Relationship between deltamethrin resistance and gut symbiotic bacteria of Aedes albopictus by 16S rDNA sequencing.

BACKGROUND: Aedes albopictus is an important vector for pathogens such as dengue, Zika, and chikungunya viruses. While insecticides is the mainstay for mosquito control, their widespread and excessive use has led to the increased resistance in Ae. albopictus globally. Gut symbiotic bacteria are believed to play a potential role in insect physiology, potentially linking to mosquitoes' metabolic resistance against insecticides. METHODS: We investigated the role of symbiotic bacteria in the development of resistance in Ae. albopictus by comparing gut symbiotic bacteria between deltamethrin-sensitive and deltamethrin-resistant populations. Adults were reared from field-collected larvae. Sensitive and resistant mosquitoes were screened using 0.03% and 0.09% deltamethrin, respectively, on the basis of the World Health Organization (WHO) tube bioassay. Sensitive and resistant field-collected larvae were screened using 5 × LC50 (lethal concentration at 50% mortality) and 20 × LC50 concentration of deltamethrin, respectively. Laboratory strain deltamethrin-sensitive adults and larvae were used as controls. The DNA of gut samples from these mosquitoes were extracted using the magnetic bead method. Bacterial 16S rDNA was sequenced using BGISEQ method. We isolated and cultured gut microorganisms from adult and larvae mosquitoes using four different media: Luria Bertani (LB), brain heart infusion (BHI), nutrient agar (NA), and salmonella shigella (SS). RESULTS: Sequencing revealed significantly higher gut microbial diversity in field-resistant larvae compared with field-sensitive and laboratory-sensitive larvae (P < 0.01). Conversely, gut microorganism diversity in field-resistant and field-sensitive adults was significantly lower compared with laboratory-sensitive adults (P < 0.01). At the species level, 25 and 12 bacterial species were isolated from the gut of field resistant larvae and adults, respectively. The abundance of Flavobacterium spp., Gemmobacter spp., and Dysgonomonas spp. was significantly higher in the gut of field-resistant larvae compared with sensitive larvae (all P < 0.05). Furthermore, the abundance of Flavobacterium spp., Pantoea spp., and Aeromonas spp. was significantly higher in the gut of field-resistant adults compared with sensitive adults (all P < 0.05). The dominant and differentially occurring microorganisms were also different between resistant larval and adult mosquitoes. These findings suggest that the gut commensal bacteria of Ae. albopictus adults and larvae may play distinct roles in their deltamethrin resistance. CONCLUSIONS: This study provides an empirical basis for further exploration of the mechanisms underlying the role of gut microbial in insecticide resistance, potentially opening a new prospect for mosquito control strategies.

CRISPR-editing of the virus vector Aedes albopictus cell line C6/36, illustrated by prohibitin 2 gene knockout.

Aedes mosquitoes are important virus vectors. We provide a toolkit for CRISPR-Cas9-editing of difficult-to-knockdown gene previously shown to be refractory to siRNA silencing in mosquito cells, which is pivotal in understanding vector biology, vector competence, host-pathogen interactions and in gene annotations. Starting from database searches of Ae. albopictus and the C6/36 cell line whole genome shotgun sequences for the prohibitin 2 (PHB2) gene, primers were designed to confirm the gene sequence in our laboratory-passaged C6/36 cell line for the correct design and cloning of CRISPR RNA into an insect plasmid vector to create a single guide RNA for the PHB2 gene target. After transfection of this plasmid vector into the C6/36 cells, cell clones selected by puromycin and/or limiting dilution were analyzed for insertions and deletions (INDELs) using PCR, sequencing and computational sequence decomposition. From this, we have identified mono-allelic and bi-allelic knockout cell clones. Using a mono-allelic knockout cell clone as an example, we characterized its INDELs by molecular cloning and computational analysis. Importantly, mono-allelic knockout was sufficient to reduce >80 % of PHB2 expression, which led to phenotypic switching and the propensity to form foci but was insufficient to affect growth rate or to inhibit Zika virus infection.•We provide a toolkit for CRISPR-Cas9-editing of the virus vector, Aedes albopictus C6/36 cell line•We validate this using a difficult-to-knockdown gene prohibitin 2•This toolkit is pivotal in understanding vector biology, vector competence, host-pathogen interactions and in gene annotations.

Integrating pyriproxyfen into the incompatible insect technique enhances mosquito population suppression efficiency and eliminates the risk of population replacement.

