In Madagascar, the health burdens caused by West Nile virus (WNV) and Rift Valley Fever virus (RVFV) vary across regions, likely resulting from the variations of their vector capacity. This study aimed to assess the degree of vector-hots contact and to develop statistical models to identify factors influencing host preferences. Bimonthly mosquito collections were performed in 25 sites of Madagascar from February to October 2019. Engorged mosquitoes were collected from indoors and outdoors resting locations within each site. Blood meal identification was performed using host-specific PCRs. Generalised linear models were developed to explain host preferences of the two most abundant species. The origin of the blood meal of 1,177 engorged females belonging to ten species was analysed. Human and cattle were the dominant vertebrate hosts identified (respectively 44.86% and 44.52%), and pigs were rarely detected (< 1%). Models were developed for Culex antennatus and Culex quinquefasciatus. For these two species, host preference was driven by climatic and environmental factors, type of habitat, indoor/outdoor location, and number of available vertebrate hosts. This study provides an essential step towards an evidence-based determination of arbovirus transmission risk and suggests to explore the use of other tools to supplement indoor vector control in Madagascar.

Background The increasing insecticide resistance of malaria vectors is an urgent concern for disease control and novel vector control strategies are needed. Wolbachia are endosymbiotic bacteria that can invade mosquito populations and reduce transmission of human pathogens. Wolbachia strains in wild Anopheles (An.) malaria vectors are rare, with only two known genuine symbioses; An. moucheti with wAnM and An. demeilloni with wAnD. In this study, we set out to determine if there was a correlation between wAnD in different An. demeilloni mosquito body parts, infective stage Plasmodium (Pl.) falciparum malaria sporozoites in cephalothoraxes and the mosquito microbiome. Methods We undertook a combination of quantitative PCR, 16S rRNA amplicon sequencing and sanger sequencing of the Wolbachia surface protein (wsp) gene after isolating An. demeilloni female body parts from wild caught individuals collected in 2021 and 2024 from the Sud Kivu region of Democratic Republic of Congo. Results Wolbachia prevalence rates were significantly higher in abdomens compared to cephalothoraxes and density was also significantly higher in abdomens (P<0.0001). Overall sporozoite prevalence was 1.3% (9/704) which was not significantly different between Wolbachia-positive and Wolbachia-negative cephalothoraxes (P=0.3630) despite Pl. falciparum only detected in Wolbachia-negative cephalothoraxes. However, Wolbachia-positive abdomens were associated with a lower sporozoites rate compared to Wolbachia-negative abdomens (P=0.0329). 16S rRNA amplicon sequencing revealed no significant difference in alpha/beta diversities between abdomens and cephalothoraxes but the cephalothorax microbiome composition between Wolbachia-positive and Wolbachia-negative was significantly different (P<0.05). Conclusions Our findings indicate a significant effect of the wAnD strain on the cephalothorax microbiome and potentially the ability of sporozoites to reach Salivary glands in mosquitoes with Wolbachia-infected abdomens. Further studies are needed to determine the mechanisms in which the wAnD strain interacts with Plasmodium sporozoites in An. demeilloni and if this strain could be used for malaria biocontrol through transinfection of major malaria vectors.

