Infected Mosquitoes Research Articles

Exploring mosquito abundance and Plasmodium infection through nested-PCR: implications for disease surveillance and control

The Plasmodium is responsible for malaria which poses a major health threat, globally. This study is based on the estimation of the relative abundance of mosquitoes, and finding out the correlations of meteorological parameters (temperature, humidity and rainfall) with the abundance of mosquitoes. In addition, this study also focused on the use of nested PCR (species-specific nucleotide sequences of 18S rRNA genes) to explore the Plasmodium spp. in female Anopheles. In the current study, the percentage relative abundance of Culex mosquitoes was 57.65% and Anopheles 42.34% among the study areas. In addition, the highest number of mosquitoes was found in March in district Mandi Bahauddin at 21 °C (Tmax = 27, Tmin = 15) average temperature, 69% average relative humidity and 131 mm rainfall, and these climatic factors were found to affect the abundance of the mosquitoes, directly or indirectly. Molecular analysis showed that overall, 41.3% of the female Anopheles pools were positive for genus Plasmodium. Among species, the prevalence of Plasmodium (P.) vivax (78.1%) was significantly higher than P. falciparum (21.9%). This study will be helpful in the estimation of future risk of mosquito-borne diseases along with population dynamic of mosquitoes to enhance the effectiveness of vector surveillance and control programs.

Web-based Deep Learning System for Malaria Parasite Detection in Granular Blood Samples

Abstract: Malaria is a serious health concern for modern humans, affecting people of all ages. Infected mosquitoes carry the fatal parasites responsible for malaria. Malaria can be diagnosed by examining a sample of the patient's blood under a microscope for parasites. The project comprises creating a web tool that employs deep learning to detect malaria parasites in blood smear photos. Convolutional neural network (CNN) models such as ResNet50, VGG19, and Customized CNN can be used to collect and categorize a set of blood smear images in order to identify patterns and characteristics. Convolutional layers, maxpooling layers, entirely linked layers, and a SoftMax layer are all utilized to create a Convolutional Neural Network (CNN) model. This technique can improve the accuracy of parasite diagnosis, increase detection rates, and reduce the disease's impact on global health.

A Scoping Review on the Epidemiology of Orthobunyaviruses of Canadian Public and Animal Health Relevance in the Context of Vector Species.

Background: Mosquito-borne orthobunyaviruses are a growing priority for public and animal health in Canada. It is anticipated that disease incidence will increase due to a warming climate, given that habitats are expanding for reservoir hosts and vectors, particularly in Canada. Little is known about the ecology of primary vectors that perpetuate these orthobunyaviruses, including the viral transmission cycle and the impact of climatic and landscape factors. Methods: A scoping review was conducted to describe the current state of knowledge on the epidemiology of orthobunyaviruses relevant to Canada. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews guidelines was used to characterize studies focused on vector species. A literature search was conducted in six databases and gray literature. Eligible studies characterized orthobunyavirus epidemiology related to vector species, including viral competency, geospatial distributions, seasonal trends, and/or risk factors. Results: A total of 1734 unique citations were identified. Screening of these citations revealed 172 relevant studies, from which 87 studies presented primary data related to vectors. The orthobunyaviruses included Cache Valley virus (CVV), Jamestown Canyon virus (JCV), Snowshoe Hare virus (SHV), and La Crosse virus (LACV). Surveillance was the predominant study focus, with most citations representing the United States, specifically, LACV surveillance in Tennessee, followed by CVV and JCV in Connecticut. Orthobunyaviruses were detected in many mosquito species across multiple genera, with high vector specificity only being reported for LACV, which included Aedes triseriatus, Aedes albopictus, and Aedes japonicus. Peridomestic areas were positively associated with infected mosquitoes compared with dense forests. Orthobunyavirus infections, coinfections, and gut microbiota affected mosquito feeding and breeding behavior. Conclusion: Knowledge gaps included Canadian surveillance data, disease modeling, and risk projections. Further research in these areas, especially accounting for climate change, is needed to guide health policy for prevention of orthobunyaviral disease.

Wolbachia invasion dynamics of a random mosquito population model with imperfect maternal transmission and incomplete CI.

In this work, we formulate a random Wolbachia invasion model incorporating the effects of imperfect maternal transmission and incomplete cytoplasmic incompatibility (CI). Under constant environments, we obtain the following results: Firstly, the complete invasion equilibrium of Wolbachia does not exist, and thus the population replacement is not achievable in the case of imperfect maternal transmission; Secondly, imperfect maternal transmission or incomplete CI may obliterate bistability and backward bifurcation, which leads to the failure of Wolbachia invasion, no matter how many infected mosquitoes would be released; Thirdly, the threshold number of the infected mosquitoes to be released would increase with the decrease of the maternal transmission rate or the intensity of CI effect. In random environments, we investigate in detail the Wolbachia invasion dynamics of the random mosquito population model and establish the initial release threshold of infected mosquitoes for successful invasion of Wolbachia into the wild mosquito population. In particular, the existence and stability of invariant probability measures for the establishment and extinction of Wolbachia are determined.

