Field Mosquitoes Research Articles

Comparative analysis of bacterial microbiota in Aedes aegypti (Diptera: Culicidae): insights from field and laboratory populations in Colombia.

Comparative studies of the microbiota in whole-body mosquitoes from natural populations and laboratory-reared specimens are scarce, particularly in tropical countries like Colombia, where understanding microbial patterns is critical for effective disease control and vector management. This study examines the bacterial microbiota of Aedes aegypti by comparing field-collected mosquitoes from 3 Colombian regions (Southern Amazon, Central Andean region, and Northern Caribbean coast) with laboratory strains (Rockefeller, Otanche, and Tolima). These regions are highly endemic for dengue and are associated with lineage 1 of Ae. aegypti, known for its elevated vector competence. Using next-generation sequencing of the 16S rRNA gene with Illumina technology, we found that field-collected mosquitoes exhibited significantly higher alpha and beta diversity compared to laboratory-reared specimens. Field mosquitoes were enriched with bacterial families such as Acetobacteraceae, Lactobacillaceae, and Bacillaceae, while laboratory mosquitoes showed a greater abundance of Enterobacteriaceae. Differential abundance analysis revealed that Acetobacter and Bacillus predominated in field mosquitoes, whereas Asaia, Cedacea, and Chryseobacterium were more common in laboratory specimens. Notably, Pseudomonas and Acinetobacter were consistently abundant across all samples. Our findings suggest that environmental factors, such as habitat and diet, significantly influence the bacterial composition and diversity of Ae. aegypti in Colombia. Future research should further explore how these factors, alongside genetic components, shape mosquito-microbiota interactions and their implications for disease transmission and vector competence.

Blood meal analysis: unveiling the feeding preferences of Aedes aegypti and Aedes albopictus in a dengue-endemic area.

Studies have suggested animals as possible reservoir hosts for flaviviruses transmitted by Aedes mosquitoes; however, there is limited evidence for the dengue virus in Malaysia. One of the possible ways to determine the zoonotic potential for any pathogen transmission is through blood meal analysis which can provide valuable insights into the feeding preferences of the mosquitoes. Unfortunately, limited information is available on the feeding preferences of Aedes mosquitoes in Malaysia. Thus, this study aimed to identify the blood-feeding preferences of Aedes aegypti and Aedes albopictus from different ecotypes in Selangor, Malaysia. The field mosquitoes were collected using a modified backpack aspirator and CDC light trap. The collected mosquitoes were initially classified based on degrees of blood digestion according to the Sella scale before extracting the DNA. The presence of vertebrate DNA was detected using nested PCR, and samples positive for vertebrate DNA were further subjected to species-specific PCR targeting the common animals found at the study locations. In general, 51 of 187 field caught Aedes mosquitoes were positive for the presence of vertebrate DNA in their blood meal. The most frequent blood meal source was human (38.2%), followed by monkey (12.7%), bovine (10.9%), chicken (7.3%) and dog (3.6%). The human blood index (HBI) of Ae. albopictus collected across the four different ecotypes revealed that, Ae. albopictus collected near human dwellings showed 100% anthropophilic tendency. Interestingly, there were two Aedes mosquitoes, Ae. aegypti (n=1) and Ae. albopictus (n=1) positive for both human and monkey blood. Since sylvatic dengue continues to flourish in Southeast Asia, this finding from blood meal analysis shows the potential for zoonotic transmission by Aedes mosquitoes in these locations. However, further research must be carried out to understand the role of animals as potential reservoir hosts for the dengue virus, especially through the detection of the virus in the blood meal.

Contrasting patterns of Asaia association with Plasmodium falciparum between field-collected Anopheles gambiae and Anopheles coluzzii from Cameroon.