Over-reliance on pyrethroid insecticides in insecticide-treated nets (ITNs) has imposed significant selection pressure for the evolution of insecticide resistance among major malaria vector species. In the Democratic Republic of Congo (DRC), the country with the second highest malaria burden globally, pyrethroid resistance is pervasive, but there is a paucity of information regarding the molecular mechanisms driving resistance. A clear understanding of the specificity of resistance mechanisms to individual insecticides and the likelihood of selecting for cross-resistance mechanisms is crucial for the development of new vector control tools. Anopheles gambiae s.l. populations from eight study sites across the DRC were phenotyped for resistance to alpha-cypermethrin, deltamethrin and permethrin, with and without pre-exposure to the synergist piperonyl butoxide (PBO), followed by multiplex amplicon sequencing. Phenotypic pyrethroid resistance and loss of PBO synergy was confirmed in all sites across the DRC. In An. gambiae s.s. four non-synonymous SNPs which have been previously associated with insecticide resistance were detected: gste2-L119V, vgsc-L995F, vgsc-L995S and rdl-A296G, while three were novel: gste2-T154S, ace1-N246T and ace1-P265L. Nationwide geographical trends in insecticide resistance mutation distribution, prevalence and selection were evident. In the West, near fixation of vgsc-L995F and almost complete absence of vgsc-L995S was observed, alongside low-moderate frequencies of rdl-A296G and gste2-L119V. Further East, the converse was apparent. Gste2-L119V was significantly associated with resistance to deltamethrin following PBO-pre-exposure, warranting functional validation to determine its putative role in reduced PBO synergy. Furthermore, gste2-T154S was implicated in deltamethrin and permethrin resistance but susceptibility to alpha-cypermethrin after PBO pre-exposure. Study findings comprise the most comprehensive overview of the prevalence of genetic markers of Anopheles insecticide resistance across the DRC and provide an important baseline for improved malaria vector control and the design of proactive insecticide resistance management strategies. Given the significant scale up in PBO-ITNs, with more than 58% of all ITNs delivered to sub-Saharan Africa in 2023 containing PBO, there is an urgent need to identify novel molecular markers to monitor changes in PBO synergy, which may be predicative of loss of intervention operational efficacy.

Malaria, a mosquito-borne disease predominantly affecting sub-Saharan Africa, remains a significant global health challenge with 263 million cases in 2023. Although progress in vaccine development has recently occurred, insecticide resistance has reduced the effectiveness of the main stay method of traditional vector control and novel innovative vector control strategies against Anopheles mosquitoes is crucial to combat the spread of malaria. Wolbachia is an endosymbiotic bacterium that can invade mosquito populations and inhibit human pathogens. Historically Wolbachia was thought to not occur naturally within wild Anopheles An .) populations, but recent evidence has confirmed the presence of at least two strains in genuine symbiosis. Detection of Wolbachia strains has pre-dominantly relied on PCR amplification of Wolbachia genes and/or sanger and illumina sequencing. In this study, we assess the use of Oxford Nanopore Technology (ONT) for detecting bacteria within wild Anopheles mosquitoes and determine the ability to detect the endosymbiotc bacterium Wolbachia . We assessed Wolbachia detection from library preparations using the ONT Field sequencing kit for MinION sequencing on the Mk1C and found a pool size of 10 mosquitoes is required preventing individual mosquito analysis. There were roughly 13 times more Wolbachia reads in pooled Anopheles demeilloni compared to An. gambiae . Utilizing the more economical Flongle flow cells and Mk1B device, we further evaluated the effectiveness of two library preparation methods that are compatible: Rapid PCR Barcoding and 16S Barcoding kits. Our findings indicate that the 16S Barcoding Kit in conjunction with the Flongle flowcell effectively identified Wolbachia in both Anopheles demeilloni and Anopheles moucheti but we found no evidence of Wolbachia in Anopheles gambiae . Our study demonstrates the feasibility of developing ONT for field-based sequencing in malaria-endemic regions, providing a potentially lower cost and ‘field friendly’ method for monitoring mosquito populations and associated endosymbionts. ![Figure][1] ### Competing Interest Statement The authors have declared no competing interest. Bill & Melinda Gates Foundation, https://ror.org/0456r8d26, INV-048598 Biotechnology and Biological Sciences Research Council, UKRI543:2023BBSRC-NSF/BIO, BB/V011278/1, BB/X018024/1, BB/W018446/1, BB/V011278/2 African research excellence fund, AREF-312-AWAN-F-C0906, AREF-312-AWAN-S-C1027 Wellcome Trust, https://ror.org/029chgv08, 101285/Z/13/Z NIHR [1]: pending:yes

