A metagenomic analysis of the phase 2 Anopheles gambiae 1000 genomes dataset reveals a wide diversity of cobionts associated with field collected mosquitoes.

The Anopheles gambiae 1000 Genomes (Ag1000G) Consortium previously utilized deep sequencing methods to catalogue genetic diversity across African An. gambiae populations. We analyzed the complete datasets of 1142 individually sequenced mosquitoes through Microsoft Premonition's Bayesian mixture model based (BMM) metagenomics pipeline. All specimens were confirmed as either An. gambiae sensu stricto (s.s.) or An. coluzzii with a high degree of confidence ( > 98% identity to reference). Homo sapiens DNA was identified in all specimens indicating contamination may have occurred either at the time of specimen collection, preparation and/or sequencing. We found evidence of vertebrate hosts in 162 specimens. 59 specimens contained validated Plasmodium falciparum reads. Human hepatitis B and primate erythroparvovirus-1 viral sequences were identified in fifteen and three mosquito specimens, respectively. 478 of the 1,142 specimens were found to contain bacterial reads and bacteriophage-related contigs were detected in 27 specimens. This analysis demonstrates the capacity of metagenomic approaches to elucidate important vector-host-pathogen interactions of epidemiological significance.

Physico-chemical characterization of Anopheles gambiae s.l. breeding sites and kdr mutations in urban areas of Cotonou and Natitingou, Benin.

BACKGROUND: This study aimed to investigate the relationship between the physicochemical characteristics of An. gambiae s.s. and An. coluzzii breeding sites, the susceptibility profiles to commonly used insecticides in public health, and the underlying insecticide resistance mechanisms. METHODS: Anopheles breeding sites surveys were conducted in Cotonou and Natitingou in September 2020, January and August 2021. Physicochemical properties and bacterial loads were determined in individual breeding sites. The WHO susceptibility assays were carried out using the female of the emerging adult mosquitoes. Anopheles species were identified through PCR techniques. Kdr L1014F/S, N1575Y and G119S mutations were investigated using TaqMan genotyping assays. RESULTS: Molecular analysis showed that all mosquitoes analyzed in Cotonou were Anopheles coluzzii, while those of Natitingou were Anopheles gambiae s.s. Fecal coliforms were identified as playing a role in this distribution through their significant influence on the presence of An. coluzzii larvae. WHO susceptibility assay indicated a high level of resistance to deltamethrin in the two cities. The resistance levels to deltamethrin were higher in Cotonou (X2 = 31.689; DF = 1; P < 0.0001). There was a suspected resistance to bendiocarb in Cotonou, whereas the mosquito population in Natitingou was resistant. The kdr L1014F mutation was highly observed in both mosquito populations (frequence: 86-91%), while the Ace-1 mutation was found in a small proportion of mosquitoes. In Cotonou, salinity was the only recorded physicochemical parameter that significantly correlated with the resistance of Anopheles mosquitoes to deltamethrin (P < 0.05). In Natitingou, significant correlations were observed between the allelic frequencies of the kdr L1014F mutation and pH, conductivity, and TDS. CONCLUSION: These results indicate a high level of pyrethroid resistance in the anopheles populations of both Cotonou and Natitingou. Moreover, this study report the involvement of abiotic factors influencing Anopheles susceptibility profile.

A DNA barcode reference library of Croatian mosquitoes (Diptera: Culicidae): implications for identification and delimitation of species, with notes on the distribution of potential vector species.

