Efficacy of a spatial repellent for control of Aedes-borne virus transmission: A cluster-randomized trial in Iquitos, Peru.

Over half the world's population is at risk for viruses transmitted by Aedes mosquitoes, such as dengue and Zika. The primary vector, Aedes aegypti, thrives in urban environments. Despite decades of effort, cases and geographic range of Aedes-borne viruses (ABVs) continue to expand. Rigorously proven vector control interventions that measure protective efficacy against ABV diseases are limited to Wolbachia in a single trial in Indonesia and do not include any chemical intervention. Spatial repellents, a new option for efficient deployment, are designed to decrease human exposure to ABVs by releasing active ingredients into the air that disrupt mosquito-human contact. A parallel, cluster-randomized controlled trial was conducted in Iquitos, Peru, to quantify the impact of a transfluthrin-based spatial repellent on human ABV infection. From 2,907 households across 26 clusters (13 per arm), 1,578 participants were assessed for seroconversion (primary endpoint) by survival analysis. Incidence of acute disease was calculated among 16,683 participants (secondary endpoint). Adult mosquito collections were conducted to compare Ae. aegypti abundance, blood-fed rate, and parity status through mixed-effect difference-in-difference analyses. The spatial repellent significantly reduced ABV infection by 34.1% (one-sided 95% CI lower limit, 6.9%; one-sided P value = 0.0236, z = 1.98). Aedes aegypti abundance and blood-fed rates were significantly reduced by 28.6 (95% CI 24.1%, ∞); z = -9.11) and 12.4% (95% CI 4.2%, ∞); z = -2.43), respectively. Our trial provides conclusive statistical evidence from an appropriately powered, preplanned cluster-randomized controlled clinical trial of the impact of a chemical intervention, in this case a spatial repellent, to reduce the risk of ABV transmission compared to a placebo.

Releasing incompatible males drives strong suppression across populations of wild and Wolbachia-carrying Aedes aegypti in Australia.

Releasing sterile or incompatible male insects is a proven method of population management in agricultural systems with the potential to revolutionize mosquito control. Through a collaborative venture with the "Debug" Verily Life Sciences team, we assessed the incompatible insect technique (IIT) with the mosquito vector Aedes aegypti in northern Australia in a replicated treatment control field trial. Backcrossing a US strain of Ae. aegypti carrying Wolbachia wAlbB from Aedes albopictus with a local strain, we generated a wAlbB2-F4 strain incompatible with both the wild-type (no Wolbachia) and wMel-Wolbachia Ae. aegypti now extant in North Queensland. The wAlbB2-F4 strain was manually mass reared with males separated from females using Verily sex-sorting technologies to obtain no detectable female contamination in the field. With community consent, we delivered a total of three million IIT males into three isolated landscapes of over 200 houses each, releasing ∼50 males per house three times a week over 20 wk. Detecting initial overflooding ratios of between 5:1 and 10:1, strong population declines well beyond 80% were detected across all treatment landscapes when compared to controls. Monitoring through the following season to observe the ongoing effect saw one treatment landscape devoid of adult Ae. aegypti early in the season. A second landscape showed reduced adults, and the third recovered fully. These encouraging results in suppressing both wild-type and wMel-Ae. aegypti confirms the utility of bidirectional incompatibility in the field setting, show the IIT to be robust, and indicate that the removal of this arbovirus vector from human-occupied landscapes may be achievable.

The invasive mosquito Aedes albopictus (Diptera, Culicidae) firstly recorded in Bohemia, Czech Republic.