BACKGROUND: The incompatible insect technique (IIT) has been used for Aedes mosquito population suppression to curb the transmission of dengue. However, its wide application is limited owing to the low output of male mosquitoes and the risk of population replacement from the release of fertile Wolbachia-infected females. This study aims to improve IIT efficiency for broader adoption. RESULTS: We assessed the impact of 10% pyriproxyfen (PPF) sticky powder exposure on Wolbachia (from Culex molestus)-transinfected Aedes albopictus Guangzhou line (GUA line) (GC) mosquitoes. We found that the exposure caused chronic toxicity in adult mosquitoes without affecting the cytoplasmic incompatibility (CI)-inducing capability of males. The PPF-contaminated GC females exhibited significant sterilization and the ability to disseminate lethal doses of PPF to breeding sites. Subsequently, we conducted a field trial combining PPF with IIT aiming to suppress the Ae. albopictus population. This combined approach, termed boosted IIT (BIIT), showed a notable enhancement in population suppression efficiency. The improved efficacy of BIIT was attributed to the dispersion of PPF particles in the field via the released PPF-contaminated male mosquitoes. During the BIIT field trial, no Wolbachia wPip-positive Ae. albopictus larvae were detected, indicating the effective elimination of the risk of Wolbachia-induced population replacement. Additionally, the field trial of BIIT against Ae. albopictus resulted in the suppression of the nontarget mosquito species Culex quinquefasciatus. CONCLUSION: Our results highlight the remarkable efficiency and feasibility of combining IIT with PPF in suppressing mosquito populations, facilitating the widespread implementation of IIT-based management of mosquito-borne diseases. © 2024 Society of Chemical Industry.

Influence of nutrition during previtellogenesis on the follicular development in the Asian Tiger mosquito, Aedes albopictus (Skuse) (Diptera: Culicidae).

Dengue fever poses a global public health threat, with 2.5 billion people at risk of infection each year. Because the Aedes albopictus is the primary vector of dengue, it is closely monitored and handled. The efficiency of Dengue eradication is strongly dependent on understanding a female mosquito's physiological age. This study addresses key entomological issues about the impact of previtellogenic nutrition on egg production mechanisms. Ovarian development included two distinct periods: previtellogenesis and vitellogenesis. Sugar intake during previtellogenesis influences the size of the blood meal. The major parameter influencing the vitellogenesis process is the presence of a hematophagous feeding event following sugar concentration. Upon subjecting female mosquitoes to sucrose, the ovarian follicles entered the third phase of previtellogenesis. Once females feed on blood following sucrose, ovarian development enters the vitellogenesis, and the oocyte cytoplasm reveals that the yolk granules are organized in one or two rows like a crown, increasing oocyte size. Females fed 15% sucrose before a blood meal, have the largest vitellogenic growth, and follicular size, which is seven times greater than those fed water only. Fecundity increased by 78.7% by adding 7% sucrose to the diet. Mitochondria within oocytes increase, most likely due to their transportation from the nurse cells, where the yolk is synthesized. This study describes in detail the histological alterations detected in the ovaries during the previtellogenesis as well as those associated with yolk formation, suggesting that yolk protein deposition in the oocyte is associated with blood meal, independent of sucrose feeding. RESEARCH HIGHLIGHTS: Adult nutrition during previtellogenesis significantly impacts various biological parameters and the physiological age of adults of Aedes albopictus. Female mosquitoes experienced significant growth in vitellogenic development, vectorial capacity, and follicular size after consuming a diet with 15% sucrose before a blood meal.

Incompatible insect technique: insights on potential outcomes of releasing contaminant females: a proof of concept under semi-field conditions.

BACKGROUND: Releasing large numbers of Aedes albopictus males, carrying the artificially introduced Wolbachia 'wPip' strain, results in a decrease in the reproductive capacity of wild females due to a phenomenon known as cytoplasmic incompatibility (CI). This vector control strategy is referred to as the incompatible insect technique (IIT). However, its widespread implementation faces various challenges, including the complexity of removing fertile females from the males intended for release. Here, we present the results of semi-field experiments comparing the impact of minimal female co-release on two IIT modes: unidirectional CI-based (UnCI IIT) and bidirectional CI-based (BiCI IIT), specifically targeting Ae. albopictus. RESULTS: The contamination of 'wPip' infected females (2%) during male releases significantly weakened the overall effectiveness of IIT, emphasizing the need for thorough sex separation. Specifically, with UnCI IIT, despite the low rate of co-released females, there was a gradual rise in 'wPip' infection frequency, resulting in more compatible mating and subsequently higher rates of egg hatching. Conversely, this pattern was effectively mitigated in BiCI IIT owing to the reciprocal sterility between the wild-type and the 'wPip' infected populations. CONCLUSION: Through an experimental approach, conducted in a semi-field setting, we have contributed to advancing scientific understanding regarding the potential outcomes of implementing the IIT strategy in the absence of a complete sexing system. The results suggest that safety measures for mitigating the potential impacts of co-released females can be tailored according to the specific type of IIT being utilized. © 2024 Society of Chemical Industry.

