Tyrosine transfer RNA levels and modifications during blood-feeding and vitellogenesis in the mosquito, Aedes aegypti.

Mosquitoes such as Aedes aegypti must consume a blood meal for the nutrients necessary for egg production. Several transcriptome and proteome changes occur post-blood meal that likely corresponds with codon usage alterations. Transfer RNA (tRNA) is the adapter molecule that reads messenger RNA codons to add the appropriate amino acid during protein synthesis. Chemical modifications to tRNA enhance codon decoding, improving the accuracy and efficiency of protein synthesis. Here, we examined tRNA modifications and transcripts associated with the blood meal and subsequent periods of vitellogenesis in A. aegypti. More specifically, we assessed tRNA transcript abundance and modification levels in the fat body at critical times post blood-feeding. Based on a combination of alternative codon usage and identification of particular modifications, we discovered that increased transcription of tyrosine tRNAs is likely critical during the synthesis of egg yolk proteins in the fat body following a blood meal. Altogether, changes in both the abundance and modification of tRNA are essential factors in the process of vitellogenin production after blood-feeding in mosquitoes.

Talking About Gene Drive in Uganda: The Need for Science Communication to Underpin Engagement.

Uganda may host the world's first field trials of gene drive mosquitoes for malaria control. Global North discourses pre-suppose African publics have access to information about gene drive and are ready to make decisions about its governance. We explore assumptions about the availability of this information in Uganda. We find a paucity of information available combined with a strong desire for information from lay publics. We discuss these findings in the context of Ugandan information infrastructures and political sensitivities to genetic technologies. If Ugandans are to decide about gene drive, they need independent information about the science to underpin engagement.

Survey of mosquito species in intensive pig farms in Hunan province, China.

Mosquitoes (Diptera: Culicidae) are one of the most studied groups of arthropods worldwide due to their high transmission capacity for pathogens, including viruses and parasites. During June to October 2022, the prevalence of mosquito species in 12 intensive pig farms from 12 representative administrative regions in Hunan province of China was investigated using traps with ultraviolet light. All collected mosquitoes were counted and identified to species according to morphological and molecular methods. A total of 4,443 mosquito specimens were collected in the pig farms, and they represented one family, four genera and nine species. Culex pipiens pipiens (24%) was the most common mosquito species, followed by Armigeres subalbatus (23.4%) and Culex tritaeniorhynchus (20.6%). Phylogenetic analyses based on mitochondrial cox1 sequences revealed all mosquito species from present study grouping into distinct monophyletic groups corresponding to nine known mosquito species with strongly supported. The results of the present investigation have implications for the ongoing control of mosquito infestation in pig farms in Hunan province, China. This is the first report of mosquito populations in intensive pig farms in Hunan province, China.

Optimization of process parameters for specific pathogen-free chicken embryonic fibroblast cultivation for yellow fever vaccine production.

The yellow fever (YF) vaccine is usually produced with egg-based methods, which has limitations, including potential adverse effects and low production yields. Alternatively, producing the vaccine using Vero cells or HEK 293 cells can overcome some of these issues, but these methods are significantly more expensive. In the current study, the YF vaccine candidate 17DD virus was produced in primary chicken embryo fibroblast (CEF) cells. The primary CEF cells isolation from eggs was optimized through a two-step process. In the first step, the important parameters that contribute to the development of the egg embryo, such as egg position, relative humidity (RH), and incubation time are optimized. In second step, primary CEF release parameters namely; trypsin volume and incubation temperature are optimized. Both steps were optimized using statistical methods. Further, the seeding cell density of isolated CEF was also optimized. It was observed that 5 x 104 cells/cm2 gave the highest virus titer of 3.89 million PFU/ml. The 17DD yields achieved in primary CEFs are much higher than egg-based production and it is an economically viable method.

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.

The bio-larvicide Bacillus thuringiensis var. israelensis is effective against Aedes koreicus, either dissolved in water or delivered through eco-compatible chitosan-based hydrogels.