STUDY OBJECTIVE: In 2016-2017, the monitoring of possible introduction of an invasive mosquito species, the Asian tiger mosquito Aedes (Stegomyia) albopictus (Skuse, 1894) (Diptera, Culicidae), was conducted in eastern, southern, central and western parts of Bohemia, Czech Republic. MATERIAL AND METHODS: The focus was placed on local major traffic arteries (motorways D1, D3 and D5 and an expressway E49), which connecting South Europe and some of Balkan countries, infested by Ae. albopictus, with the Czech capital Prague. In total, more than 100 ovitraps were placed on 16 study sites - close surroundings of refuelling gas stations and neighbouring parking lots. RESULTS: In August and September 2017, totally eight specimens of Ae. albopictus were collected at the ovitrap site near Mezno/Mitrovice, Central Bohemia on D3 motorway and other two specimens were recovered at the gas station near Rozvadov, West Bohemia on D5 motorway. On the other hand, Ae. albopictus was not recorded on a main Czech motorway D1 connecting Prague and Bratislava capitals during the monitoring. CONCLUSION: The introduction of this mosquito into the Czech Republic is known since 2012 from surroundings of Mikulov town (South Moravian Region), our records were then the first in the region of Bohemia. Moreover, the distance between positive localities shows the potential for Ae. albopictus to be introduced by ground transport anywhere within the Czech Republic.

Alternative patterns of sex chromosome differentiation in Aedes aegypti (L).

BACKGROUND: Some populations of West African Aedes aegypti, the dengue and zika vector, are reproductively incompatible; our earlier study showed that divergence and rearrangements of genes on chromosome 1, which bears the sex locus (M), may be involved. We also previously described a proposed cryptic subspecies SenAae (PK10, Senegal) that had many more high inter-sex FST genes on chromosome 1 than did Ae.aegypti aegypti (Aaa, Pai Lom, Thailand). The current work more thoroughly explores the significance of those findings. RESULTS: Intersex standardized variance (FST) of single nucleotide polymorphisms (SNPs) was characterized from genomic exome capture libraries of both sexes in representative natural populations of Aaa and SenAae. Our goal was to identify SNPs that varied in frequency between males and females, and most were expected to occur on chromosome 1. Use of the assembled AaegL4 reference alleviated the previous problem of unmapped genes. Because the M locus gene nix was not captured and not present in AaegL4, the male-determining locus, per se, was not explored. Sex-associated genes were those with FST values ≥ 0.100 and/or with increased expected heterozygosity (H exp , one-sided T-test, p < 0.05) in males. There were 85 genes common to both collections with high inter-sex FST values; all genes but one were located on chromosome 1. Aaa showed the expected cluster of high inter-sex FST genes proximal to the M locus, whereas SenAae had inter-sex FST genes along the length of chromosome 1. In addition, the Aaa M-locus proximal region showed increased H exp levels in males, whereas SenAae did not. In SenAae, chromosomal rearrangements and subsequent suppressed recombination may have accelerated X-Y differentiation. CONCLUSIONS: The evidence presented here is consistent with differential evolution of proto-Y chromosomes in Aaa and SenAae.

Complete mitogenome sequence of Aedes (Hulecoeteomyia) japonicus japonicus from Hawai'i Island.

We report the first complete mitogenome (Mt) sequence of Aedes japonicus japonicus (Diptera: Culicidae). The sequence was extracted from one adult from the Big Island of Hawai'i Island. The length of the Ae. japonicus japonicus Mt was 16,528bp with 78.1% AT content. Its sequence is most similar to the Mt sequence of Aedes koreicus with 90.81% sequence identity. This is the first full Mt sequence available for this species and provides important genetic resource for studying population genetics and dynamics of this important invasive mosquito species.

Neem-based products as potential eco-friendly mosquito control agents over conventional eco-toxic chemical pesticides-A review.