Leaf and Flower Extracts from the Dwarf Elder (Sambucus ebulus): Toxicity and Repellence against Cosmopolitan Mosquito-Borne Diseases Vectors.

As there has been no scientific evidence of the bioactivity of Sambucus ebulus (Adoxaceae) extracts against insects, we chemically characterized S. ebulus leaves and flowers extracted in methanol and water. The crude extracts, phenolic compounds, and amino acids isolated were tested as larvicides against the fourth-instar larvae of Aedes albopictus and Culex pipiens (Diptera: Culicidae). To understand their mode of action, we evaluated the in vitro acetylcholinesterase (AChE) inhibitor effect of the crude extracts on the two mosquito larvae through a colorimetric method. Furthermore, the deterrent effect of the crude extracts against ovipositing Ae. albopictus females was assessed in the open field. Twelve phenylpropanoids and fourteen amino acids were detected in the extracts, with a prevalence of hydroxycinnamic acids and nonaromatic amino acids. The most toxic compound to Ae. albopictus larvae after 24 h was gallic acid, followed by the crude S. ebulus leaf extract; on Cx. pipiens, it was the crude flower extract. The AChE test showed higher inhibition on both mosquito species exerted by the leaf extract if compared to the flower extract, and it also deterred oviposition by Ae. albopictus females starting from the third day. The results indicated that vegetal extracts could effectively help in the integrated vector management of mosquitoes.

The origin and insecticide resistance of Aedes albopictus mosquitoes established in southern Mozambique.

BACKGROUND: The Aedes albopictus mosquito is of medical concern due to its ability to transmit viral diseases, such as dengue and chikungunya. Aedes albopictus originated in Asia and is now present on all continents, with the exception of Antarctica. In Mozambique, Ae. albopictus was first reported in 2015 within the capital city of Maputo, and by 2019, it had become established in the surrounding area. It was suspected that the mosquito population originated in Madagascar or islands of the Western Indian Ocean (IWIO). The aim of this study was to determine its origin. Given the risk of spreading insecticide resistance, we also examined relevant mutations in the voltage-sensitive sodium channel (VSSC). METHODS: Eggs of Ae. albopictus were collected in Matola-Rio, a municipality adjacent to Maputo, and reared to adults in the laboratory. Cytochrome c oxidase subunit I (COI) sequences and microsatellite loci were analyzed to estimate origins. The presence of knockdown resistance (kdr) mutations within domain II and III of the VSSC were examined using Sanger sequencing. RESULTS: The COI network analysis denied the hypothesis that the Ae. albopictus population originated in Madagascar or IWIO; rather both the COI network and microsatellites analyses showed that the population was genetically similar to those in continental Southeast Asia and Hangzhou, China. Sanger sequencing determined the presence of the F1534C knockdown mutation, which is widely distributed among Asian populations, with a high allele frequency (46%). CONCLUSIONS: These results do not support the hypothesis that the Mozambique Ae. albopictus population originated in Madagascar or IWIO. Instead, they suggest that the origin is continental Southeast Asia or a coastal town in China.

Detection and population genetic analysis of Aedes albopictus (Diptera: Culicidae) based on knockdown resistance (kdr) mutations in the Yangtze River basin of China.