Mosquito control, which is not always easily accomplished, is further complicated by the spread of invasive species. This is the case of Aedes koreicus, a mosquito native to East Asia, whose presence has been recorded in several European countries, including Italy. This mosquito found suitable ecological conditions in central Europe in general, and in northern Italy in particular, as shown by the ongoing expansion of its distribution. While basic knowledge on feeding habits of Ae. koreicus have already been acquired, information on its vectorial competence is scarce. Therefore, active monitoring on the presence of this mosquito, and the pre-planning of future control actions, are of paramount importance. Currently, there are no specific guidelines for controlling this mosquito, both in its native regions and in invaded countries. Here we present the first study on the efficacy of a bioinsecticide based on Bacillus thuringiensis on Ae. koreicus larvae, with a comparison with results obtained on the tiger mosquito Aedes albopictus. Our results proved that this bioinsecticide is effective on Ae. koreicus, both dissolved in water and incorporated into MosChito raft, a hydrogel-based matrix that has recently been developed for the delivery of insecticides to other mosquito species and suitable for safe and eco-compatible applications.

Early rise of West Nile fever in Israel, June 2024.

This report describes an unusual surge of West Nile fever in Israel in June 2024, during which 125 cases were diagnosed, compared with 4 cases on average during June in previous years (2014-23). Of the cases, 64 (62.1%) had neuroinvasive disease and 12 (9.6%) died; the 2024 case fatality rate was not significantly elevated vs the average rate in 2014-23. The early rise could be related to a temperature increase in spring and early summer of 2024.

Mark-Release-Recapture (MRR) of Sterile Male Aedes albopictus (Skuse) in Sri Lanka: Field Performance of Sterile Males and Estimation of the Wild Mosquito Population Density.

Dengue is an important mosquito-borne disease in Sri Lanka. The Sterile Insect Technique (SIT) is an environment-friendly and novel method that can suppress dengue vector mosquitoes in Sri Lanka. This study aimed to evaluate the field performance of sterile males and the density of wild male Aedes albopictus (Skuse) using a Mark-Release-Recapture (MRR) assay. Laboratory-colonized male pupae were exposed to 50 Gy gamma using a Co60 source. Sterile males (approx. 10,000) marked with fluorescent dust were released weekly for 4 consecutive weeks (January-February 2021) in a geographically isolated 30 ha site in Gampaha. Results show sterile males could disperse up to 543.8 m with a mean distance of 255.1 ± 44.6 m and survive up to 6 days with a mean life expectancy of 3.55 ± 2.32 days. A high field mating competitiveness of sterile males based on a Fried value of 0.47 ± 0.007 and significant induced sterility in the wild eggs in the second generation were found. The mean wild male mosquito population density was 163 males/ha. The data generated will be useful for designing future trials in Sri Lanka and other countries with similar situations.

Arthropod-Borne Viruses of Human and Animal Importance: Overwintering in Temperate Regions of Europe during an Era of Climate Change.

The past three decades have seen an increasing number of emerging arthropod-borne viruses in temperate regions This process is ongoing, driven by human activities such as inter-continental travel, combined with the parallel emergence of invasive arthropods and an underlying change in climate that can increase the risk of virus transmission and persistence. In addition, natural events such as bird migration can introduce viruses to new regions. Despite the apparent regularity of virus emergence, arthropod-borne viruses circulating in temperate regions face the challenge of the late autumn and winter months where the arthropod vector is inactive. Viruses therefore need mechanisms to overwinter or they will fail to establish in temperate zones. Prolonged survival of arthropod-borne viruses within the environment, outside of both vertebrate host and arthropod vector, is not thought to occur and therefore is unlikely to contribute to overwintering in temperate zones. One potential mechanism is continued infection of a vertebrate host. However, infection is generally acute, with the host either dying or producing an effective immune response that rapidly clears the virus. There are few exceptions to this, although prolonged infection associated with orbiviruses such as bluetongue virus occurs in certain mammals, and viraemic vertebrate hosts therefore can, in certain circumstances, provide a route for long-term viral persistence in the absence of active vectors. Alternatively, a virus can persist in the arthropod vector as a mechanism for overwintering. However, this is entirely dependent on the ecology of the vector itself and can be influenced by changes in the climate during the winter months. This review considers the mechanisms for virus overwintering in several key arthropod vectors in temperate areas. We also consider how this will be influenced in a warming climate.

Transovarial Transmission of Cell-Fusing Agent Virus in Naturally Infected Aedes aegypti Mosquitoes.