The widespread prevalence of Asaia in mosquitoes makes it a potential candidate for paratrangenic control in Anopheles. To better understand whether this bacterium could be used for malaria control, we quantified Asaia in An. gambiae s.l populations in malaria endemic regions examining co-infection with Plasmodium falciparum. Adult Anopheles mosquitoes were collected across two different eco-geographical localities in Cameroon, during both the dry and wet seasons. DNA was extracted from whole individual mosquitoes, and real time-qPCR amplification of the 16S ribosomal RNA was used to quantify Asaia in both An. gambiae and An. coluzzii samples. We also detected and quantified P. falciparum infection in the same mosquitoes. The density of Asaia was successfully quantified in a total of 864 field mosquitoes, comprising of 439 An. gambiae from Bankeng and 424 An. coluzii collected from Gounougou. Interestingly, a higher prevalence of Asaia in An. gambiae (88.3%) compared to An. coluzzii (80.9%) was observed. Moreover, the density of Asaia in both species was significantly affected by seasonal changes in the two localities. Furthermore, a significant difference between the infection densities of Asaia and the Plasmodium infection status in the two species was recorded. However, no correlation was observed between the number of Asaia and P. falciparum infections. This study provides evidence that naturally occurring Asaia infection is not correlated to P. falciparum development within An. gambiae and An. coluzzii. Nevertheless, further studies incorporating experimental infections are required to better investigate the correlation between Anopheles mosquitoes, Asaia, and Plasmodium.IMPORTANCEThe symbiont Asaia has emerged as a promising candidate for paratransgenic control of malaria, but further analysis of its biology and genetics across Africa is necessary. In this study, we investigated and quantified the influence of Asaia in naturally infected An. gambiae s.l. populations with the malaria parasite Plasmodium falciparum. Genomic DNA was extracted from whole individual mosquitoes collected from two localities, and Asaia was quantified using real-time qPCR by amplification of the 16S ribosomal RNA gene. We also detected and quantified Plasmodium falciparum infection in the same mosquitoes and established the correlation between Asaia and Plasmodium coinfection. This study provides evidence that naturally occurring Asaia infection is not correlated with P. falciparum development within An. gambiae and An. coluzzii mosquitoes.

A naturally isolated symbiotic bacterium suppresses flavivirus transmission by Aedes mosquitoes.

The commensal microbiota of the mosquito gut plays a complex role in determining the vector competence for arboviruses. In this study, we identified a bacterium from the gut of field Aedes albopictus mosquitoes named Rosenbergiella sp. YN46 (Rosenbergiella_YN46) that rendered mosquitoes refractory to infection with dengue and Zika viruses. Inoculation of 1.6 × 103 colony forming units (CFUs) of Rosenbergiella_YN46 into A. albopictus mosquitoes effectively prevents viral infection. Mechanistically, this bacterium secretes glucose dehydrogenase (RyGDH), which acidifies the gut lumen of fed mosquitoes, causing irreversible conformational changes in the flavivirus envelope protein that prevent viral entry into cells. In semifield conditions, Rosenbergiella_YN46 exhibits effective transstadial transmission in field mosquitoes, which blocks transmission of dengue virus by newly emerged adult mosquitoes. The prevalence of Rosenbergiella_YN46 is greater in mosquitoes from low-dengue areas (52.9 to ~91.7%) than in those from dengue-endemic regions (0 to ~6.7%). Rosenbergiella_YN46 may offer an effective and safe lead for flavivirus biocontrol.

Development of an Enzyme-Linked Immunosorbent Assay (ELISA) as a tool to detect NS1 of dengue virus serotype 2 in female Aedes aegypti eggs for the surveillance of dengue fever transmission

Dengue is a significant disease transmitted by Aedes mosquitoes in the tropics and subtropics worldwide. The disease is caused by four virus (DENV) serotypes and is transmitted to humans by female Aedes aegypti mosquito bites infected with the virus and vertically to their progeny. Current strategies to control dengue transmission focus on the vector. In this study, we describe an indirect Enzyme-Linked Immunosorbent Assay (ELISA), using a monoclonal antibody against the non-structural dengue virus protein 1 (NS1), to detect DENV2 in Ae. aegypti eggs. The assay detects NS1 in eggs homogenates with 87.5% sensitivity and 75.0% specificity and it is proposed as a tool for the routine entomovirological surveillance of DENV 2 in field mosquito populations.

Larvicidal potential, toxicological assessment, and molecular docking studies of four Egyptian bacterial strains against Culex pipiens L. (Diptera: Culicidae)