BACKGROUND: Aedes albopictus is an important vector of chikungunya, dengue, yellow fever and Zika viruses. Insecticides are often the most effective tools for rapidly decreasing the density of vector populations, especially during arbovirus disease outbreaks. However, the intense use of insecticides, particularly pyrethroids, has led to the selection of resistant mosquito populations worldwide. Mutations in the voltage-gated sodium channel (VGSC) gene are one of the main drivers of insecticide resistance in Ae. albopictus and are also known as "knockdown resistance" (kdr) mutations. Knowledge about genetic mutations associated with insecticide resistance is a prerequisite for developing techniques for rapid resistance diagnosis. Here, we report studies on the origin and dispersion of kdr haplotypes in samples of Ae. albopictus from the Yangtze River Basin, China; METHODS: Here, we report the results of PCR genotyping of kdr mutations in 541 Ae. albopictus specimens from 22 sampling sites in 7 provinces and municipalities in the Yangtze River Basin. Partial DNA sequences of domain II and domain III of the VGSC gene were amplified. These DNA fragments were subsequently sequenced to discover the possible genetic mutations mediating knockdown resistance (kdr) to pyrethroids. The frequency and distribution of kdr mutations were assessed in 22 Ae. albopictus populations. Phylogenetic relationships among the haplotypes were used to infer whether the kdr mutations had a single or multiple origins; RESULTS: The kdr mutation at the 1016 locus had 2 alleles with 3 genotypes: V/V (73.38%), V/G (26.43%) and G/G (0.18%). The 1016G homozygous mutation was found in only one case in the CQSL strain in Chongqing, and no 1016G mutations were detected in the SHJD (Shanghai), NJDX (Jiangsu) or HBQN (Hubei) strains. A total of 1532 locus had two alleles and three genotypes, I/I (88.35%), I/T (8.50%) and T/T (3.14%). A total of 1534 locus had four alleles and six genotypes: F/F (49.35%), F/S (19.96%), F/C (1.48%) and F/L (0.18%); S/S (23.66%); and C/C (5.36%). Haplotypes with the F1534C mutation were found only in Ae. albopictus populations in Chongqing and Hubei, and C1534C was found only in three geographic strains in Chongqing. Haplotypes with the 1534S mutation were found only in Ae. albopictus populations in Sichuan and Shanghai. F1534L was found only in HBYC. The Ae. albopictus populations in Shanghai were more genetically differentiated from those in the other regions (except Sichuan), and the genetic differentiation between the populations in Chongqing and those in the middle-lower reaches of the Yangtze River (Huber, Jiangsu, Jiangxi, and Anhui) was lower. Shanghai and Sichuan displayed low haplotype diversity and low nucleotide diversity. Phylogenetic analysis and sequence comparison revealed that the 1016 locus was divided into three branches, with the Clade A and Clade B branches bearing the 1016 mutation occurring mostly in Jiangsu and the Clade C branch bearing the 1016 mutation occurring mostly in Chongqing, suggesting at least two origins for 1016G. IIIS6 phylogenetic analysis and sequence comparison revealed that F1534S, F1534C and I1532T can be divided into two branches, indicating that IIIS6 has two origins; CONCLUSIONS: Combined with the distribution of kdr mutations and the analysis of population genetics, we infer that besides the local selection of pyrethroid resistance mutations, dispersal and colonization of Ae. albopictus from other regions may explain why kdr mutations are present in some Ae. albopictus populations in the Yangtze River Basin.

Assessment of sublethal effects of permethrin on adult life characteristics and resistance dynamics in Aedes albopictus (Diptera: Culicidae).

Mosquitoes are regularly exposed to adverse effects of insecticides employed in field during vector control campaigns. Its primary goal is to eliminate the vector population; nevertheless, this practise typically ignores the residual impacts and long-term repercussions on the remaining population. Here, the current study analysed how sublethal exposure of insecticides alter the life qualities, genotypic and biochemical characteristics of mosquitoes. The resistance ratio value in Laboratory Resistant (Lab-R) larvae increased 10 times (0.010 mg/l to 0.108 mg/l) compared to Laboratory Susceptible (LabS) larvae. It also revealed that the surviving mosquitoes had 50% reduction in hatchability but had longer larval and pupal periods (15 days and 2 days), respectively. The survival rates decrease in female by 2 days but increase in male by 7 days which is of concern and necessitates additional study. Moreover, major role of monooxygenase was confirmed behind resistance development which was further supported by piperonyl butoxide assay where reduction in Tolerance Ratio (TR50) by 12-fold occurred and gene expression profile also showed high expression level of CYP6P12 gene. In resistant strain, cuticular thickness increased by 1.23 times and alteration at codon 1532 (ATC to TTC) on VGSC gene leads to mutation I1532F. The data gleaned from our work highlights the threat of sublethal insecticides on vector control techniques and offers ample evidence that the larval selection alters adult life qualities, metabolic properties and transgenerational features which contributes to the damage caused by resistance.

Mosquito population dynamics is shaped by the interaction among larval density, season, and land use.