Since the detection of the Asian mosquito Anopheles stephensi in Dijbouti in 2012, it has spread throughout the Horn of Africa. This invasive vector continues to expand across the continent and is a significant threat to malaria control programs. Vector control methods, including insecticide-treated nets and indoor residual spraying, have substantially reduced the malaria burden. However, the increasing prevalence of mosquitoes resistant to insecticides, including An. stephensi populations, undermines ongoing malaria elimination efforts. Understanding population structure, gene flow between populations, and the distribution of insecticide resistance mutations is essential for guiding effective malaria control strategies. Here, we generated whole genome sequencing data for An. stephensi sourced from Awash Sebat Kilo, Ethiopia (n = 27) and compared with South Asian populations (n = 45; India and Pakistan) to assess genomic diversity, population structure, and uncovering insecticide resistance mutations. Population structure analysis using genome-wide single nucleotide polymorphisms (n = 15,533,476) revealed Ethiopian isolates clustering as a distinct ancestral group, separate from South Asian isolates. Three insecticide resistance-associated SNPs (gaba gene: A296S and V327I; vgsc L1014F) were detected. Evidence of ongoing selection was found in several loci, including genes previously associated with neonicotinoids, ivermectin, DDT, and pyrethroid resistance. This study represents the first whole genome population genetics study of invasive An. stephensi, revealing genomic differences from South Asian populations, which can be used for future assessments of vector population dispersal and detection of insecticide resistance mechanisms.

Hothouse alien mollusc species, defined as those with breeding populations only able to live in heated environments, are under-recorded in Britain and Ireland. A project extending over 10 years visited 56 heated glasshouses and identified 15 such species, while another two are known to be present in British hothouses. One species, Liardetia samoensis, is new to the British list. Another 12 species which have previously been recorded as hothouse aliens are no longer extant in Britain or Ireland. Consideration is given to whether these aliens could lie in the open in our climate, and the consequences if they were able to do so. An Appendix lists all records of hothouse alien molluscs found in literature and on databases and will be a useful tool for future research.

Abstract Robust diagnostic tools and surveillance are crucial for malaria control and elimination efforts. Malaria caused by neglected Plasmodium parasites is often underestimated due to the lack of rapid diagnostic tools that can accurately detect these species. While nucleic-acid amplification technologies stand out as the most sensitive methods for detecting and confirming Plasmodium species, their implementation in resource-constrained settings poses significant challenges. Here, we present a Pan Plasmodium recombinase polymerase amplification lateral flow (RPA–LF) assay, capable of detecting all six human infecting Plasmodium species in low resource settings. The Pan Plasmodium RPA-LF assay successfully detected low density clinical infections with a preliminary limit of detection between 10–100 fg/µl for P. falciparum. When combined with crude nucleic acid extraction, the assay can serve as a point-of-need tool for molecular xenomonitoring. This utility was demonstrated by screening laboratory-reared Anopheles stephensi mosquitoes fed with Plasmodium-infected blood, as well as field samples of An. funestus s.l. and An. gambiae s.l. collected from central Africa. Overall, our proof-of-concept Pan Plasmodium diagnostic tool has the potential to be applied for clinical and xenomonitoring field surveillance, and after further evaluation, could become an essential tool to assist malaria control and elimination.