BACKGROUND: Mosquitoes pose a risk to human health worldwide, and correct species identification and detection of cryptic species are the most important keys for surveillance and control of mosquito vectors. In addition to traditional identification based on morphology, DNA barcoding has recently been widely used as a complementary tool for reliable identification of mosquito species. The main objective of this study was to create a reference DNA barcode library for the Croatian mosquito fauna, which should contribute to more accurate and faster identification of species, including cryptic species, and recognition of relevant vector species. METHODS: Sampling was carried out in three biogeographical regions of Croatia over six years (2017-2022). The mosquitoes were morphologically identified; molecular identification was based on the standard barcoding region of the mitochondrial COI gene and the nuclear ITS2 region, the latter to identify species within the Anopheles maculipennis complex. The BIN-RESL algorithm assigned the COI sequences to the corresponding BINs (Barcode Index Number clusters) in BOLD, i.e. to putative MOTUs (Molecular Operational Taxonomic Units). The bPTP and ASAP species delimitation methods were applied to the genus datasets in order to verify/confirm the assignment of specimens to specific MOTUs. RESULTS: A total of 405 mosquito specimens belonging to six genera and 30 morphospecies were collected and processed. Species delimitation methods assigned the samples to 31 (BIN-RESL), 30 (bPTP) and 28 (ASAP) MOTUs, with most delimited MOTUs matching the morphological identification. Some species of the genera Culex, Aedes and Anopheles were assigned to the same MOTUs, especially species that are difficult to distinguish morphologically and/or represent species complexes. In total, COI barcode sequences for 34 mosquito species and ITS2 sequences for three species of the genus Anopheles were added to the mosquito sequence database for Croatia, including one individual from the Intrudens Group, which represents a new record for the Croatian mosquito fauna. CONCLUSION: We present the results of the first comprehensive study combining morphological and molecular identification of most mosquito species present in Croatia, including several invasive and vector species. With the exception of some closely related species, this study confirmed that DNA barcoding based on COI provides a reliable basis for the identification of mosquito species in Croatia.

MGSurvE: A framework to optimize trap placement for genetic surveillance of mosquito populations.

Genetic surveillance of mosquito populations is becoming increasingly relevant as genetics-based mosquito control strategies advance from laboratory to field testing. Especially applicable are mosquito gene drive projects, the potential scale of which leads monitoring to be a significant cost driver. For these projects, monitoring will be required to detect unintended spread of gene drive mosquitoes beyond field sites, and the emergence of alternative alleles, such as drive-resistant alleles or non-functional effector genes, within intervention sites. This entails the need to distribute mosquito traps efficiently such that an allele of interest is detected as quickly as possible-ideally when remediation is still viable. Additionally, insecticide-based tools such as bednets are compromised by insecticide-resistance alleles for which there is also a need to detect as quickly as possible. To this end, we present MGSurvE (Mosquito Gene SurveillancE): a computational framework that optimizes trap placement for genetic surveillance of mosquito populations such that the time to detection of an allele of interest is minimized. A key strength of MGSurvE is that it allows important biological features of mosquitoes and the landscapes they inhabit to be accounted for, namely: i) resources required by mosquitoes (e.g., food sources and aquatic breeding sites) can be explicitly distributed through a landscape, ii) movement of mosquitoes may depend on their sex, the current state of their gonotrophic cycle (if female) and resource attractiveness, and iii) traps may differ in their attractiveness profile. Example MGSurvE analyses are presented to demonstrate optimal trap placement for: i) an Aedes aegypti population in a suburban landscape in Queensland, Australia, and ii) an Anopheles gambiae population on the island of São Tomé, São Tomé and Príncipe. Further documentation and use examples are provided in project's documentation. MGSurvE is intended as a resource for both field and computational researchers interested in mosquito gene surveillance.

Fine-scale adaptive divergence and population genetic structure of Aedes aegypti in Metropolitan Manila, Philippines.

BACKGROUND: The adaptive divergence of Aedes aegypti populations to heterogeneous environments can be a driving force behind the recent expansion of their habitat distribution and outbreaks of dengue disease in urbanized areas. In this study, we investigated the population genomics of Ae. aegypti at a regional scale in Metropolitan Manila, Philippines. METHODS: We used the Pool-Seq double digestion restriction-site association DNA sequencing (ddRAD-Seq) approach to generate a high number of single nucleotide polymorphisms (SNPs), with the aim to determine local adaptation and compare the population structure with 11 microsatellite markers. A total of 217 Ae. aegypti individuals from seven female and seven male populations collected from Metropolitan Manila were used in the assays. RESULTS: We detected 65,473 SNPs across the populations, of which 76 were non-neutral SNPs. Of these non-neutral SNPs, the multivariate regression test associated 50 with eight landscape variables (e.g. open space, forest, etc.) and 29 with five climate variables (e.g. air temperature, humidity, etc.) (P-value range 0.005-0.045) in female and male populations separately. Male and female populations exhibited contrasting spatial divergence, with males exhibiting greater divergence than females, most likely reflecting the different dispersal abilities of male and female mosquitoes. In the comparative analysis of the same Ae. aegypti individuals, the pairwise FST values of 11 microsatellite markers were lower than those of the neutral SNPs, indicating that the neutral SNPs generated via pool ddRAD-Seq were more sensitive in terms of detecting genetic differences between populations at fine-spatial scales. CONCLUSIONS: Overall, our study demonstrates the utility of pool ddRAD-Seq for examining genetic differences in Ae. aegypti populations in areas at fine-spatial scales that could inform vector control programs such as Wolbachia-infected mosquito mass-release programs. This in turn would provide information on mosquito population dispersal patterns and the potential barriers to mosquito movement within and around the release area. In addition, the potential of environmental adaptability observed in Ae. aegypti could help population control efforts.