Understanding how variation in key abiotic and biotic factors interact at spatial scales relevant for mosquito fitness and population dynamics is crucial for predicting current and future mosquito distributions and abundances, and the transmission potential for human pathogens. However, studies investigating the effects of environmental variation on mosquito traits have investigated environmental factors in isolation or in laboratory experiments that examine constant environmental conditions that often do not occur in the field. To address these limitations, we conducted a semi-field experiment in Athens, Georgia using the invasive Asian tiger mosquito (Aedes albopictus). We selected nine sites that spanned natural variation in impervious surface and vegetation cover to explore effects of the microclimate (temperature and humidity) on mosquitoes. On these sites, we manipulated conspecific larval density at each site. We repeated the experiment in the summer and fall. We then evaluated the effects of land cover, larval density, and time of season, as well as interactive effects, on the mean proportion of females emerging, juvenile development time, size upon emergence, and predicted per capita population growth (i.e., fitness). We found significant effects of larval density, land cover, and season on all response variables. Of most note, we saw strong interactive effects of season and intra-specific density on each response variable, including a non-intuitive decrease in development time with increasing intra-specific competition in the fall. Our study demonstrates that ignoring the interaction between variation in biotic and abiotic variables could reduce the accuracy and precision of models used to predict mosquito population and pathogen transmission dynamics, especially those inferring dynamics at finer-spatial scales across which transmission and control occur.

Para poder predecir la distribución y abundancia de las poblaciones de mosquitos y la transmisión potencial de patógenos a humanos, es crucial comprender cómo factores abióticos y bióticos clave para el éxito reproductivo y la dinámica poblacional de los mosquitos interactúan a escalas relevantes. Sin embargo, los estudios que han investigado los efectos de variables ambientales en las características demográficas de los mosquitos han considerado su efecto de forma aislada o en experimentos de laboratorio bajo condiciones ambientales constantes que, a menudo, no reflejan lo que ocurre en el campo. Para abordar estas limitaciones, llevamos a cabo un experimento de semi-campo en Athens, Georgia, utilizando el mosquito invasor tigre asiático (Aedes albopictus). Seleccionamos nueve sitios que abarcaban variaciones naturales en la superficie impermeable y cobertura vegetal para explorar los efectos del microclima (temperatura y humedad) en los mosquitos. También manipulamos la densidad de larvas de tigre asiático en dos experimentos que fueron realizados en el verano y otoño. Evaluamos los efectos de la cobertura vegetal, la densidad de larvas, la temporada climática, y la interacción entre estas variables en la proporción de hembras que emergieron, el tiempo de desarrollo de las larvas, el tamaño al momento de la emergencia, y el crecimiento demográfico per cápita previsto (éxito reproductivo). Encontramos efectos significativos de la densidad de larvas, la variación de la cobertura vegetal y la estación del año en todas las variables de respuesta. Más notablemente, observamos un fuerte efecto de la interacción entre la temporada climática y la densidad de larvas en todas las variables de respuesta, incluyendo una disminución no intuitiva en el tiempo de desarrollo con el aumento de la competencia intraespecífica en el otoño. Nuestro estudio evidencia que ignorar la interacción entre variables abióticas y bióticas podría reducir la exactitud y precisión de los modelos utilizados para predecir las dinámicas de las poblaciones de mosquitos, y por tanto, de la transmisión de patógenos. Esto, especialmente en modelos que infieren estas dinámicas a escalas espaciales más finas, en las cuales ocurre la transmisión y el control.

Complete coding sequence of an Aedes flavivirus strain isolated from Aedes albopictus collected in Northern Italy.

Complete genome data for the globally distributed Aedes flavivirus (AEFV) is scarce. We analyzed a new Italian AEFV strain isolated from Aedes albopictus. The results demonstrated genetic diversity among Italian AEFVs. The high similarity between AEFV genomes across geographically distant regions suggests long distance spreading via invasive host mosquito species.

Sampling efficiency and screening of Aedes albopictus for yellow fever virus in Niger Delta region of Nigeria.

Introduction: Aedes albopictus, like Aedes aegypti, is a virulent vector of arboviruses especially the well-documented spread of yellow fever around the world. Although yellow fever is prevalent in Nigeria, there is a paucity of information in the Niger Delta region on the distribution of Aedes mosquito vectors and molecular detection of the virus in infected mosquitoes. This study sampled Aedes mosquitoes around houses associated with farms from four communities (Otolokpo, Ute-Okpu, Umunede, and Ute Alohen) in Ika North-East Local Government Area of Delta State, Nigeria. Methods: various sampling methods were used in Aedes mosquito collection to test their efficacy in the survey. Mosquitoes in holding cages were killed by freezing and morphologically identified. A pool of 15 mosquitoes per Eppendorf tube was preserved in RNAi later for yellow fever virus screening. Two samples were molecularly screened for each location. Results: seven hundred and twenty-five (725) mosquitoes were obtained from the various traps. The mean abundance of the mosquitoes was highest in m-HLC (42.9) compared to the mosquitoes sampled using other techniques (p<0.0001). The mean abundance of mosquitoes was lowest in Center for Disease Control (CDC) light traps without attractant (0.29). No yellow fever virus strain was detected in all the mosquitoes sampled at the four locations. Conclusion: this study suggests that Aedes albopictus are the mosquitoes commonly biting around houses associated with farms. More so, yellow fever virus was not detected in the mosquitoes probably due to the mass vaccination exercise that was carried out the previous year in the study area. More studies are required using the m-HLC to determine the infection rate in this endemic area.