Background Culex (Cx.) tritaeniorhynchus is an invasive mosquito species with an extensive and expanding inter-continental distribution, currently reported across Asia, Africa, the Middle East, Europe and now Australia. It is an important vector of medical and veterinary pathogens which cause significant morbidity and mortality in human and animal populations. Across regions endemic for Japanese encephalitis virus (JEV), Cx. tritaeniorhynchus is considered the major vector and has also been shown to contribute to the transmission of several other zoonotic arboviruses including Rift Valley fever virus (RVFV) and West Nile virus (WNV). Methods In this study, we used laboratory vector competence experiments to determine if Cx. tritaeniorhynchus from a Southern European population were competent JEV vectors. We also obtained samples from multiple geographically dispersed Cx. tritaeniorhynchus populations from countries within Europe, Africa, Eurasia and Asia to perform phylogenetic analysis to measure the level of mitochondrial divergence using the cytochrome oxidase subunit 1 (CO1) gene. We also undertook bacterial 16S rRNA gene amplicon sequencing to determine microbial diversity and used multi-locus sequence typing (MLST) to determine any evidence for the presence of strains of the naturally occurring endosymbiotic bacterium Wolbachia. Results Cx. tritaeniorhynchus from a Greek population were shown be be competent vectors of JEV with high levels of virus present in saliva. We found a signficant level of mitochondrial genetic diversity using the mosquito CO1 gene between geographically dispersed populations. Furthermore, we report diverse microbiomes identified by 16S rRNA gene amplicon sequencing within and between geographical populations. Evidence for the detection of the endosymbiotic bacteria Wolbachia was confirmed using Wolbachia-specific PCR and MLST. Conclusions This study enhances our understanding of the diversity of Cx. tritaeniorhynchus and the associated microbiome across its inter-continental range and highlights the need for greater surveillance of this invasive vector species in Europe.

Wolbachia is an endosymbiotic bacterium that naturally infects many insect species, including mosquitoes that transmit human diseases. Wolbachia strains have been shown to inhibit the transmission of both arboviruses and malaria Plasmodium parasites. The existence of natural strains in wild Anopheles (An.) mosquitoes, the vectors of malaria parasites, in an endosymbiotic relationship is still to be fully determined. Although Wolbachia has been reported to be present in wild populations of the An. gambiae complex, the primary vectors of malaria in Sub-Saharan Africa, Wolbachia DNA sequence density and infection frequencies are low. As most studies have used highly sensitive nested PCR as the only detection method, more robust evidence is required to determine whether Wolbachia strains are established as endosymbionts in Anopheles species. Techniques such as fluorescent in situ hybridization, microbiome sequencing, and Wolbachia whole genome sequencing have provided concrete evidence for genuine Wolbachia strains in two mosquito species: An. moucheti and An. demeilloni. In this chapter, the current methodology used to determine if resident strains exist in Anopheles mosquitoes will be reviewed, including both PCR- and non-PCR-based protocols.

Lymphatic filariasis (LF) is a leading cause of disability due to infectious disease worldwide. The Recife Metropolitan Region (RMR) is the only remaining focus of LF in Brazil, where the parasite Wuchereria bancrofti is transmitted solely by the mosquito Culex quinquefasciatus . This study reports the results of transmission assessment surveys and molecular xenomonitoring in the city of Olinda, RMR, after nearly 15 years (2015-2016) of interventions for LF elimination. Participants were screened for W. bancrofti antigen via immunochromatographic card tests (ICT) in: 1) door-todoor surveys conducted for all children aged 5-7 years from 4 out of 17 intervention areas treated with at least five annual doses of mass drug administration (MDA), and 2) a two -stage cluster sampling survey of residents aged 5 years and older in non-MDA areas. Mosquitoes were collected via handheld aspirators in four MDA areas, differentiated by species, sex, and physiological status, pooled into groups of up to 10 blood -fed, semigravid, and gravid mosquitoes, and screened for W. bancrofti infection by real-time quantitative polymerase chain reaction (RT-qPCR). All 1,170 children from MDA areas and the entire population sample of 990 residents in non-MDA areas were ICT negative. In MDA areas, a total of 3,152 female C X . quinquefasciatus mosquitoes in 277 households (range, 0-296 mosquitoes per house) were collected via aspiration. RT-qPCR of 233 pools of mosquitos were negative for W. bancrofti RNA; an independent reference laboratory confirmed these results. These results provide evidence that LF transmission has been halted in this setting.