Mosquito-borne arboviruses include several pathogens that are responsible for many diseases of significant public health burden. Mosquitoes also host many insect-specific viruses that cannot replicate in vertebrate cells. These insect-specific viruses persist in nature predominantly via vertical transmission (VT), and they exhibit high VT rates (VTRs). Cell-fusing agent virus (CFAV), an insect-specific orthoflavivirus, shows high VTRs in naturally infected mosquitoes but not in artificially infected mosquitoes. To determine whether the high VTRs are due to transovarial transmission, we investigated VT and ovary infection patterns in naturally CFAV-infected Aedes aegypti (Bangkok) mosquitoes. VT was monitored by detecting CFAV among the progeny by reverse-transcription polymerase chain reaction and ovary infection was determined by in situ hybridization using a virus-specific probe. We showed that in CFAV-positive mosquitoes, ovarian follicles were infected, suggesting that VT occurs by transovarial transmission in naturally infected mosquitoes. Additionally, mosquitoes harbored dormant, non-replicative CFAV that remained below the detection level. These results suggested that CFAV persists via VT in nature and has the potential to remain dormant in diapausing mosquitoes during unfavorable conditions. Understanding this VT mechanism is crucial for comprehending the persistence of insect-specific viruses (and potentially dual-host arboviruses) in their natural environment.

Culex quinquefasciatus Mosquitoes Resist Acquisition of Methicillin-Resistant Staphylococcus aureus: Insights from Field and Laboratory Studies.

The emergence of antimicrobial resistance (AMR) in pathogens and their spillover into the environment have become a global public health menace. Insects can acquire these pathogens from the environment and would serve as mechanical and biological vectors. The current study assessed the ability of Culex quinquefasciatus mosquitoes to acquire methicillin-resistant Staphylococcus aureus (MRSA) through the exposure of the mosquitoes to the pathogen via rearing water, blood feed, or a feeding membrane under laboratory conditions. In addition, mosquito immatures collected from their habitat in the vicinity of hospitals, veterinary dispensaries, and butcher shops at 15 study sites in Puducherry were screened for MRSA infection. All samples were subjected to the culture-based isolation of S. aureus from the surface and homogenate. The presence of the S. aureus-specific nuc gene and the genes that confer resistance to methicillin (mecA and mecC) were screened using PCR tests. MRSA was not evident either on the external surface or in the homogenate of the mosquitoes emerging from the larvae reared in the MRSA-spiked water or those fed with MRSA through blood or smeared membranes. Furthermore, the presence of MRSA was not evident in any of the field-caught mosquitoes. Hence, we conclude that C. quinquefasciatus mosquitoes are impervious to MRSA colonization.

Taxonomy, Bio-Ecology and Insecticide Resistance of Anopheline Vectors of Malaria in Sri Lanka.

The objective of this review was to update the current knowledge on major malaria vectors in Sri Lanka and their bio-ecology and insecticide resistance status. Relevant data were collected through a comprehensive literature search performed using databases such as PubMed, NIH, Google Scholar and Web of Science. Sri Lanka had been endemic to malaria for centuries. However, due to a coordinated public health effort last indigenous malaria case was reported in 2012 and the island nation was declared free of malaria in 2016. Although 25 anopheline mosquitoes have been reported so far on the island, only Anopheles culicifacies and An. subpictus have been established as primary and secondary vectors of malaria respectively. Both vector species exist as a species complex, and the sibling species of each complex differ in their bio-ecology and susceptibility to malaria parasites and insecticides. The article provides a comprehensive and updated account of the bio-ecology and insecticide resistance of malaria vectors and highlights the challenges ahead of retaining a malaria-free status.

Diversity and relative abundance of immature mosquitoes (Diptera: Culicidae) in Hohenbergia catingae Ule bromeliads and larvitraps in the Nísia Floresta National Forest, northeast Brazil.