Gene silencing in the aedine cell lines C6/36 and U4.4 using long double-stranded RNA.

BACKGROUND: RNA interference (RNAi) is a target-specific gene silencing method that can be used to determine gene functions and investigate host-pathogen interactions, as well as facilitating the development of ecofriendly pesticides. Commercially available transfection reagents (TRs) can improve the efficacy of RNAi. However, we currently lack a product and protocol for the transfection of insect cell lines with long double-stranded RNA (dsRNA). METHODS: We used agarose gel electrophoresis to determine the capacity of eight TRs to form complexes with long dsRNA. A CellTiter-Glo assay was then used to assess the cytotoxicity of the resulting lipoplexes. We also measured the cellular uptake of dsRNA by fluorescence microscopy using the fluorophore Cy3 as a label. Finally, we analyzed the TRs based on their transfection efficacy and compared the RNAi responses of Aedes albopictus C6/36 and U4.4 cells by knocking down an mCherry reporter Semliki Forest virus in both cell lines. RESULTS: The TRs from Biontex (K4, Metafectene Pro, and Metafectene SI+) showed the best complexing capacity and the lowest dsRNA:TR ratio needed for complete complex formation. Only HiPerFect was unable to complex the dsRNA completely, even at a ratio of 1:9. Most of the complexes containing mCherry-dsRNA were nontoxic at 2 ng/µL, but Lipofectamine 2000 was toxic at 1 ng/µL in U4.4 cells and at 2 ng/µL in C6/36 cells. The transfection of U4.4 cells with mCherry-dsRNA/TR complexes achieved significant knockdown of the virus reporter. Comparison of the RNAi response in C6/36 and U4.4 cells suggested that C6/36 cells lack the antiviral RNAi response because there was no significant knockdown of the virus reporter in any of the treatments. CONCLUSIONS: C6/36 cells have an impaired RNAi response as previously reported. This investigation provides valuable information for future RNAi experiments by showing how to mitigate the adverse effects attributed to TRs. This will facilitate the judicious selection of TRs and transfection conditions conducive to RNAi research in mosquitoes.

Design and evaluation of an efficient mosquito trap.

BACKGROUND: Vector mosquito control is important for preventing and controlling mosquito-borne infectious diseases. This study designed and developed a mosquito killer (MK) with a specific light wavelength, simulated human body temperature, human odor, and a photocatalyst to stimulate CO2 based on the physiological characteristics and ecological habits of mosquitoes. We tested the trapping effect of individual and multiple mosquito-trapping elements of the MK through two-way selection experiments and compared them with several commercial mosquito traps. RESULTS: The 365 nm wavelength MK was significantly more effective than the 395 nm (Cx. quinquefasciatus: 62.00% vs. 34.25%; Ae. albopictus: 50.75% vs 45.00%, An. sinensis: 49.75% vs 39.00%). Mosquitoes captured by the MK with heaters at 365 nm were significantly more than those captured by the MK without heaters at 365 nm. A trap with a 365 nm wavelength, heating element, and lure showed significantly better capture effectiveness than MK with a 365 nm wavelength, heating element, but without lure (Cx. quinquefasciatus: 67.00% vs. 29.75%, Ae. albopictus: 60.25% vs 36.25%, An. sinensis: 49.75% vs 39.75%). The coated photocatalyst trap with a 365 nm wavelength, heating element, and lure showed significantly better capture effectiveness than the trap without coating (Cx. quinquefasciatus: 54.25% vs. 42.50%; Ae. albopictus: 53.50% vs 44.00%, An. sinensis: 50.00% vs 41.25%). This trap demonstrated a significantly better capture advantage for Cx. quinquefasciatus and Ae. albopictus compared to the three commercial products. CONCLUSION: The developed mosquito trap with multiple attractant factors significantly enhanced the capture effectiveness of common mosquitoes. © 2024 Society of Chemical Industry.