Mosquitoes cause serious health hazards for millions of people across the globe by acting as vectors of deadly communicable diseases like malaria, filariasis, dengue and yellow fever. Use of conventional chemical insecticides to control mosquito vectors has led to the development of biological resistance in them along with adverse environmental consequences. In this light, the recent years have witnessed enormous efforts of researchers to develop eco-friendly and cost-effective alternatives with special emphasis on plant-derived mosquitocidal compounds. Neem oil, derived from neem seeds (Azadirachta indica A. Juss, Meliaceae), has been proved to be an excellent candidate against a wide range of vectors of medical and veterinary importance including mosquitoes. It is environment-friendly, and target-specific at the same time. The active ingredients of neem oil include limonoids like azadirachtin A, nimbin, salannin and numerous other substances that are still waiting to be discovered. Of these, azadirachtin has been shown to be very effective and is mainly responsible for its toxic effects. The quality of the neem oil depends on its azadirachtin content which, in turn, depends on its manufacturing process. Neem oil can be used directly or as nanoemulsions or nanoparticles or even in the form of effervescent tablets. When added to natural breeding habitat waters they exert their mosquitocidal effects by acting as ovicides, larvicides, pupicides and/or oviposition repellents. The effects are generated by impairing the physiological pathways of the immature stages of mosquitoes or directly by causing physical deformities that impede their development. Neem oil when used directly has certain disadvantages mainly related to its disintegration under atmospheric conditions rendering it ineffective. However, many of its formulations have been reported to remain stable under environmental conditions retaining its efficiency for a long time. Similarly, neem seed cake has also been found to be effective against the mosquito vectors. The greatest advantage is that the target species do not develop resistance against neem-based products mainly because of the innumerable number of chemicals present in neem and their combinations. This makes neem-based products highly potential yet unexplored candidates of mosquito control agents. The current review helps to elucidate the roles of neem oil and its various derivatives on mosquito vectors of public health concern.

Consumption of endemic arbovirus mosquito vectors by bats in the southeastern United States.

Mosquitoes affect human health and well-being globally through their roles as disease-causing pathogen vectors. Utilizing genetic techniques, we conducted a large-scale dietary study of three bat species common to the southeastern U.S.A., Lasiurus seminolus (Seminole bat), Nycticeius humeralis (evening bat), and Myotis austroriparius (southeastern myotis). Through next-generation sequencing of a 180 bp portion of cytochrome oxidase subunit I (COI) of mitochondrial DNA from 180 bat guano samples, we documented consumption of 17 species of mosquitoes by bats, including six endemic arbovirus vectors. Culex quinquefasciatus, Culex coronator, Culiseta melanura, Culex salinarius, Culex erraticus, and Coquillettidia perturbans were consumed by 51.3%, 43.7%, 27.2%, 22.8%, 18.0%, and 12.7% of bats sampled, respectively. Consumption of two of these mosquito species was explained by spatial variables reflecting the prevalence of mosquito larval habitat, five were explained by bat traits (bat mass, bat species), and two were explained by these factors plus temporal variables (maximum daily temperature, time since sunset, date), making it challenging to offer specific guidance on how best to promote bats as a means of reducing arbovirus vector species. Our results show that common bat species of the southeastern U.S.A. consume endemic, but not exotic, arbovirus mosquito vectors. Future studies are needed to understand the impact of bat consumption on mosquito numbers and public health.

Geographic distribution of the V1016G knockdown resistance mutation in Aedes albopictus: a warning bell for Europe.

BACKGROUND: Colonization of large part of Europe by the Asian tiger mosquito Aedes albopictus is causing autochthonous transmission of chikungunya and dengue exotic arboviruses. While pyrethroids are recommended only to reduce/limit transmission, they are widely implemented to reduce biting nuisance and to control agricultural pests, increasing the risk of insurgence of resistance mechanisms. Worryingly, pyrethroid resistance (with mortality < 70%) was recently reported in Ae. albopictus populations from Italy and Spain and associated with the V1016G point mutation in the voltage-sensitive sodium channel gene conferring knockdown resistance (kdr). Genotyping pyrethroid resistance-associated kdr mutations in field mosquito samples represents a powerful approach to detect early signs of resistance without the need for carrying out phenotypic bioassays which require availability of live mosquitoes, dedicated facilities and appropriate expertise. METHODS: Here we report results on the PCR-genotyping of the V1016G mutation in 2530 Ae. albopictus specimens from 69 sampling sites in 19 European countries. RESULTS: The mutation was identified in 12 sites from nine countries (with allele frequencies ranging from 1 to 8%), mostly distributed in two geographical clusters. The western cluster includes Mediterranean coastal sites from Italy, France and Malta as well as single sites from both Spain and Switzerland. The eastern cluster includes sites on both sides of the Black Sea in Bulgaria, Turkey and Georgia as well as one site from Romania. These results are consistent with genomic data showing high connectivity and close genetic relationship among West European populations and a major barrier to gene flow between West European and Balkan populations. CONCLUSIONS: The results of this first effort to map kdr mutations in Ae. albopictus on a continental scale show a widespread presence of the V1016G allele in Europe, although at lower frequencies than those previously reported from Italy. This represents a wake-up call for mosquito surveillance programs in Europe to include PCR-genotyping of pyrethroid resistance alleles, as well as phenotypic resistance assessments, in their routine activities.