Potential of MALDI-TOF MS biotyping to detect deltamethrin resistance in the dengue vector Aedes aegypti.

Insecticide resistance in mosquitoes is spreading worldwide and represents a growing threat to vector control. Insecticide resistance is caused by different mechanisms including higher metabolic detoxication, target-site modification, reduced penetration and behavioral changes that are not easily detectable with simple diagnostic methods. Indeed, most molecular resistance diagnostic tools are costly and labor intensive and then difficult to use for routine monitoring of insecticide resistance. The present study aims to determine whether mosquito susceptibility status against the pyrethroid insecticides (mostly used for mosquito control) could be established by the protein signatures of legs and/or thoraxes submitted to MALDI-TOF Mass Spectrometry (MS). The quality of MS spectra for both body parts was controlled to avoid any bias due to unconformity protein profiling. The comparison of MS profiles from three inbreeds Ae. aegypti lines from French Guiana (IRF, IR03, IR13), with distinct deltamethrin resistance genotype / phenotype and the susceptible reference laboratory line BORA (French Polynesia), showed different protein signatures. On both body parts, the analysis of whole protein profiles revealed a singularity of BORA line compared to the three inbreeding lines from French Guiana origin, suggesting that the first criteria of differentiation is the geographical origin and/or the breeding history rather than the insecticide susceptibility profile. However, a deeper analysis of the protein profiles allowed to identify 10 and 11 discriminating peaks from leg and thorax spectra, respectively. Among them, a specific peak around 4870 Da was detected in legs and thoraxes of pyrethroid resistant lines compared to the susceptible counterparts hence suggesting that MS profiling may be promising to rapidly distinguish resistant and susceptible phenotypes. Further work is needed to confirm the nature of this peak as a deltamethrin resistant marker and to validate the routine use of MS profiling to track insecticide resistance in Ae. aegypti field populations.

Can neonicotinoid and pyrrole insecticides manage malaria vector resistance in high pyrethroid resistance areas in Côte d'Ivoire?

BACKGROUND: Anopheles mosquito resistance to insecticide remains a serious threat to malaria vector control affecting several sub-Sahara African countries, including Côte d'Ivoire, where high pyrethroid, carbamate and organophosphate resistance have been reported. Since 2017, new insecticides, namely neonicotinoids (e.g.; clothianidin) and pyrroles (e.g.; chlorfenapyr) have been pre-qualified by the World Health Organization (WHO) for use in public health to manage insecticide resistance for disease vector control. METHODS: Clothianidin and chlorfenapyr were tested against the field-collected Anopheles gambiae populations from Gagnoa, Daloa and Abengourou using the WHO standard insecticide susceptibility biossays. Anopheles gambiae larvae were collected from several larval habitats, pooled and reared to adulthood in each site in July 2020. Non-blood-fed adult female mosquitoes aged 2 to 5 days were exposed to diagnostic concentration deltamethrin, permethrin, alpha-cypermethrin, bendiocarb, and pirimiphos-methyl. Clothianidin 2% treated papers were locally made and tested using WHO tube bioassay while chlorfenapyr (100 µg/bottle) was evaluated using WHO bottle assays. Furthermore, subsamples of exposed mosquitoes were identified to species and genotyped for insecticide resistance markers including the knock-down resistance (kdr) west and east, and acetylcholinesterase (Ace-1) using molecular techniques. RESULTS: High pyrethroid resistance was recorded with diagnostic dose in Abengourou (1.1 to 3.4% mortality), in Daloa (15.5 to 33.8%) and in Gagnoa (10.3 to 41.6%). With bendiocarb, mortality rates ranged from 49.5 to 62.3%. Complete mortality (100% mortality) was recorded with clothianidin in Gagnoa, 94.9% in Daloa and 96.6% in Abengourou, while susceptibility (mortality > 98%) to chlorfenapyr 100 µg/bottle was recorded at all sites and to pirimiphos-methyl in Gagnoa and Abengourou. Kdr-west mutation was present at high frequency (0.58 to 0.73) in the three sites and Kdr-east mutation frequency was recorded at a very low frequency of 0.02 in both Abengourou and Daloa samples and absent in Gagnoa. The Ace-1 mutation was present at frequencies between 0.19 and 0.29 in these areas. Anopheles coluzzii represented 100% of mosquitoes collected in Daloa and Gagnoa, and 72% in Abengourou. CONCLUSIONS: This study showed that clothianidin and chlorfenapyr insecticides induce high mortality in the natural and pyrethroid-resistant An. gambiae populations in Côte d'Ivoire. These results could support a resistance management plan by proposing an insecticide rotation strategy for vector control interventions.