Mosquito control in Egypt depends on applying chemical synthetic pesticides that impact negatively on human health and the environment as well as the development of antibiotic and chemical resistance. This study aims to control the 3rd and 4th instars of Culex pipiens larvae using four bacterial strains. According to Phenotypic and molecular identification, the four isolates were identified as Bacillus subtilis MICUL D2023, Serratia marcescens MICUL A2023, Streptomyces albus LARVICID, and Pseudomonas fluorescens MICUL B2023. All strains were deposited in GenBank under accession numbers OQ764791, OQ729954, OQ726575, and OQ891356, respectively. Larvicidal activity of all microbial strain metabolites against a field strain of C. pipiens explored low LC50 results and reached its lowest values on the 3rd day with values of 6.40%, 38.4%, and 46.33% for P. fluorescens, S. albus, and S. marcescens, respectively. In addition, metabolites of P. fluorescence were more toxic than those of S. albus, followed by S. marcescens. B. subtilis shows no larvicidal effect on both field and lab mosquito strains. Microscopic alterations of 3rd and 4th instars showed toxic effects on different body parts (thorax, midgut, and anal gills), including losing external hairs, abdominal breakage, and larvae shrinkage, as well as different histological malformations in the digestive tract, midgut, and cortex. GC–MS analysis detected 51, 30, and 32 different active compounds from S. albus, S. marcescens, and P. fluorescens, respectively. GC detected 1, 2-BENZEA2:A52NEDICARBOXYLIC ACID, 2-Cyclohexene-1-carboxylic-acid-5-2-butenyl-methyl ester, and 3 octadecahydro2R3S4Z9Z-11R-12S from S. albus, S. marcesens, and P. fluorescens, respectively. Total protein, Total carbohydrate, and Acetylcholine esterase activity indicated significantly low levels on the 3rd day. All strain metabolites were safe against HSF cell lines. The docking results confirmed the role of the produced metabolites as larvicidal agents and Acetylcholine esterase inhibition. Such a problem need more studies on applying more and more natural pesticides.

Gut Bacterial Diversity of Field and Laboratory-Reared Aedes albopictus Populations of Rio de Janeiro, Brazil.

The mosquito microbiota impacts different parameters in host biology, such as development, metabolism, immune response and vector competence to pathogens. As the environment is an important source of acquisition of host associate microbes, we described the microbiota and the vector competence to Zika virus (ZIKV) of Aedes albopictus from three areas with distinct landscapes. Adult females were collected during two different seasons, while eggs were used to rear F1 colonies. Midgut bacterial communities were described in field and F1 mosquitoes as well as in insects from a laboratory colony (>30 generations, LAB) using 16S rRNA gene sequencing. F1 mosquitoes were infected with ZIKV to determine virus infection rates (IRs) and dissemination rates (DRs). Collection season significantly affected the bacterial microbiota diversity and composition, e.g., diversity levels decreased from the wet to the dry season. Field-collected and LAB mosquitoes' microbiota had similar diversity levels, which were higher compared to F1 mosquitoes. However, the gut microbiota composition of field mosquitoes was distinct from that of laboratory-reared mosquitoes (LAB and F1), regardless of the collection season and location. A possible negative correlation was detected between Acetobacteraceae and Wolbachia, with the former dominating the gut microbiota of F1 Ae. albopictus, while the latter was absent/undetectable. Furthermore, we detected significant differences in infection and dissemination rates (but not in the viral load) between the mosquito populations, but it does not seem to be related to gut microbiota composition, as it was similar between F1 mosquitoes regardless of their population. Our results indicate that the environment and the collection season play a significant role in shaping mosquitoes' bacterial microbiota.

Arbovirus surveillance on the Mexico-USA border: West Nile virus identification in various species of field mosquitoes.

West Nile virus (WNV) has been documented in human and/or mosquito samples near the border with Mexico in El Paso, Texas, and Doña Ana County, New Mexico. However, on the Mexican side of the border, particularly in the State of Chihuahua, no such cases of WNV-infected mosquitoes have been documented. We tested 367 mosquitoes of four species (Culex quinquefasciatus, Cx. tarsalis, Aedes aegypti, and Aedes (Ochlerotatus) epactius) and found a high rate of WNV-positivity, including the first record of Ae. (Ochlerotatus) epactius infection with WNV. These results call for intensifying WNV surveillance efforts on the border between the United States and Mexico, with particular emphasis on vector control and monitoring of the species included in this study.

Dynamic of Mayaro Virus Transmission in Aedes aegypti, Culex quinquefasciatus Mosquitoes, and a Mice Model.