Towards modernizing the taxonomy of Mediterranean Culicoides using classical morphology, mtDNA barcoding, and MALDI-TOF MS protein profiling.

Culicoides biting midges (Diptera: Ceratopogonidae) are a highly successful group of small (1-3 mm) hematophagous flies, infamous for the role they play as biological vectors for numerous pathogens of veterinary significance. The principal aim of the national animal disease surveillance program of Israel is to be able to rapidly sort and identify live field-captured insects including Culicoides for arbovirus screening. In this exploratory study, three identification methods-classical morphology, DNA barcoding, and MALDI-TOF MS-were applied simultaneously to individuals of 10 Culicoides species that commonly attack livestock in Israel. The strengths and limitations of the three methods are compared and evaluated. In essence, the CO1 barcoding and MALDI-TOF MS results closely matched those of classical morphology. Furthermore, at a higher level and in strong accordance with recognized subgenera, the 10 species, in the reconstructed phylogenies, coalesced into multiple deeper-branched monophyletic clades. However, some discrepancies between the molecular and protein profiling results did occur and proved difficult to assess in terms of taxonomic significance. This difficulty underscores how tricky it is to establish clear species limits when methods involving borderline cutoff values and similarity indices are used as a taxonomic aid. An added shortcoming of the pluralistic triple-method approach is that a significant percentage of the species-level depositions in the GenBank and BOLD databases are misidentified, hindering structured comparison and interpretation of the morphological and molecular results obtained. Aspects of the unresolved taxonomy of various biting midge assemblages within the Mediterranean basin, including minor changes to the Israeli Culicoides checklist, are discussed in light of the methods applied. It is observed that the direct access that classical morphology provides to the external environment (or species niche) is indispensable to the full and correct interpretation (and application) of concomitant molecular and protein profiling results. The Culicoides taxonomy of the future ought to be fully integrative, during which the assimilation of modern methodological advances should strengthen-rather than undermine-the morphological foundations laid down during the 260-year Linnaean epoch.

Phenotypic and genotypic pyrethroid resistance of Aedes albopictus, with focus on the 2017 chikungunya outbreak in Italy.

BACKGROUND: The highly invasive mosquito species Aedes albopictus has become a major health concern in temperate areas due to its role as vector of exotic arboviruses. Pyrethroid insecticides represent the main tools for limiting the circulation of such mosquito-borne viruses. The present work aim to extend previous reports on phenotypic pyrethroid-resistance in European Ae. albopictus, to identify its genetic basis and to monitor the geographical distribution of resistant genotypes, with a particular focus on sites experiencing the 2017 chikungunya outbreak in Italy. RESULTS: Bioassays, performed according to World Health Organization protocols, showed full susceptibility to deltamethrin (concentration = 0.05%) and varying levels of resistance to permethrin (0.75%) and/or α-cypermethrin (0.05%) across Italy, with highest levels in the core of the 2017 chikungunya outbreak. Partial genotyping of the VSSC gene revealed widespread distribution of V1016G mutation and confirmed its association with pyrethroid resistance. CONCLUSION: The results obtained show that the condition for the spread of pyrethroid resistance in Ae. albopictus in Europe exists under strong selective pressure due to intensive insecticide spraying to control exotic arbovirus outbreak or high levels of nuisance. The results draw attention to the need for an evidence-based implementation of mosquito nuisance control, taking insecticide resistance management into consideration. © 2019 Society of Chemical Industry.