BACKGROUND:Ovarian cancers are hallmarked by chromosomal instability. New therapies deliver improved patient outcomes in relevant phenotypes, however therapy resistance and poor long-term survival signal requirements for better patient preselection. An impaired DNA damage response (DDR) is a major chemosensitivity determinant. Comprising five pathways, DDR redundancy is complex and rarely studied alongside chemoresistance influence from mitochondrial dysfunction. We developed functional assays to monitor DDR and mitochondrial states and trialled this suite on patient explants.METHODS:We profiled DDR and mitochondrial signatures in cultures from 16 primary-setting ovarian cancer patients receiving platinum chemotherapy. Explant signature relationships to patient progression-free (PFS) and overall survival (OS) were assessed by multiple statistical and machine-learning methods.RESULTS:DR dysregulation was wide-ranging. Defective HR (HRD) and NHEJ were near-mutually exclusive. HRD patients (44%) had increased SSB abrogation. HR competence was associated with perturbed mitochondria (78% vs 57% HRD) while every relapse patient harboured dysfunctional mitochondria. DDR signatures classified explant platinum cytotoxicity and mitochondrial dysregulation. Importantly, explant signatures classified patient PFS and OS.CONCLUSIONS:Whilst individual pathway scores are mechanistically insufficient to describe resistance, holistic DDR and mitochondrial states accurately predict patient survival. Our assay suite demonstrates promise for translational chemosensitivity prediction.

BACKGROUND:Insecticide resistance among malaria-vector species is a pervasive problem that might jeopardise global disease-control efforts. Novel vector-control tools with different modes of action, including long-lasting insecticidal nets (LLINs) incorporating new active ingredients, are urgently needed to delay the evolution and spread of insecticide resistance. We aimed to measure phenotypic and genotypic insecticide-resistance profiles among wild Anopheles collected over 3 years to assess the longitudinal effects of dual-active-ingredient LLINs on insecticide resistance. METHODS:For this analysis, data nested in a 3-year, four parallel-arm, superiority cluster-randomised controlled trial (cRCT) in Tanzania, collected from 84 clusters (39 307 households) formed of 72 villages in the Misungwi district, were used to measure insecticide-resistance profiles among female Anopheles mosquitoes via insecticide-resistance bioassays and quantitative RT-PCR of metabolic-resistance genes. Wild, blood-fed, indoor-resting mosquitoes were collected annually during the rainy seasons from house walls in clusters from all four trial groups. Mosquitoes were morphologically identified as An gambiae sensu lato (SL) or An funestus SL before separate bioassay testing. The primary outcomes were lethal-dose values for α-cypermethrin, permethrin, and piperonyl butoxide pre-exposure plus permethrin-resistance intensity bioassays, mortality 72 h after insecticidal exposure for chlorfenapyr bioassays, fertility reduction 72 h after insecticidal exposure for pyriproxyfen bioassays, and fold change in metabolic-enzyme expression relative to an insecticide-susceptible laboratory strain. All primary outcomes were measured in An funestus SL 1 year, 2 years, and 3 years after LLIN distribution. Primary outcomes were also assessed in An gambiae SL if enough mosquitoes were collected. The cRCT is registered with ClinicalTrials.gov (NCT03554616). FINDINGS:Between May 24, 2019, and Oct 25, 2021, 47 224 female Anopheles were collected for resistance monitoring. In the pyrethroid (PY)-LLIN group, there were significant increases in α-cypermethrin-resistance intensity (year 1 LD50=9·52 vs year 2 76·20, p<0·0001) and permethrin-resistance intensity (year 1 13·27 vs year 2 35·83, p=0·0019) in An funestus SL. In the pyriproxyfen PY-LLIN group, there was similar increase in α-cypermethrin-resistance intensity (year 1 0·71 vs year 2 81·56, p<0·0001) and permethrin-resistance intensity (year 1 5·68 vs year 2 50·14, p<0·0001). In the piperonyl butoxide PY-LLIN group, α-cypermethrin-resistance intensity (year 1 33·26 vs year 3 70·22, p=0·0071) and permethrin-resistance intensity (year 1 47·09 vs year 3 2635·29, p<0·0001) also increased over time. In the chlorfenapyr PY-LLIN group, there were no effects on α-cypermethrin-resistance intensity (year 1 0·42 vs year 3 0·99, p=0·54) or permethrin-resistance intensity (data were not estimable due to nearly 100% mortality). There were also minimal reductions in chlorfenapyr susceptibility. However, in the chlorfenapyr PY-LLIN group, a significant decline in piperonyl-butoxide synergy was seen by year 3 (year 1 0·02 vs year 3 0·26, p=0·020). Highly over-expressed detoxification enzymes showed dynamic patterns of selection throughout the trial. INTERPRETATION:Our phenotypic data supports trial epidemiological findings; chlorfenapyr PY-LLINs provided superior protection from malaria across multiple transmission seasons, with few effects on insecticide-resistance selection. Rapid pyrethroid-resistance intensification in the piperonyl butoxide PY-LLIN group and pre-existing tolerance of pyriproxyfen in vector populations might explain the poorer performance of these two interventions regarding malaria outcomes. Further work is required to elucidate the potential mechanisms driving cross-resistance between pyrethroids and novel active ingredients to better inform the design of pre-emptive resistance-management strategies. FUNDING:UK Department for International Development; UK Medical Research Council; Wellcome Trust; UK Department of Health and Social Care; UK Foreign, Commonwealth and Development Office; and The Bill and Melinda Gates Foundation via the Innovative Vector Control Consortium.