Mayaro virus (MAYV) is transmitted by Haemagogus spp. mosquitoes and has been circulating in Amazon areas in the North and Central West regions of Brazil since the 1980s, with an increase in human case notifications in the last 10 years. MAYV introduction in urban areas is a public health concern as infections can cause severe symptoms similar to other alphaviruses. Studies with Aedes aegypti have demonstrated the potential vector competence of the species and the detection of MAYV in urban populations of mosquitoes. Considering the two most abundant urban mosquito species in Brazil, we investigated the dynamics of MAYV transmission by Ae. aegypti and Culex quinquefasciatus in a mice model. Mosquito colonies were artificially fed with blood containing MAYV and infection (IR) and dissemination rates (DR) were evaluated. On the 7th day post-infection (dpi), IFNAR BL/6 mice were made available as a blood source to both mosquito species. After the appearance of clinical signs of infection, a second blood feeding was performed with a new group of non-infected mosquitoes. RT-qPCR and plaque assays were carried out with animal and mosquito tissues to determine IR and DR. For Ae. aegypti, we found an IR of 97.5-100% and a DR reached 100% in both 7 and 14 dpi. While IR and DR for Cx. quinquefasciatus was 13.1-14.81% and 60% to 80%, respectively. A total of 18 mice were used (test = 12 and control = 6) for Ae. aegypti and 12 (test = 8 and control = 4) for Cx. quinquefasciatus to evaluate the mosquito-mice transmission rate. All mice that were bitten by infected Ae. aegypti showed clinical signs of infection while all mice exposed to infected Cx. quinquefasciatus mosquitoes remained healthy. Viremia in the mice from Ae. aegypti group ranged from 2.5 × 108 to 5 × 109 PFU/mL. Ae. aegypti from the second blood feeding showed a 50% IR. Our study showed the applicability of an efficient model to complete arbovirus transmission cycle studies and suggests that the Ae. aegypti population evaluated is a competent vector for MAYV, while highlighting the vectorial capacity of Ae. aegypti and the possible introduction into urban areas. The mice model employed here is an important tool for arthropod-vector transmission studies with laboratory and field mosquito populations, as well as with other arboviruses.

Livestock fatalities attributed to a massive attack of Psorophora columbiae following Hurricane Laura

The objective of this case report is to characterize the sudden fatality of livestock associated with a massive attack of dark rice field mosquitoes (Psorophora columbiae) after the passage of Hurricane Laura in southwest Louisiana late August 2020. In 3 days, approximately 30 deer, 600 beef cattle, 100 sheep and 30 horses died following a brief history of weakness or, in most cases, no clinical signs prior to death. Several on-site visits were performed by veterinarians to evaluate animals and perform necropsies. Two deer were received for necropsy, and tissue samples from 1 bull and 1 horse were evaluated at the Louisiana Animal Diagnostic Disease Laboratory. In addition, postmortem examination was performed on another 15 animals by veterinarians. Similar gross alterations were observed in all necropsies. Lesions included multifocal petechiae and ecchymoses in the skin and the subcutaneous tissues. Both deer also had pale discoloration and accentuated lobular patterns in their livers. Histologically, the livers of the deer and the bull exhibited centrilobular degeneration and hepatocyte dissociation suggestive of severe anemia. The description of this animal fatality event associated with Psorophora colum- biae attack demonstrates the need to establish prevention and/or intervention protocols in areas prone to hurricanes and tropical depressions.

Comparative analysis of the larvicidal activity of temephos (EC50) and novaluaron (EC10) to control Anopheles stephensi in Sri Lanka

BackgroundAnopheles stephensi was first recorded in the coastal area of Mannar District, Sri Lanka, in December 2016. Since then, this vector has been isolated from other districts in the Northern and Eastern Provinces of Sri Lanka. Chemical control is the main arm of vector control that can be used to reduce the vector densities within a short period. Thus, the present study aimed at evaluating the efficacy of using selected insecticides for the control of An. stephensi larvae. MethodThe third and fourth instar larval stages of An. stephensi (F2 generation) of field mosquitoes that were caught using cattle baited net trap collections from Columbuthurai, Kurunagar, and Navanthurai areas in Jaffna District, Sri Lanka, were obtained from the laboratory colony established at Jaffna. Batches of 100 larvae were taken for experiments and introduced separately to a concentration series of temephos and novaluron (0.04–400 ppm). A control test was also performed at each setup without introducing insecticides. The mortality rates of An. stephensi larvae exposed to different concentrations of larvicides were recorded at 1, 24 and 48-h intervals. The experiment was replicated five times at individual concentrations for each selected chemical. Data were analyzed using the General Linear Model (GLM) and Probit analysis. ResultsThe highest mortality rate (100%) at a 1-h exposure period was observed from temephos at >100 ppm. The mortality rates varied significantly for different concentrations and larvicides (p < 0.05). At 24-h of the exposure period, the 100% mortality of An. stephensi larvae were observed from both temephos and novaluron even at 0.04 ppm. ConclusionBoth temephos and novaluron reported 100% mortality rates in An. stephensi larvae at 1-h and 24-h exposure periods. Based on the findings, temephos and novaluron can be recommended as effective larvicides for chemical-based control of An. stephensi in Jaffna, Sri Lanka. Further, it is recommended to conduct a field-based study, where habitat types and water quality are highly heterogeneous and may affect the residual activity.