The monsoon-associated equine South African pointy mosquito 'Aedes caballus'; the first comprehensive record from southeastern Iran with a description of ecological, morphological, and molecular aspects.

The equine South African pointy vector mosquito, Aedes caballus, poses a significant threat to human health due to its capacity for transmitting arboviruses. Despite favorable climate for its existence in southeast Iran, previous records of this species in the area have indicated very low abundance. This comprehensive field and laboratory study aimed to assess its current adult population status in this region, utilizing a combination of ecological, morphological and molecular techniques. Four distinct types of traps were strategically placed in three fixed and two variable mosquito sampling sites in the southern strip of Sistan and Baluchistan Province. Subsequently, DNA was extracted from trapped mosquitoes and subjected to PCR amplification using the molecular markers COI, ITS2, and ANT. In total, 1734 adult Ae. caballus specimens were collected from rural areas, with the majority being captured by CO2-baited bednet traps. A notable increase in the abundance of this species was observed following rainfall in February. The genetic analysis revealed multiple haplotypes based on COI and ITS2 sequences, with COI gene divergence at 0.89%, and ITS2 sequence divergence at 1.6%. This suggests that previous challenges in morphological identification may have led to misidentifications, with many adults previously classified as Ae. vexans potentially being Ae. caballus. The findings of this study hold significant implications for public health authorities, providing valuable insights for integrated and targeted vector control and disease management efforts.

Effects of Arboviral Infections on Transposable Element Transcript Levels in Aedes aegypti.

Transposable elements are mobile repeated sequences found in all genomes. Transposable elements are controlled by RNA interference pathways in most organisms, and this control involves the PIWI-interacting RNA pathway and the small interfering RNA pathway, which is also known to be the first line of antiviral defense in invertebrates. Using Drosophila, we recently showed that viral infections result in the modulation of transposable element transcript levels through modulation of the small RNA repertoire. The Aedes aegypti mosquito is of particular interest because almost half of its genome is made of transposable elements, and it is described as a major vector of viruses (such as the dengue [DENV], Zika [ZIKV], and chikungunya [CHIKV] arboviruses). Moreover, Aedes mosquitoes are unique among insects in that the PIWI-interacting RNA pathway is also involved in the somatic antiviral response, in addition to the transposable element control and PIWI-interacting RNA pathway genes expanded in the mosquito genome. For these reasons, we studied the impacts of viral infections on transposable element transcript levels in A. aegypti samples. We retrieved public datasets corresponding to RNA-seq data obtained from viral infections by DENV, ZIKV, and CHIKV in various tissues. We found that transposable element transcripts are moderately modulated following viral infection and that the direction of the modulation varies greatly across tissues and viruses. These results highlight the need for an in-depth investigation of the tightly intertwined interactions between transposable elements and viruses.

Genetic variability of Aedes aegypti (Diptera: Culicidae) in El Salvador and Honduras: presence of a widespread haplotype and implications for mosquito control.