Landscape and meteorological variables associated with Aedes aegypti and Aedes albopictus mosquito infestation in two southeastern USA coastal cities.

Aedes transmitted arboviral human cases are increasing worldwide and spreading to new areas of the United States of America (USA). These diseases continue to re-emerge likely due to changes in vector ecology, urbanization, human migration, and larger range of climatic suitability. Recent shifts in landscape and weather variables are predicted to impact the habitat patterns of urban mosquitoes such as Aedes aegypti and Aedes albopictus. Miami (FL) is in the tropical zone and an established hotspot for arboviruses, while Charleston (SC) is in the humid subtropical zone and newly vulnerable. Although these coastal cities have distinct climates, both have hot summers. To understand mosquito infestation in both cities and potentiate our surveillance effort, we performed egg collections in the warmest season. We applied remote sensing with land-use cover and weather variation to identify mosquito infestation patterns. Our study found predominant occurrence of Ae. aegypti and, to a lesser extent, Ae. albopictus in both cities. We detected statistically significant positive and negative associations between entomological indicators and most weather variables in combined data from both cities. For all entomological indices, weekly wind speed and relative humidity were significantly positively associated, while precipitation and maximum temperature were significantly negatively associated. Aedes egg abundance was significantly positively associated with open land in Charleston but was negatively associated with vegetation cover in combined data. There is a clear need for further observational studies to determine the impact of climate change on Ae. aegypti and Ae. albopictus infestation in the Southeastern region of the USA.

Aedes albopictus saliva contains a richer microbial community than the midgut.

BACKGROUND: Past findings demonstrate that arthropods can egest midgut microbiota into the host skin leading to dual colonization of the vertebrate host with pathogens and saliva microbiome. A knowledge gap exists on how the saliva microbiome interacts with the pathogen in the saliva. To fill this gap, we need to first define the microbial composition of mosquito saliva. METHODS: The current study aimed at analyzing and comparing the microbial profile of Aedes albopictus saliva and midgut as well as assessing the impact of Zika virus (ZIKV) infection on the midgut and saliva microbial composition. Colony-reared Ae. albopictus strains were either exposed to ZIKV infectious or noninfectious bloodmeal. At 14 ays postinfection, the 16S V3-V4 hypervariable rRNA region was amplified from midgut and saliva samples and sequenced on an Illumina MiSeq platform. The relative abundance and diversity of midgut and saliva microbial taxa were assessed. RESULTS: We observed a richer microbial community in the saliva compared with the midgut, yet some of the microbial taxa were common in the midgut and saliva. ZIKV infection did not impact the microbial diversity of midgut or saliva. Further, we identified Elizabethkingia spp. in the Ae. albopictus saliva. CONCLUSIONS: This study provides insights into the microbial community of the Ae. albopictus saliva as well as the influence of ZIKV infection on the microbial composition of its midgut and saliva. The identification of Elizabethkingia spp., an emerging pathogen of global health significance, in Ae. albopictus saliva is of medical importance. Future studies to assess the interactions between Ae. albopictus saliva microbiome and ZIKV could lead to novel strategies for developing transmission barrier tools.

LA CROSSE VIRUS VECTOR RESTING BEHAVIORS - FIELD STUDIES WITH PROKOPAK AND RESTING SHELTER COLLECTIONS PROVIDE LOW YIELD.

Resting adult mosquito collections provide opportunities to sample broad physiological conditions (e.g., blood-engorged, gravid, nectar-engorged, and/or parous) that yield important biological information necessary to understand vector and pathogen transmission ecology. In this study, we evaluated Prokopak aspirations of Rhododendron spp. and human-powered pop-up resting shelter collections at 4 residences with historical evidence of proximal La Crosse virus (LACV) transmission from May through September 2022. The goal of this study was to investigate these sampling methods in the context of LACV vector biology-focused principally on Aedes triseriatus (primary LACV vector) and 2 invasive species (Ae. albopictus and Ae. japonicus) that likely serve as secondary LACV vectors. Overall, 304 resting shelters and 80 Prokopak collections yielded a grand total of 33 mosquitoes, of which a third were LACV vectors (Ae. triseriatus [n = 1, 3.0%], Ae. albopictus [n = 4, 12.1%], and Ae. japonicus [n = 6, 18.2%]). Anopheles punctipennis (n = 9, 27.2%) was the most frequently collected species followed by Culex erraticus (n = 7, 21.2%), whereas the least frequently collected species were Ae. triseriatus and Cx. pipiens (n = 1, 3.0%). Despite substantial collection efforts, and concurrent gravid-trap evidence of LACV vectors at the collection sites, Prokopak aspiration of Rhododendron spp. and human-powered pop-up resting shelters did not yield a meaningful number of LACV vectors and thus, as described within, may not be useful adjuncts for the evaluation of LACV ecology and disease risk. Additional approaches to evaluate the resting behavior of these vectors in LACV endemic areas are needed.