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

BackgroundColonization 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.MethodsHere we report results on the PCR-genotyping of the V1016G mutation in 2530 Ae. albopictus specimens from 69 sampling sites in 19 European countries.ResultsThe 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.ConclusionsThe 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.Graphical

Elevated expression of odorant receptors and odorant-binding proteins genes detected in antennae of Culex quinquefasciatus field females

Culex quinquefasciatus is responsible for the transmission of filarial worms and several arboviruses. Olfaction plays a crucial role in disease transmission as it influences behaviors that are essential for the survival and reproduction of the mosquito, such as the host-seeking behavior, courtship, and oviposition. Understanding the molecular events that coordinate how mosquitoes find their host may lead to alternative methods to reduce diseases transmission. Our aim was to investigate the differential expression profile of odorant receptor (ORs) and odorant-binding proteins (OBPs) genes in Cx. quinquefasciatus field females compared with CqSLab laboratory mosquito colony. Seventeen genes of interest were evaluated for their qualitative and specific expression by RT-PCR on RNAs extracted from female antennae, female legs, complete male bodies, incomplete female bodies (no head and no legs), and L4 larvae. The general expression mapping of olfactory genes revealed that all analyzed genes were expressed in antennae. Some genes showed different qualitative expression profiles, such as CquiOR2, CquiOR64, CquiOR93, CquiOBP11, and CquiOBP16, which were expressed exclusively in female antennae. On the other hand, CquiOR37, CquiOBP2, and CquiOBP43 are expressed in all sample types, and CquiOBP10 was expressed in female antennae and legs and in the complete male bodies. The expression of CquiOBP5 was detected in the female’s antennae and body, but it was absent in the legs. The quantitative differential expression analysis of six of the 17 genes by RT-qPCR was performed from RNA samples from antenna pools collected in three physiological states, post-emergence, post-mating, and post-blood feeding of the field females and CqSLab. A total of 3,600 antennae were analyzed, in pools containing 100 pairs. Most genes screened showed a higher expression level in field mosquitoes when compared with the laboratory strain CqSLab. The expression of CquiOBP5 and CquiOBP10 genes was significantly different between the post-mating and post blood-meal samples of laboratory females (p &amp;lt; 0.05). Our results suggest specialization of the function of the genes studied and divergence in the expression pattern of field mosquitoes compared with laboratory mosquitoes, and therefore, caution should be exercised in the interpretation of data from laboratory mosquito studies.

Molecular Drivers of Multiple and Elevated Resistance to Insecticides in a Population of the Malaria Vector Anopheles gambiae in Agriculture Hotspot of West Cameroon.

(1) Background: Malaria remains a global public health problem. Unfortunately, the resistance of malaria vectors to commonly used insecticides threatens disease control and elimination efforts. Field mosquitoes have been shown to survive upon exposure to high insecticide concentrations. The molecular mechanisms driving this pronounced resistance remain poorly understood. Here, we elucidated the pattern of resistance escalation in the main malaria vector Anopheles gambiae in a pesticide-driven agricultural hotspot in Cameroon and its impact on vector control tools; (2) Methods: Larval stages and indoor blood-fed female mosquitoes (F0) were collected in Mangoum in May and November and forced to lay eggs; the emerged mosquitoes were used for WHO tube, synergist and cone tests. Molecular identification was performed using SINE PCR, whereas TaqMan-based PCR was used for genotyping of L1014F/S and N1575Y kdr and the G119S-ACE1 resistance markers. The transcription profile of candidate resistance genes was performed using qRT-PCR methods. Characterization of the breeding water and soil from Mangoum was achieved using the HPLC technique; (3) Results: An. gambiae s.s. was the only species in Mangoum with 4.10% infection with Plasmodium. These mosquitoes were resistant to all the four classes of insecticides with mortality rates <7% for pyrethroids and DDT and <54% for carbamates and organophophates. This population also exhibited high resistance intensity to pyrethroids (permethrin, alpha-cypermethrin and deltamethrin) after exposure to 5× and 10× discriminating doses. Synergist assays with PBO revealed only a partial recovery of susceptibility to permethrin, alpha-cypermethrin and deltamethrin. Only PBO-based nets (Olyset plus and permaNet 3.0) and Royal Guard showed an optimal efficacy. A high amount of alpha-cypermethrin was detected in breeding sites (5.16-fold LOD) suggesting ongoing selection from agricultural pesticides. The 1014F-kdr allele was fixed (100%) whereas the 1575Y-kdr (37.5%) and the 119S Ace-1R (51.1%) were moderately present. Elevated expression of P450s, respectively, in permethrin and deltamethrin resistant mosquitoes [CYP6M2 (10 and 34-fold), CYP6Z1(17 and 29-fold), CYP6Z2 (13 and 65-fold), CYP9K1 (13 and 87-fold)] supports their role in the observed resistance besides other mechanisms including chemosensory genes as SAP1 (28 and 13-fold), SAP2 (5 and 5-fold), SAP3 (24 and 8-fold) and cuticular genes as CYP4G16 (6 and 8-fold) and CYP4G17 (5 and 27-fold). However, these candidate genes were not associated with resistance escalation as the expression levels did not differ significantly between 1×, 5× and 10× surviving mosquitoes; (4) Conclusions: Intensive and multiple resistance is being selected in malaria vectors from a pesticide-based agricultural hotspot of Cameroon leading to loss in the efficacy of pyrethroid-only nets. Further studies are needed to decipher the molecular basis underlying such resistance escalation to better assess its impact on control interventions.