BACKGROUND: This study examined population genetics of Aedes aegypti in El Salvador and Honduras, two adjacent countries in Central America. Aedes aegypti is associated with yellow fever, dengue, chikungunya, and Zika. Each year, thousands of cases of dengue are typically reported in El Salvador and Honduras. METHODS: In El Salvador, collections were obtained from five Departments. In Honduras, samples were obtained from six municipalities in four Departments. Mitochondrial DNA cytochrome oxidase I (COI) was sequenced, and consensus sequences were combined with available sequences from El Salvador to determine haplotype number, haplotype diversity, nucleotide diversity, and Tajima's D. A haplotype network was produced to examine the relationship between genotypes. RESULTS: In El Salvador, there were 17 haplotypes, while in Honduras there were 4 haplotypes. In both El Salvador and Honduras, Haplotype 1 is most abundant and widespread. In El Salvador, haplotype H2 was also widespread in 10 of 11 sampled municipalities, but it was not present in Honduras. The capital of El Salvador (San Salvador) and the eastern region of ES had the highest haplotype diversity of regions sampled. CONCLUSIONS: Haplotype 1 and H2 each belong to different phylogenetic lineages of Ae. aegypti. The most geographically widespread haplotype (H1) may have been present the longest and could be a remnant from previous eradication programs. These data may contribute to future control programs for Ae. aegypti in the two countries.

Transgene removal using an in cis programmed homing endonuclease via single-strand annealing in the mosquito Aedes aegypti.

While gene drive strategies have been proposed to aid in the control of mosquito-borne diseases, additional genome engineering technologies may be required to establish a defined end-of-product-life timeline. We previously demonstrated that single-strand annealing (SSA) was sufficient to program the scarless elimination of a transgene while restoring a disrupted gene in the disease vector mosquito Aedes aegypti. Here, we extend these findings by establishing that complete transgene removal (four gene cassettes comprising ~8-kb) can be programmed in cis. Reducing the length of the direct repeat from 700-bp to 200-bp reduces, but does not eliminate, SSA activity. In contrast, increasing direct repeat length to 1.5-kb does not increase SSA rates, suggesting diminishing returns above a certain threshold size. Finally, we show that while the homing endonuclease Y2-I-AniI triggered both SSA and NHEJ at significantly higher rates than I-SceI at one genomic locus (P5-EGFP), repair events are heavily skewed towards NHEJ at another locus (kmo), suggesting the nuclease used and the genomic region targeted have a substantial influence on repair outcomes. Taken together, this work establishes the feasibility of engineering temporary transgenes in disease vector mosquitoes, while providing critical details concerning important operational parameters.

First report of V1016I, F1534C and V410L kdr mutations associated with pyrethroid resistance in Aedes aegypti populations from Niamey, Niger.

BACKGROUND: Ae. aegypti is the vector of important µ arboviruses, including dengue, Zika, chikungunya and yellow fever. Despite not being specifically targeted by insecticide-based control programs in West Africa, resistance to insecticides in Ae. aegypti has been reported in countries within this region. In this study, we investigated the status and mechanisms of Ae. aegypti resistance in Niamey, the capital of Niger. This research aims to provide baseline data necessary for arbovirus outbreak prevention and preparedness in the country. METHODS: Ovitraps were used to collect Ae. aegypti eggs, which were subsequently hatched in the insectary for bioassay tests. The hatched larvae were then reared to 3-5-day-old adults for WHO tube and CDC bottle bioassays, including synergist tests. The kdr mutations F1534C, V1016I, and V410L were genotyped using allele-specific PCR and TaqMan qPCR methods. RESULTS: Ae. aegypti from Niamey exhibited moderate resistance to pyrethroids but susceptibility to organophosphates and carbamates. The kdr mutations, F1534C, V1016I and V410L were detected with the resistant tri-locus haplotype 1534C+1016L+410L associated with both permethrin and deltamethrin resistance. Whereas the homozygote tri-locus resistant genotype 1534CC+1016LL+410LL was linked only to permethrin resistance. The involvement of oxidase and esterase enzymes in resistance mechanisms was suggested by partial restoration of mosquitoes' susceptibility to pyrethroids in synergist bioassays. CONCLUSION: This study is the first report of Ae. aegypti resistance to pyrethroid insecticides in Niamey. The resistance is underpinned by target site mutations and potentially involves metabolic enzymes. The observed resistance to pyrethroids coupled with susceptibility to other insecticides, provides data to support evidence-based decision-making for Ae. aegypti control in Niger.