The invasion and establishment of An. stephensi mosquitoes in the Horn of Africa represents a significant regional threat, which may jeopardise malaria control, particularly in urban areas which were formally free from disease transmission. Novel vector surveillance methods are urgently needed, both agnostic to mosquito larval morphology, and simple to implement at the sampling stage. Using new multiplex TaqMan assays, specifically targeting An. stephensi and Ae. aegypti, we validated the use of environmental DNA (eDNA) for simultaneous vector detection in shared artificial breeding sites. Study findings demonstrated that An. stephensi and Ae. aegypti eDNA deposited by as few as one second instar larva in 1L of water was detectable. Characterization of molecular insecticide resistance mechanisms, using novel amplicon-sequencing panels for both vector species, was possible from eDNA shed by as few as 16–32 s instar larvae in 50 ml of water. An. stephensi eDNA, derived from emergent pupae for 24 h, was remarkably stable, and still detectable ~ 2 weeks later. eDNA surveillance has the potential to be implemented in local endemic communities and at points of country entry, to monitor the spread of invasive vector species. Further studies are required to validate the feasibility of this technique under field conditions.

The introduction into Britain of two helicid species, Cochlicella acuta and C. barbara, is discussed in the knowledge that old carbon may have influenced the chronology; modern specimens of C. acuta from Cornish sand dunes gave radiocarbon dates which are 600−800 years old. The introduction of C. acuta is confirmed to the early Bronze Age and it may have been present in the late Neolithic. C. barbara arrived in Cornwall prior to its first observation in the 1960s but, in view of the old carbon problem, a precise date cannot be determined. Xerocrassa geyeri became extinct in most of southern Britain in the early Holocene, but survived at Gwithian in Cornwall for several more millennia, with radiocarbon dating suggesting that it could have been present there until the later part of the early Bronze Age.

The distribution of the black rat (Rattus rattus) has been heavily influenced by its association with humans. The dispersal history of this non-native commensal rodent across Europe, however, remains poorly understood, and different introductions may have occurred during the Roman and medieval periods. Here, in order to reconstruct the population history of European black rats, we first generate a de novo genome assembly of the black rat. We then sequence 67 ancient and three modern black rat mitogenomes, and 36 ancient and three modern nuclear genomes from archaeological sites spanning the 1st-17th centuries CE in Europe and North Africa. Analyses of our newly reported sequences, together with published mitochondrial DNA sequences, confirm that black rats were introduced into the Mediterranean and Europe from Southwest Asia. Genomic analyses of the ancient rats reveal a population turnover in temperate Europe between the 6th and 10th centuries CE, coincident with an archaeologically attested decline in the black rat population. The near disappearance and re-emergence of black rats in Europe may have been the result of the breakdown of the Roman Empire, the First Plague Pandemic, and/or post-Roman climatic cooling.