Reduced performance of community bednets against pyrethroid-resistant Anopheles funestus and Anopheles gambiae, major malaria vectors in Cameroon

BackgroundLong-lasting insecticidal nets (LLINs) are a vital tool in the fight against malaria vectors. However, their efficacy in the field can be impacted by several factors, including patterns of usage, net age, mosquito resistance and the delayed mortality effect, all of which could influence malaria transmission. We have investigated the effectiveness of the various brands of LLINs available in markets and households in Cameroon on pyrethroid-resistant mosquitoes and assessed their post-exposure effect.MethodsFollowing quality control assessment on a susceptible laboratory mosquito strain, we evaluated the immediate and delayed mortality effects of exposure to LLINs (both newly bough LLINst and used ones collected from households in Elende village, Cameroon, in 2019) using standard WHO cone tests on Anopheles gambiae and Anopheles funestus populations collected from the Centre region of Cameroon. Alive female mosquitoes were genotyped for various resistance markers at different time points post-exposure to evaluate the impact of insecticide resistance on the efficacy of bednets.ResultsThe laboratory-susceptible strain experienced high mortality rates when exposed to all pyrethroid-only brands of purchased nets (Olyset® Net, Super Net, PermaNet® 2.0, Yorkool®, Royal Sentry®) (Mean±SEM: 68.66 ± 8.35% to 93.33 ± 2.90%). However, low mortality was observed among wild An. funestus mosquitoes exposed to the bednets (0 ± 0 to 28 ± 6.7%), indicating a reduced performance of these nets against field mosquitoes. Bednets collected from households also showed reduced efficacy on the laboratory strain (mortality: 19–66%), as well as displaying a significant loss of efficacy against the local wild strains (mortality: 0 ± 0% to 4 ± 2.6% for An. gambiae sensu lato and 0 ± 0% to 8 ± 3.2% for An. funestus). However, compared to the unexposed group, mosquitoes exposed to bednets showed a significantly reduced longevity, indicating that the efficacy of these nets was not completely lost. Mosquitoes with the CYP6P9a-RR and L119F-GSTe2 mutations conferring pyrethroid resistance showed greater longevity after exposure to the Olyset net than their susceptible counterparts, indicating the impact of resistance on bednet efficacy and delayed mortality.ConclusionThese findings show that although standard bednets drastically lose their efficacy against pyrethroid-resistant field mosquitoes, they still are able to induce delayed mortality in exposed populations. The results of this study also provide evidence of the actual impact of resistance on the quality and efficacy of LLINs in use in the community, with mosquitoes carrying the CYP6P9a-RR and L119F-GSTe2 mutations conferring pyrethroid resistance living longer than their susceptible counterparts. These results highlight the need to use new-generation nets that do not rely solely on pyrethroids.Graphical

Metagenomic sequencing characterizes a wide diversity of viruses in field mosquito samples in Nigeria