Resistance to pyrethroids and the relationship between adult resistance and knockdown resistance (kdr) mutations in Aedes albopictus in dengue surveillance areas of Guizhou Province, China.

The Ae. albopictus mosquito has gained global attention due to its ability to transmit viruses, including the dengue and zika. Mosquito control is the only effective way to manage dengue fever, as no effective treatments or vaccines are available. Insecticides are highly effective in controlling mosquito densities, which reduces the chances of virus transmission. However, Ae. albopictus has developed resistance to pyrethroids in several provinces in China. Pyrethroids target the voltage-gated sodium channel gene (VGSC), and mutations in this gene may result in knockdown resistance (kdr). Correlation studies between resistance and mutations can assist viruses in managing Ae. albopictus, which has not been studied in Guizhou province. Nine field populations of Ae. albopictus at the larval stage were collected from Guizhou Province in 2022 and reared to F1 to F2 generations. Resistance bioassays were conducted against permethrin, beta-cypermethrin, and deltamethrin for both larvae and adults of Ae. albopictus. Kdr mutations were characterized by PCR and sequencing. Additionally, the correlation between the kdr allele and pyrethroid resistance was analyzed. All nine populations of Ae. albopictus larvae and adults were found to be resistant to three pyrethroid insecticides. One kdr mutant allele at codon 1016, one at 1532 and three at 1534 were identified with frequencies of 13.86% (V1016G), 0.53% (I1532T), 58.02% (F1534S), 11.69% (F1534C), 0.06% (F1534L) and 0.99% (F1534P), respectively. Both V1016G and F1534S mutation mosquitoes were found in all populations. The kdr mutation F1534S was positively correlated with three pyrethroid resistance phenotypes (OR > 1, P < 0.05), V1016G with deltamethrin and beta-cypermethrin resistance (OR > 1, P < 0.05) and F1534C only with beta-cypermethrin resistance (OR > 1, P < 0.05). Current susceptibility status of wild populations of Ae. albopictus to insecticides and a higher frequency of kdr mutations from dengue-monitored areas in Guizhou Province are reported in this paper. Outcomes of this study can serve as data support for further research and development of effective insecticidal interventions against Ae. albopictus populations in Guizhou Province.

Considerations for first field trials of low-threshold gene drive for malaria vector control.

Sustainable reductions in African malaria transmission require innovative tools for mosquito control. One proposal involves the use of low-threshold gene drive in Anopheles vector species, where a 'causal pathway' would be initiated by (i) the release of a gene drive system in target mosquito vector species, leading to (ii) its transmission to subsequent generations, (iii) its increase in frequency and spread in target mosquito populations, (iv) its simultaneous propagation of a linked genetic trait aimed at reducing vectorial capacity for Plasmodium, and (v) reduced vectorial capacity for parasites in target mosquito populations as the gene drive system reaches fixation in target mosquito populations, causing (vi) decreased malaria incidence and prevalence. Here the scope, objectives, trial design elements, and approaches to monitoring for initial field releases of such gene dive systems are considered, informed by the successful implementation of field trials of biological control agents, as well as other vector control tools, including insecticides, Wolbachia, larvicides, and attractive-toxic sugar bait systems. Specific research questions to be addressed in initial gene drive field trials are identified, and adaptive trial design is explored as a potentially constructive and flexible approach to facilitate testing of the causal pathway. A fundamental question for decision-makers for the first field trials will be whether there should be a selective focus on earlier points of the pathway, such as genetic efficacy via measurement of the increase in frequency and spread of the gene drive system in target populations, or on wider interrogation of the entire pathway including entomological and epidemiological efficacy. How and when epidemiological efficacy will eventually be assessed will be an essential consideration before decisions on any field trial protocols are finalized and implemented, regardless of whether initial field trials focus exclusively on the measurement of genetic efficacy, or on broader aspects of the causal pathway. Statistical and modelling tools are currently under active development and will inform such decisions on initial trial design, locations, and endpoints. Collectively, the considerations here advance the realization of developer ambitions for the first field trials of low-threshold gene drive for malaria vector control within the next 5 years.