The Nísia Floresta National Forest, located in Rio Grande do Norte (RN), is an important remnant of the Atlantic Forest biome in Brazil. Bromeliad tanks in this forest offer suitable breeding sites for mosquito species that may act as viral vectors, thus posing an epidemiological concern. However, studies investigating the presence of immature Culicidae in natural breeding sites in RN have thus far been restricted to Caatinga vegetation. This study investigated mosquitoes and their natural breeding sites in bromeliads growing in the Nísia Floresta National Forest. From March 2013 to February 2014, monthly samples were collected from the tanks of five randomly selected bromeliads and larvitraps placed in each of the three forest management areas. Hohenbergia catingae Ule (Hohenbergia bromeliad) is an important shelter for immature mosquitoes. Culex (Microculex) was the predominant species, representing 86% of the immature mosquitoes collected. A rare occurrence of Aedes (Stegomyia) aegypti (Linnaeus, 1762) (generally associated with urban areas under high anthropogenic influence) was observed, highlighting the importance of investigating the presence of mosquitoes in different natural habitats. An analysis of species diversity revealed that species such as Culex imitator Theobald, 1903 and Culex davisi Kumm, 1933, have a strong association with bromeliads. In tire traps (larvitraps) Aedes (Stegomyia) albopictus Skuse, 1894 was predominant. Environmental changes, such as deforestation, removal of bromeliads, and climate change in the area, can influence the migration of species and adaptation to new habitats in a peridomiciliary environment around the forest, consequently the possibility of transmission of virus and other pathogens.

Disclosing the virome of Aedes, Anopheles and Culex female mosquitoes, Alto Pantanal of Mato Grosso, Brazil, 2019.

Using Illumina NextSeq sequencing and bioinformatics, we identified and characterized thirty-three viral sequences of unsegmented and multipartite viral families in Aedes spp., Culex sp. and Anopheles darlingi female mosquito pools from Porto São Luiz and Pirizal, Alto Pantanal. Seventeen sequences belong to unsegmented viral families, twelve represent putative novel insect-specific viruses (ISVs) within families Chuviridae (3/33; partial genomes) and coding-complete sequences of Xinmoviridae (1/33), Rhabdoviridae (2/33) and Metaviridae (6/33); and five coding-complete sequences of already-known ISVs. Notably, two putative novel rhabdoviruses, Corixo rhabdovirus 1 and 2, were phylogenetically related to Coxipo dielmovirus, but separated from other Alpharhabdovirinae genera, sharing Anopheles spp. as host. Regarding multipartite families, sixteen segments of different putative novel viruses were identified (13 coding-complete segments) within Durnavirales (4/33), Elliovirales (1/33), Hareavirales (3/33) and Reovirales (8/33) orders. Overall, this study describes twenty-eight (28/33) putative novel ISVs and five (5/33) already described viruses using metagenomics approach.

Sequencing and Description of the Mitochondrial Genome of Orthopodomyia fascipes (Diptera: Culicidae).

The genus Orthopodomyia Theobald, 1904 (Diptera: Culicidae) comprises 36 wild mosquito species, with distribution largely restricted to tropical and temperate areas, most of which are not recognized as vectors of epidemiological importance due to the lack of information related to their bionomy and involvement in the cycle transmission of infectious agents. Furthermore, their evolutionary relationships are not completely understood, reflecting the scarcity of genetic information about the genus. Therefore, in this study, we report the first complete description of the mitochondrial genome of a Neotropical species representing the genus, Orthopodomyia fascipes Coquillet, 1906, collected in the Brazilian Amazon region. Using High Throughput Sequencing, we obtained a mitochondrial sequence of 15,598 bp, with an average coverage of 418.5×, comprising 37 functional subunits and a final portion rich in A + T, corresponding to the control region. The phylogenetic analysis, using Maximum Likelihood and Bayesian Inference based on the 13 protein-coding genes, corroborated the monophyly of Culicidae and its two subfamilies, supporting the proximity between the tribes Orthopodomyiini and Mansoniini, partially disagreeing with previous studies based on the use of molecular and morphological markers. The information generated in this study contributes to a better understanding of the taxonomy and evolutionary history of the genus and other groups of Culicidae.

Identification and molecular characterization of Wanghe virus: A novel member of the Solinviviridae family from mosquitoes, China.