Mosquito vectors are a tremendous public health threat. One in six diseases worldwide is vector-borne transmitted mainly by mosquitoes. In the last couple of years, there have been active Yellow fever virus (YFV) outbreaks in many settings in Nigeria, and nationwide, entomological surveillance has been a significant effort geared towards understanding these outbreaks. In this study, we used a metagenomic sequencing approach to characterize viruses present in vector samples collected during various outbreaks of Yellow fever (YF) in Nigeria between 2017 and 2020. Mosquito samples were grouped into pools of 1 to 50 mosquitoes, each based on species, sex and location. Twenty-five pools of Aedes spp and one pool of Anopheles spp collected from nine states were sequenced and metagenomic analysis was carried out. We identified a wide diversity of viruses belonging to various families in this sample set. Seven different viruses detected included: Fako virus, Phasi Charoen-like virus, Verdadero virus, Chaq like-virus, Aedes aegypti totivirus, cell fusing agent virus and Tesano Aedes virus. Although there are no reports of these viruses being pathogenic, they are an understudied group in the same families and closely related to known pathogenic arboviruses. Our study highlights the power of next generation sequencing in identifying Insect specific viruses (ISVs), and provide insight into mosquito vectors virome in Nigeria.

Rapid Differential Detection of Japanese Encephalitis Virus and Getah Virus in Pigs or Mosquitos by a Duplex TaqMan Real-Time RT-PCR Assay.

Both JEV (Japanese encephalitis virus) and GETV (Getah virus) pose huge threats to the safety of animals and public health. Pigs and mosquitoes play a primary role in JEV and GETV transmission. However, there is no way to quickly distinguish between JEV and GETV. In this study, we established a one-step duplex TaqMan RT-qPCR for rapid identification and detection of JEV and GETV. Primers and probes located in the NS1 gene of JEV and the E2 gene of GETV that could specifically distinguish JEV from GETV were selected for duplex TaqMan RT-qPCR. In duplex real-time RT-qPCR detection, the correlation coefficients (R2) of the two viruses were higher than 0.999. The RT-qPCR assay demonstrated high sensitivity, extreme specificity, and excellent repeatability. Detection of JEV and GETV in field mosquito and pig samples was 100 times and 10 times more sensitive than using traditional PCR, respectively. In addition, the new test took less time and could be completed in under an hour. Clinical sample testing revealed the prevalence of JEV and GETV in mosquitoes and pig herds in China. This complete duplex TaqMan RT-qPCR assay provided a fast, efficient, specific, and sensitive tool for the detection and differentiation of JEV and GETV.

Detection of NS1 protein from dengue virus in excreta and homogenates of wild-caught Aedes aegypti mosquitoes using monoclonal antibodies.

Dengue fever is one of the most devastating infectious diseases worldwide. Development of methods for dengue virus (DENV) detection in mosquitoes to assess prevalence as a preliminary screen for entomological surveillance in endemic regions of DENV will certainly contribute to the control of the disease. A monoclonal antibody against the NS1 (nonstructural protein 1) viral protein was generated using recombinant NS1 protein and used to detect and analyze DENV in both excreta and total homogenates from Aedes aegypti mosquitoes. Results demonstrated expression of NS1 in excreta of DENV laboratory-infected mosquitoes and homogenates from field mosquitoes infected with DENV. The immunodetection method reported here represents a first-line strategy for assessing the prevalence of DENV in mosquitoes, for entomological surveillance in endemic regions of dengue. Detection of DENV prevalence in field mosquitoes could have an impact on vector surveillance measures to interrupt dengue transmission.

Efficacy of the Microbial Larvicide VectoMax&amp;lt;sup&amp;gt;&amp;#174;&amp;lt;/sup&amp;gt;G against &amp;lt;i&amp;gt;Anopheles gambiae&amp;lt;/i&amp;gt; s.l. and &amp;lt;i&amp;gt;Culex&amp;lt;/i&amp;gt; spp. Larvae under Laboratory and Open Field Trial Experiments in the City of Yaound&amp;#233;, Cameroon