Genetic markers associated with the widespread insecticide resistance in malaria vector Anopheles funestus populations across Tanzania.

BACKGROUND: Anopheles funestus is a leading vector of malaria in most parts of East and Southern Africa, yet its ecology and responses to vector control remain poorly understood compared with other vectors such as Anopheles gambiae and Anopheles arabiensis. This study presents the first large-scale survey of the genetic and phenotypic expression of insecticide resistance in An. funestus populations in Tanzania. METHODS: We performed insecticide susceptibility bioassays on An. funestus mosquitoes in nine regions with moderate-to-high malaria prevalence in Tanzania, followed by genotyping for resistance-associated mutations (CYP6P9a, CYP6P9b, L119F-GSTe2) and structural variants (SV4.3 kb, SV6.5 kb). Generalized linear models were used to assess relationships between genetic markers and phenotypic resistance. An interactive R Shiny tool was created to visualize the data and support evidence-based interventions. RESULTS: Pyrethroid resistance was universal but reversible by piperonyl-butoxide (PBO). However, carbamate resistance was observed in only five of the nine districts, and dichloro-diphenyl-trichloroethane (DDT) resistance was found only in the Kilombero valley, south-eastern Tanzania. Conversely, there was universal susceptibility to the organophosphate pirimiphos-methyl in all sites. Genetic markers of resistance had distinct geographical patterns, with CYP6P9a-R and CYP6P9b-R alleles, and the SV6.5 kb structural variant absent or undetectable in the north-west but prevalent in all other sites, while SV4.3 kb was prevalent in the north-western and western regions but absent elsewhere. Emergent L119F-GSTe2, associated with deltamethrin resistance, was detected in heterozygous form in districts bordering Mozambique, Malawi and the Democratic Republic of Congo. The resistance landscape was most complex in western Tanzania, in Tanganyika district, where all five genetic markers were detected. There was a notable south-to-north spread of resistance genes, especially CYP6P9a-R, though this appears to be interrupted, possibly by the Rift Valley. CONCLUSIONS: This study underscores the need to expand resistance monitoring to include An. funestus alongside other vector species, and to screen for both the genetic and phenotypic signatures of resistance. The findings can be visualized online via an interactive user interface and could inform data-driven decision-making for resistance management and vector control. Since this was the first large-scale survey of resistance in Tanzania's An. funestus, we recommend regular updates with greater geographical and temporal coverage.

Spatio-temporal characterization of phenotypic resistance in malaria vector species.

BACKGROUND: Malaria, a deadly disease caused by Plasmodium protozoa parasite and transmitted through bites of infected female Anopheles mosquitoes, remains a significant public health challenge in sub-Saharan Africa. Efforts to eliminate malaria have increasingly focused on vector control using insecticides. However, the emergence of insecticide resistance (IR) in malaria vectors pose a formidable obstacle, and the current IR mapping models remain static, relying on fixed coefficients. This study introduces a dynamic spatio-temporal approach to characterize phenotypic resistance in Anopheles gambiae complex and Anopheles arabiensis. We developed a cellular automata (CA) model and applied it to data collected from Ethiopia, Nigeria, Cameroon, Chad, and Burkina Faso. The data encompasses georeferenced records detailing IR levels in mosquito vector populations across various classes of insecticides. In characterizing the dynamic patterns of confirmed resistance, we identified key driving factors through correlation analysis, chi-square tests, and extensive literature review. RESULTS: The CA model demonstrated robustness in capturing the spatio-temporal dynamics of confirmed IR states in the vector populations. In our model, the key driving factors included insecticide usage, agricultural activities, human population density, Land Use and Land Cover (LULC) characteristics, and environmental variables. CONCLUSIONS: The CA model developed offers a robust tool for countries that have limited data on confirmed IR in malaria vectors. The embrace of a dynamical modeling approach and accounting for evolving conditions and influences, contribute to deeper understanding of IR dynamics, and can inform effective strategies for malaria vector control, and prevention in regions facing this critical health challenge.

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.