Novel insecticides are urgently needed to control insecticide-resistant populations of Anopheles malaria vectors. Broflanilide acts as a non-competitive antagonist of the gamma-aminobutyric acid receptor and has shown prolonged effectiveness as an indoor residual spraying product (VECTRON T500) in experimental hut trials against pyrethroid-resistant vector populations. This multi-centre study expanded upon initial discriminating concentration testing of broflanilide, using six Anopheles insectary colonies ( An. gambiae Kisumu KCMUCo, An. gambiae Kisumu NIMR, An. arabiensis KGB, An. arabiensis SENN, An. coluzzii N’Gousso and An. stephensi SK), representing major malaria vector species, to facilitate prospective susceptibility monitoring of this new insecticide; and investigated the potential for cross-resistance to broflanilide via the A296S mutation associated with dieldrin resistance ( rdl ). Across all vector species tested, the discriminating concentration for broflanilide ranged between LC 99 × 2 = 1.126–54.00 μg/ml or LC 95 × 3 = 0.7437–17.82 μg/ml. Lower concentrations of broflanilide were required to induce complete mortality of An. arabiensis SENN (dieldrin-resistant), compared to its susceptible counterpart, An. arabiensis KGB, and there was no association between the presence of the rdl mechanism of resistance and survival in broflanilide bioassays, demonstrating a lack of cross-resistance to broflanilide. Study findings provide a benchmark for broflanilide susceptibility monitoring as part of ongoing VECTRON T500 community trials in Tanzania and Benin.

Background Resistance to major public health insecticides in Côte d’Ivoire has intensified and now threatens the long-term effectiveness of malaria vector control interventions. Methods This study evaluated the bioefficacy of conventional and next-generation long-lasting insecticidal nets (LLINs), determined resistance profiles, and characterized molecular and metabolic mechanisms in wild Anopheles coluzzii from South-East Côte d’Ivoire in 2019. Results Phenotypic resistance was intense: more than 25% of mosquitoes survived exposure to ten times the doses of pyrethroids required to kill susceptible populations. Similarly, 24-hour mortality to deltamethrin-only LLINs was very low and not significantly different to an untreated net. Sub-lethal pyrethroid exposure did not induce significant delayed vector mortality 72 hours later. In contrast, LLINs containing the synergist piperonyl butoxide (PBO), or new insecticides, clothianidin and chlorfenapyr, were highly toxic to An. coluzzii. Pyrethroid-susceptible An. coluzzii were significantly more likely to be infected with malaria, compared to those that survived insecticidal exposure. Pyrethroid resistance was associated with significant over-expression of CYP6P4, CPY6Z1 and CYP6P3. Conclusions Study findings raise concerns regarding the operational failure of standard LLINs and support the urgent deployment of vector control interventions incorporating PBO, chlorfenapyr or clothianidin in areas of high resistance intensity in Côte d’Ivoire.