Background: With the rapid expansion of insecticide resistance limiting the effectiveness of insecticide-based vector control interventions, integrated control strategies associating larviciding could be appropriate to improve current control efforts. The present experimental study assesses laboratory and field efficacy of the larvicide VectoMax®G on Anopheline and Culicine larval stages in Yaoundé. Methods: The effect of the larvicide VectoMax®G, a combination of Bacillus thuringiensis var. israelensis (Bti) and Bacillus sphaericus (Bs), on larval development was assessed during both laboratory and open field trial experiments. Laboratory experiments permitted the evaluation of five different concentrations with four replicates/experiments. Laboratory experiments were conducted with Anopheles coluzzii “Ngousso” and Culex quinquefasciatus laboratory strains. Open field trials were conducted using sixteen plastic containers with a diameter of 0.31 m buried in an array of four rows with 4 containers each. Distance between rows and between containers in a row was 1 meter. This experiment permitted to test the effect of the microbial larvicide VectoMax®G under operational application conditions on field mosquito populations. Results: The time to induce 100% mortality after exposure to serial concentrations of the larvicide varied according to the dose from 4 - 12 hours for An. coluzzii and 6 - 9 hours for Cx. quinquefasciatus in laboratory experiments. Measurements of the residual activity indicated that all VectoMax®G concentrations were still active after 35 days and killed 86% - 100% of larvae. Lethal dose of VectoMax®G killing 50% of larvae was estimated at 5.24 × 10-8 mg/m2 for An. coluzzii and 1.25 × 10-8 mg/m2 for Cx. quinquefasciatus. The lethal concentration inducing 95% mortality was estimated at 3.13 × 10-7 mg/m2 for An. coluzzii and 2.5 × 10-8 mg/m2 for Cx. quinquefasciatus. Open field trials tests indicated that sub-lethal concentrations of VectoMax®G successfully killed 100% An. gambiae s.l. larvae within 24 hours, while with Culex spp. larvae, 100% mortality was recorded after 48 hours post-treatment. Natural recolonization of water containers by larvae was recorded between 3 and 6 days respectively after the treatment with sublethal doses. Late instar larvae were recorded 5 and 6 days after treatment. When the jars were treated with reference dosage or supra doses of VectoMax®G, recolonization of water containers was observed six days after treatments. No pupae of both species were found 6 and 7 days post-treatment. Conclusions: The study indicated high efficacy of the microbial larvicide VectoMax®G against Anopheline and Culex larvae. Microbial larvicides such as VectoMax®G could be appropriate for controlling mosquito population particularly in areas experiencing high insecticide resistance or outdoor biting mosquitoes.

Urban mosquitoes and filamentous green algae: their biomonitoring role in heavy metal pollution in open drainage channels in Nairobi industrial area, Kenya

BackgroundIndustrial wastewater is a human health hazard upon exposure. Aquatic organisms in contaminated wastewater may accumulate the toxic elements with time. Human population living in informal settlements in Nairobi industrial area risk exposure to such toxic elements. Biomonitoring using aquatic organisms in open drainage channels can be key in metal exposure assessment. Levels of Mercury (Hg), Lead (Pb), Chromium (Cr), Cadmium (Cd), Thallium (Tl), and Nickel (Ni) were established in samples of wastewater, filamentous green algae (Spirogyra) and mosquitoes obtained from open drainage channels in Nairobi industrial area, Kenya.ResultsPb, Cr, & Ni levels ranged from 3.08 to 15.31 µg/l while Tl, Hg, & Cd ranged from 0.05 to 0.12 µg/l in wastewater. The Pb, Cr, Ni, & Cd levels were above WHO, Kenya & US EPA limits for wastewater but Hg was not. Pb, Cr, Tl, & Ni levels in assorted field mosquitoes were 1.3–2.4 times higher than in assorted laboratory-reared mosquitoes. Hg & Cd concentrations in laboratory-reared mosquitoes (0.26 mg/kg & 1.8 mg/kg respectively) were higher than in field mosquitoes (0.048 mg/kg & 0.12 mg/kg respectively). The levels of Pb, Cr, & Ni were distinctively higher in field mosquito samples than in wastewater samples from the same site. Pb, Cr, Ni, Cd & Hg levels in green filamentous Spirogyra algae were 110.62, 29.75, 14.45, 0.44, & 0.057 mg/kg respectively. Correlation for Pb & Hg (r (2) = 0.957; P < 0.05); Cd & Cr (r (2) = 0.985; P < 0.05) in algae samples was noted. The metal concentrations in the samples analyzed were highest in filamentous green algae and least in wastewater.ConclusionWastewater, mosquitoes, and filamentous green algae from open drainage channels and immediate vicinity, in Nairobi industrial area (Kenya) contained Hg, Pb, Cr, Cd, Tl, and Ni. Mosquitoes in urban areas and filamentous green algae in open drainage channels can play a role of metal biomonitoring in wastewater. The potential of urban mosquitoes transferring heavy metals to human population from the contaminated wastewater should be investigated.