolomic techniques. The results provide functional evidence for the role of the upregulated gene PNP in gut physiological activities. Our findings highlight key molecular changes in miR-1174-depleted Ae. aegypti mosquitoes and thus provide a basis and novel insights for increased understanding of the molecular mechanism involved in a lineage-specific miRNA in mosquito vectors.Graphical