Complete mitogenome sequence of Aedes (Stegomyia) aegypti derived from field isolates from California and South Africa.

The Aedes aegypti mitogenome (Mt) sequences of field isolates from California and South Africa revealed a deletion between position 14,522 and 14,659 of the Mt contig of the AaegL5 reference genome. The length of the mitogenome of the California isolate was 16,659 bp and had 99.0% similarity with the AaegL5 Mt contig. The South African isolate sequence was 16,600 bp long and had 97.9% similarity with the reference. The region between 1496 and 1664 bp is similar to a nuclear pseudogene that might be a copy of a portion of the mitochondrial genome.

Monitoring arbovirus in Thailand: Surveillance of dengue, chikungunya and zika virus, with a focus on coinfections.

Infections caused by arboviruses such as dengue virus (DENV), chikungunya virus (CHIKV), and Zika virus (ZIKV) frequently occur in tropical and subtropical regions. These three viruses are transmitted by Aedes (Ae.) aegypti and Ae. albopictus. In Thailand, the highest incidence of arbovirus infection and the high circulation of Aedes mosquito mainly occurs in the Southern provinces of the country. Few studies have focused on the incidence of co-infection of arboviruses in this region. In the present study, a cross-sectional study was conducted on a cohort of 182 febrile patients from three hospitals located in Southern Thailand. Surveillance of DENV, CHIKV and ZIKV was conducted from May to October 2016 during the rainy season. The serological analysis and molecular detection of arboviruses were performed by ELISA and multiplex RT-PCR respectively. The results demonstrated that 163 cases out of 182 patients (89.56%) were infected with DENV, with a predominance of DENV-2. Among these DENV positive cases, a co-infection with CHIKV for 6 patients (3.68%) and with ZIKV for 1 patient (0.61%) were found. 19 patients out of 182 were negative for arboviruses. This study provides evidence of co-infection of arboviruses in Southern Thailand and highlight the importance of testing DENV and other medically important arboviruses, such as CHIKV and ZIKV simultaneously.

Aedes aegypti in the Black Sea: recent introduction or ancient remnant?

BACKGROUND: The yellow fever mosquito Aedes aegypti transmits viral diseases that have plagued humans for centuries. Its ancestral home are forests of Africa and ~400-600 years ago it invaded the New World and later Europe and Asia, causing some of the largest epidemics in human history. The species was rarely detected in countries surrounding the Mediterranean Sea after the 1950s, but during the last 16 years it re-appeared in Madeira, Russia and in the eastern coast of the Black Sea. We genotyped Ae. aegypti populations from the Black Sea region to investigate whether this is a recent invasion (and if so, where it came from) or a remnant of pre-eradication populations that extended across the Mediterranean. We also use the Black Sea populations together with a world reference panel of populations to shed more light into the phylogeographical history of this species. RESULTS: Microsatellites and ~19,000 genome-wide single nucleotide polymorphisms (SNPs) support the monophyletic origin of all populations outside Africa, with the New World as the site of first colonization. Considering the phylogenetic relationships, the Black Sea populations are basal to all Asian populations sampled. Bayesian analyses combined with multivariate analyses on both types of markers suggest that the Black Sea population is a remnant of an older population. Approximate Bayesian Computation Analysis indicates with equal probability, that the origin of Black Sea populations was Asia or New World and assignment tests favor the New World. CONCLUSIONS: Our results confirmed that Ae. aegypti left Africa and arrived in New World ~500 years ago. The lineage that returned to the Old World and gave rise to present day Asia and the Black Sea populations split from the New World approximately 100-150 years ago. Globally, the Black Sea population is genetically closer to Asia, but still highly differentiated from both New World and Asian populations. This evidence, combined with bottleneck signatures and divergence time estimates, support the hypothesis of present day Black Sea populations being remnants of older populations, likely the now extinct Mediterranean populations that, consistent with the historic epidemiological record, likely represent the original return of Ae. aegypti to the Old World.

First record of Aedes albopictus (Skuse 1894) on São tomé island.