Epidemiological Trends of Dengue in One of the Western Hilly Areas of Himachal Pradesh.

Background: Dengue is one of the neglected tropical diseases, with a wide spectrum of diseases, ranging from acute febrile illness dengue fever to life-threatening dengue hemorrhagic fever or dengue shock syndrome. In recent years, it has become a major public health concern in many nonendemic areas as well. Materials and Methods: A secondary data analysis of records available with district Integrated Disease Surveillance Programme cell was conducted to study distribution (time, place, and person) of dengue from 2017 to 2022 in Kangra, a sub-Himalayan district of Himachal Pradesh (HP). Results: In the evaluated period (2017-2022), a total of 6008 cases suspected of dengue were tested and test positivity of 7% (441) with male gender predominance was found. Mean age of the diagnosed cases was 37.7 ± 16.8 years. A seasonal trend was observed starting from late August to November in all study years. Conclusion: Dengue is still a neglected disease, but it has shown its presence especially in this part of HP, indicating the need for better preparation and sensitization of vector-borne disease control program activities, especially in post-monsoon, to prevent future epidemics.

Mayaro virus, a potential threat for Europe: vector competence of autochthonous vector species.

BACKGROUND: Mayaro virus (MAYV) is an emerging alphavirus, primarily transmitted by the mosquito Haemagogus janthinomys in Central and South America. However, recent studies have shown that Aedes aegypti, Aedes albopictus and various Anopheles mosquitoes can also transmit the virus under laboratory conditions. MAYV causes sporadic outbreaks across the South American region, particularly in areas near forests. Recently, cases have been reported in European and North American travelers returning from endemic areas, raising concerns about potential introductions into new regions. This study aims to assess the vector competence of three potential vectors for MAYV present in Europe. METHODS: Aedes albopictus from Italy, Anopheles atroparvus from Spain and Culex pipiens biotype molestus from Belgium were exposed to MAYV and maintained under controlled environmental conditions. Saliva was collected through a salivation assay at 7 and 14 days post-infection (dpi), followed by vector dissection. Viral titers were determined using focus forming assays, and infection rates, dissemination rates, and transmission efficiency were calculated. RESULTS: Results indicate that Ae. albopictus and An. atroparvus from Italy and Spain, respectively, are competent vectors for MAYV, with transmission possible starting from 7 dpi under laboratory conditions. In contrast, Cx. pipiens bioform molestus was unable to support MAYV infection, indicating its inability to contribute to the transmission cycle. CONCLUSIONS: In the event of accidental MAYV introduction in European territories, autochthonous outbreaks could potentially be sustained by two European species: Ae. albopictus and An. atroparvus. Entomological surveillance should also consider certain Anopheles species when monitoring MAYV transmission.

Challenges of Robust RNAi-Mediated Gene Silencing in Aedes Mosquitoes.

In this study, we report the complexities and challenges associated with achieving robust RNA interference (RNAi)-mediated gene knockdown in the mosquitoes Aedes aegypti and Aedes albopictus, a pivotal approach for genetic analysis and vector control. Despite RNAi's potential for species-specific gene targeting, our independent efforts to establish oral delivery of RNAi for identifying genes critical for mosquito development and fitness encountered significant challenges, failing to reproduce previously reported potent RNAi effects. We independently evaluated a range of RNAi-inducing molecules (siRNAs, shRNAs, and dsRNAs) and administration methods (oral delivery, immersion, and microinjection) in three different laboratories. We also tested various mosquito strains and utilized microorganisms for RNA delivery. Our results reveal a pronounced inconsistency in RNAi efficacy, characterized by minimal effects on larval survival and gene expression levels in most instances despite strong published effects for the tested targets. One or multiple factors, including RNase activity in the gut, the cellular internalization and processing of RNA molecules, and the systemic dissemination of the RNAi signal, could be involved in this variability, all of which are barely understood in mosquitoes. The challenges identified in this study highlight the necessity for additional research into the underlying mechanisms of mosquito RNAi to develop more robust RNAi-based methodologies. Our findings emphasize the intricacies of RNAi application in mosquitoes, which present a substantial barrier to its utilization in genetic control strategies.