Arboviruses have always been a significant public health concern. Metagenomic surveillance has expanded the number of novel, often unclassified arboviruses, especially mosquito-borne and mosquito-specific viruses. This report presents the first description of a novel single-stranded RNA virus, Wanghe virus, identified from mosquitoes that were collected in Shandong Province in 2022. In this study, a total of 4,795 mosquitoes were collected and then divided into 105 pools according to location and species. QRT-PCR and nested PCR were performed to confirm the presence of Wanghe virus, and its genomic features and phylogenetic relationships were further analyzed. Our results revealed that Wanghe virus was detected in 9 out of the 105 mosquito pools, resulting in a minimum infection rate (MIR) of 0.19 % (9/4,795). One complete genome sequence and three viral partial sequences were obtained from the Wanghe virus-positive pools. Pairwise distance analysis indicated that these amplified sequences shared high nucleotide identity. Phylogenetic analysis demonstrated that Wanghe virus is most closely related to Guiyang Solinvi-like virus 3, which belongs to Solinviviridae. Further analyses indicated that Wanghe virus is a new, unclassified member of Solinviviridae.

Loop-Mediated Isothermal Amplification Assay to Detect Invasive Malaria Vector Anopheles stephensi Mosquitoes.

Spread of the Anopheles stephensi mosquito, an invasive malaria vector, threatens to put an additional 126 million persons per year in Africa at risk for malaria. To accelerate the early detection and rapid response to this mosquito species, confirming its presence and geographic extent is critical. However, existing molecular species assays require specialized laboratory equipment, interpretation, and sequencing confirmation. We developed and optimized a colorimetric rapid loop-mediated isothermal amplification assay for molecular An. stephensi species identification. The assay requires only a heat source and reagents and can be used with or without DNA extraction, resulting in positive color change in 30-35 minutes. We validated the assay against existing PCR techniques and found 100% specificity and analytical sensitivity down to 0.0003 nanograms of genomic DNA. The assay can successfully amplify single mosquito legs. Initial testing on samples from Marsabit, Kenya, illustrate its potential as an early vector detection and malaria mitigation tool.

Evaluation of a mass trapping strategy to prevent mosquito nuisance in campsites of southern France.

Mosquito traps, historically used for surveillance and research, have gained prominence as a tool for mosquito control, amidst concern over the environmental impact and increased resistance to insecticide-based methods. In this study, we tested the effectiveness of a mass trapping barrier design with two types of traps, Mosquito Magnet (MM) traps and BG-Protector (BGP) traps. This experiment was conducted in three coastal camping areas in southern France between summer and autumn 2022, where the presence of floodwater mosquito species with anthropophilic preferences like Aedes caspius represents a year-long nuisance. MM traps were set around the campsite as a barrier to interfere with mosquitoes from entering the campsites, whereas BGP traps were set within the campsites, with the aim of diverting mosquitoes away from humans at peak activity hours. Over 210,000 mosquitoes of 11 species from 4 genera were collected by both trap types across treatment campsites, with no significant differences in mosquito community samplings between BGP and MM traps. Barrier traps effectively targeted Ae. caspius, reducing total mosquito abundance in two of the three study sites by 34% and 55%. This study provides valuable insights into the efficacy and feasibility of using mass trapping barriers as a complementary control strategy for mosquito species in wetlands.

CRISPR-Cas9 and Cas12a target site richness reflects genomic diversity in natural populations of Anopheles gambiae and Aedes aegypti mosquitoes.

Due to limitations in conventional disease vector control strategies including the rise of insecticide resistance in natural populations of mosquitoes, genetic control strategies using CRISPR gene drive systems have been under serious consideration. The identification of CRISPR target sites in mosquito populations is a key aspect for developing efficient genetic vector control strategies. While genome-wide Cas9 target sites have been explored in mosquitoes, a precise evaluation of target sites focused on coding sequence (CDS) is lacking. Additionally, target site polymorphisms have not been characterized for other nucleases such as Cas12a, which require a different DNA recognition site (PAM) and would expand the accessibility of mosquito genomes for genetic engineering. We undertook a comprehensive analysis of potential target sites for both Cas9 and Cas12a nucleases within the genomes of natural populations of Anopheles gambiae and Aedes aegypti from multiple continents. We demonstrate that using two nucleases increases the number of targets per gene. Also, we identified differences in nucleotide diversity between North American and African Aedes populations, impacting the abundance of good target sites with a minimal degree of polymorphisms that can affect the binding of gRNA. Lastly, we screened for gRNAs targeting sex-determination genes that could be widely applicable for developing field genetic control strategies. Overall, this work highlights the utility of employing both Cas9 and Cas12a nucleases and underscores the importance of designing universal genetic strategies adaptable to diverse mosquito populations.