Integrated de novo transcriptome of Culex pipiens mosquito larvae as a resource for genetic control strategies.

We present a de novo transcriptome of the mosquito vector Culex pipiens, assembled by sequences of susceptible and insecticide resistant larvae. The high quality of the assembly was confirmed by TransRate and BUSCO. A mapping percentage until 94.8% was obtained by aligning contigs to Nr, SwissProt, and TrEMBL, with 27,281 sequences that simultaneously mapped on the three databases. A total of 14,966 ORFs were also functionally annotated by using the eggNOG database. Among them, we identified ORF sequences of the main gene families involved in insecticide resistance. Therefore, this resource stands as a valuable reference for further studies of differential gene expression as well as to identify genes of interest for genetic-based control tools.

Insecticide susceptibility status of Anopheles albimanus populations in historical malaria foci in Quintana Roo, Mexico.

BACKGROUND: Mexico has experienced a significant reduction in malaria cases over the past two decades. Certification of localities as malaria-free areas (MFAs) has been proposed as a steppingstone before elimination is achieved throughout the country. The Mexican state of Quintana Roo is a candidate for MFA certification. Monitoring the status of insecticide susceptibility of major vectors is crucial for MFA certification. This study describes the susceptibility status of Anopheles albimanus, main malaria vector, from historically important malaria foci in Quintana Roo, using both phenotypic and genotypic approaches. METHODS: Adult mosquito collections were carried out at three localities: Palmar (Municipality of Othon P. Blanco), Buenavista (Bacalar) and Puerto Morelos (Puerto Morelos). Outdoor human-landing catches were performed by pairs of trained staff from 18:00 to 22:00 during 3-night periods at each locality during the rainy season of 2022. Wild-caught female mosquitoes were exposed to diagnostic doses of deltamethrin, permethrin, malathion, pirimiphos-methyl or bendiocarb using CDC bottle bioassays. Mortality was registered at the diagnostic time and recovery was assessed 24 h after exposure. Molecular analyses targeting the Voltage-Gated Sodium Channel (vgsc) gene and acetylcholinesterase (ace-1) gene were used to screen for target site polymorphisms. An SNP analysis was carried out to identify mutations at position 995 in the vgsc gene and at position 280 in the ace-1 gene. RESULTS: A total of 2828 anophelines were collected. The main species identified were Anopheles albimanus (82%) and Anopheles vestitipennis (16%). Mortalities in the CDC bottle bioassay ranged from 99% to 100% for all the insecticides and mosquito species. Sequence analysis was performed on 35 An. albimanus across the three localities; of those, 25 were analysed for vgsc and 10 for ace-1 mutations. All individuals showed wild type alleles. CONCLUSION: The results demonstrated that An. albimanus populations from historical malaria foci in Quintana Roo are susceptible to the main insecticides used by the Ministry of Health.

Island mosquitoes of Thailand: an update on species diversity and DNA barcoding.

Mosquitoes (Diptera: Culicidae) are among the most medically significant insects, with several species acting as vectors for human pathogens. Although there are frequent reports of mosquito-borne diseases in the border island areas of Thailand, comprehensive data on the diversity and DNA barcoding of these mosquito species remain limited. This study investigated mosquito diversity in two main archipelagos in Thailand-the Trat archipelago (comprising Chang Island and Kood Island) and the Ranong archipelago (comprising Chang Island and Phayam Island)-and generated DNA barcode data from the mosquitoes found there. The survey across these islands discovered a total of 41 species, highlighting the presence of several species known to be vectors for human diseases. Thirty-seven mosquito species from the island areas were documented to provide reference DNA barcode sequences for mosquitoes in Thailand's island regions. Two species, Aedes fumidus and Finlaya flavipennis, have been added as new COI sequence records in the database. DNA barcoding was highly effective in classifying almost all species by identifying barcoding gaps, except for Anopheles baimaii and Anopheles dirus, which could not be distinguished. Additionally, the study noted that geographical variations might influence certain mosquito species, such as Anopheles barbirostris A3 and Mansonia dives, causing them to be split into two distinct subgroups. The findings of this study are crucial, as they aid in classifying mosquito species using molecular techniques and expand our knowledge of disease vectors in these biodiverse regions.