Biological invasions have critical impacts on native biodiversity and human societies and especially on oceanic islands that are fragile and threatened ecosystems. The invasive tiger mosquito Aedes (Stegomyia) albopictus (Skuse, 1894) native to Southeast Asia has been introduced during the past 30 years almost everywhere in the world, including the Americas, the Pacific, Europe and Africa. It has been reported for the first time in the Gulf of Guinea in 2000, first in Cameroon, then in Bioko Island in 2003 and more recently in Gabon in 2007. Here we report the first record of Ae. albopictus on São Tomé Island. Although we cannot estimate precisely the year of introduction on São Tomé Island, it most likely arrived within the last 10 years. By sequencing the mitochondrial cytochrome c oxidase gene from individual adults, we detected three haplotypes already present in mainland Africa. More studies are needed to explore the dynamics of its expansion and competition with insular native mosquitoes.

The complete mitochondrial DNA genomes for two lineages of Aedes notoscriptus (Diptera: Culicidae).

The complete mitochondrial genomes for two deeply divergent lineages of the urban adapted mosquito Aedes notoscriptus Skuse (Diptera: Culicidae) in Australia were sequenced using a combination of next generation Illumina and traditional Sanger sequencing. The 15,846 and 15,851 bp circular genomes share 95.0% nucleotide identity. They both have the full complement of 37 metazoan genes and identical gene arrangements to previously published Culicidae species with the one non-coding A + T rich control region present between rns and tRNA-Ile. All protein initiation codons are ATN apart from COX1 (TCG). Eight protein coding genes encode full TAA stop codons, one uses an incomplete TA and four use T. Typical cloverleaf structures containing DHU and TΨC stem and loops can be inferred for all 22 tRNAs.

The complete mitochondrial DNA genome of Aedes vigilax (Diptera: Culicidae).

The complete mitochondrial genomes of two main clades of the medically significant saltmarsh mosquito Aedes vigilax Skuse (Diptera: Culicidae) were obtained using combined Illumina and Sanger sequencing. The two 15,877 bp circular genomes share 99.0% nucleotide identity and encode 37 genes with identical gene arrangement similar to previously published Culicidae species with a non-coding A + T rich region between rns and tRNA-Ile. Protein initiation codon is ATN apart from ND5 (GTG) and COX1 (TCG). Eight protein-coding genes encode full TAA stop codon, while five are completed by mRNA polyadenylation. Typical cloverleaf structures containing DHU and TΨC stem and loops can be inferred for all 22 tRNAs.

Enhancement of the BG-sentinel trap with varying number of mice for field sampling of male and female Aedes albopictus mosquitoes.

BACKGROUND: Trapping male mosquitoes in the field is essential for the development of area-wide vector control programs with a sterile insect technique (SIT) component. To determine the optimal temporal and spatial release strategy, an estimation of the wild population density and its temporal dynamics is essential. Among the traps available for such data collection, the BG-Sentinel trap developed by the Biogents company uses a combination of visual cues, convection currents and olfactory signals. Although in numerous cases, this trap has shown high efficiency in sampling Aedes albopictus, in some cases low capture rates of Ae. albopictus males were recorded for the BG-sentinel mosquito trap baited with synthetic attractants. METHODS: The effects of modifying the BG-sentinel trap (by adding one mouse, two or three live mice to the trap) on the efficiency of trapping Ae. albopictus males and females was tested. The experiment was carried out in three distinct areas on La Réunion that have been selected for pilot field testing of the release of sterile male Ae. albopictus mosquitoes. The effect of four types of attractant (including the generic BG-Lure, one mouse or two to three mice) in baited BGS traps was tested with a Latin square design in order to control for the variability of different sampling positions and dates. RESULTS: At the three studied sites, the number of Ae. albopictus adults caught and the proportion of males per trap consistently increased with the number of mice present in the trap. CONCLUSION: The results from this study suggest that some new attractants derived from, or similar to, mouse odors could be developed and tested in combination with other existing attractive components, such as CO2 and heat, in order to provide a reliable estimation method for Ae. albopictus adult male abundance in the wild.