AbstractCulex (Cx.) tritaeniorhynchusis a mosquito species with an extensive and expanding inter-continental geographic distribution, currently reported in over 50 countries, across Asia, Africa, the Middle East, Europe and now Australia. It is an important vector of medical and veterinary concern, capable of transmitting multiple arboviruses which cause significant morbidity and mortality in human and animal populations. In regions endemic for Japanese encephalitis virus (JEV) in Asia,Cx. tritaeniorhynchusis considered the major vector and this species has also been shown to contribute to the transmission of several other significant zoonotic arboviruses, including Rift Valley fever virus and West Nile virus.Significant variation in vectorial capacity can occur between different vector populations. Obtaining knowledge of a species from across its geographic range is crucial to understanding its significance for pathogen transmission across diverse environments and localities. Vectorial capacity can be influenced by factors including the mosquito genetic background, composition of the microbiota associated with the mosquito and the co-infection of human or animal pathogens. In addition to enhancing information on vector surveillance and potential risks for pathogen transmission, determining the genetic and microbial diversity of distinct populations of a vector species is also critical for the development and application of effective control strategies.In this study, multiple geographically dispersed populations ofCx. tritaeniorhynchusfrom countries within Europe, Africa, Eurasia and Asia were sampled. Molecular analysis demonstrated a high level of genetic and microbial diversity within and between populations, including genetic divergence in the mosquitoCO1gene, as well as diverse microbiomes identified by16S rRNAgene amplicon sequencing. Evidence for the detection of the endosymbiotic bacteriaWolbachiain some populations was confirmed usingWolbachia-specific PCR detection and sequencing ofWolbachiaMLST genes; in addition to PCR-based detection of insect-specific viruses. Laboratory vector competence showedCx. tritaeniorhynchusfrom a Greek population are likely to be competent vectors of JEV. This study expands understanding of the diversity ofCx. tritaeniorhynchusacross its inter-continental range, highlights the need for a greater focus on this invasive vector species and helps to inform potential future directions for development of vector control strategies.

Chagas disease vector control relies on prompt, accurate identification of houses infested with triatomine bugs for targeted insecticide spraying. However, most current detection methods are laborious, lack standardization, have substantial operational costs and limited sensitivity, especially when triatomine bug densities are low or highly focal. We evaluated the use of FTA cards or cotton-tipped swabs to develop a low-technology, non-invasive method of detecting environmental DNA (eDNA) from both triatomine bugs and Trypanosoma cruzi for use in household surveillance in eastern Colombia, an endemic region for Chagas disease. Study findings demonstrated that Rhodnius prolixus eDNA, collected on FTA cards, can be detected at temperatures between 21 and 32 °C, when deposited by individual, recently blood-fed nymphs. Additionally, cotton-tipped swabs are a feasible tool for field sampling of both T. cruzi and R. prolixus eDNA in infested households and may be preferable due to their lower cost. eDNA detection should not yet replace current surveillance tools, but instead be evaluated in parallel as a more sensitive, higher-throughput, lower cost alternative. eDNA collection requires virtually no skills or resources in situ and therefore has the potential to be implemented in endemic communities as part of citizen science initiatives to control Chagas disease transmission.

Wolbachia is a genus of obligate bacterial endosymbionts that infect a diverse range of arthropod species as well as filarial nematodes, with its single described species, Wolbachia pipientis , divided into several ‘supergroups’ based on multilocus sequence typing. Wolbachia strains in mosquitoes have been shown to inhibit the transmission of human pathogens, including Plasmodium malaria parasites and arboviruses. Despite their large host range, Wolbachia strains within the major malaria vectors of the Anopheles gambiae and Anopheles funestus complexes appear at low density, established solely on PCR-based methods. Questions have been raised as to whether this represents a true endosymbiotic relationship. However, recent definitive evidence for two distinct, high-density strains of supergroup B Wolbachia within Anopheles demeilloni and Anopheles moucheti has opened exciting possibilities to explore naturally occurring Wolbachia endosymbionts in Anopheles for biocontrol strategies to block Plasmodium transmission. Here, we utilize genomic analyses to demonstrate that both Wolbachia strains have retained all key metabolic and transport pathways despite their smaller genome size, with this reduction potentially attributable to degenerated prophage regions. Even with this reduction, we confirmed the presence of cytoplasmic incompatibility (CI) factor genes within both strains, with wAnD maintaining intact copies of these genes while the cifB gene was interrupted in wAnM, so functional analysis is required to determine whether wAnM can induce CI. Additionally, phylogenetic analysis indicates that these Wolbachia strains may have been introduced into these two Anopheles species via horizontal transmission events, rather than by ancestral acquisition and subsequent loss events in the Anopheles gambiae species complex. These are the first Wolbachia genomes, to our knowledge, that enable us to study the relationship between natural strain Plasmodium malaria parasites and their anopheline hosts.