How does the enhanced mortality of Wolbachia-infected immature mosquitoes affect dengue transmission?

How does the enhanced mortality of <i>Wolbachia</i>-infected immature mosquitoes affect dengue transmission?

Estimating the effects of temperature on transmission of the human malaria parasite, Plasmodium falciparum

Despite concern that climate change could increase the human risk to malaria in certain areas, the temperature dependency of malaria transmission is poorly characterized. Here, we use a mechanistic model fitted to experimental data to describe how Plasmodium falciparum infection of the African malaria vector, Anopheles gambiae, is modulated by temperature, including its influences on parasite establishment, conversion efficiency through parasite developmental stages, parasite development rate, and overall vector competence. We use these data, together with estimates of the survival of infected blood-fed mosquitoes, to explore the theoretical influence of temperature on transmission in four locations in Kenya, considering recent conditions and future climate change. Results provide insights into factors limiting transmission in cooler environments and indicate that increases in malaria transmission due to climate warming in areas like the Kenyan Highlands, might be less than previously predicted.

A Comparative Study of Five-Year Dengue Mortality in Madhya Pradesh and Chhattisgarh

Introduction: Dengue is the most widely dispersed mosquito-borne illness, and infected Aedes species mosquitoes convey it. The four dengue virus serotypes (DEN-1, DEN-2, DEN-3, and DEN-4) of the Flavivirus genus cause dengue infection in humans. According to the WHO classification from 1997, there are three different types of symptomatic dengue virus infection: dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS). Dengue fever is prevalent in more than 100 nations, with the majority of cases coming from the WHO’s Western Pacific, South-east Asia, and Americas areas.Methods: This retrospective analytical study was done using the Global Burden of Disease Compare (GBD Compare) tool, where the data of Madhya-Pradesh and Chhattisgarh State for the five-year duration from year 2013 to year 2017 was collected. Factors studied included Dengue Mortality as a percentage of total-all-cause mortalities, its range, and annual percent change (APC).Result: Despite of resonating year-wise results of percentage of total all cause mortalities in MP (0.17, 0.34, 0.19, 0.35, 0.21%) and Chhattisgarh (0.17, 0.38, 0.19, 0.39, 0.21%), there is alarming difference of APC in MP (2.702, 6.11, 2.72, 6.11, 2.72%) and Chhattisgarh (4.19, 4.75, 4.19%,4.75%,4.19%). The mortality and its range are proportionate to APC in MP, whereas APC is found high despite of the mortalities of dengue being same in MP and Chhattisgarh.Conclusion: Addressing the complexities of morbidity and mortality trends requires a multifaceted approach that considers age, gender, and regional differences. By doing so, researchers can contribute to a more nuanced understanding of disease profiles, paving the way for targeted interventions and policies that address the specific health needs of diverse populations.

An evolutionarily conserved ubiquitin ligase drives infection and transmission of flaviviruses

Mosquito-borne flaviviruses such as dengue (DENV) and Zika (ZIKV) cause hundreds of millions of infections annually. The single-stranded RNA genome of flaviviruses is translated into a polyprotein, which is cleaved equally into individual functional proteins. While structural proteins are packaged into progeny virions and released, most of the nonstructural proteins remain intracellular and could become cytotoxic if accumulated over time. However, the mechanism by which nonstructural proteins are maintained at the levels optimal for cellular fitness and viral replication remains unknown. Here, we identified that the ubiquitin E3 ligase HRD1 is essential for flaviviruses infections in both mammalian hosts and mosquitoes. HRD1 directly interacts with flavivirus NS4A and ubiquitylates a conserved lysine residue for ER-associated degradation. This mechanism avoids excessive accumulation of NS4A, which otherwise interrupts the expression of processed flavivirus proteins in the ER. Furthermore, a small-molecule inhibitor of HRD1 named LS-102 effectively interrupts DENV2 infection in both mice and Aedes aegypti mosquitoes, and significantly disturbs DENV transmission from the infected hosts to mosquitoes owing to reduced viremia. Taken together, this study demonstrates that flaviviruses have evolved a sophisticated mechanism to exploit the ubiquitination system to balance the homeostasis of viral proteins for their own advantage and provides a potential therapeutic target to interrupt flavivirus infection and transmission.

The first adolescent case infected with chikungunya virus in South Korea

Chikungunya fever, a viral illness transmitted to humans through the bites of infected mosquitoes, presents with symptoms such as high fever, severe myalgia, headache, arthralgia, rash, and vomiting. This disease predominantly manifests in Southeast Asia, Africa, and Central and South America, with a limited occurrence in Northeast Asia. To date, no such a documented case has been reported in South Korea. Herein, we present the first adolescent case of chikungunya fever in South Korea following a travel to Bali, Indonesia.

Prevalence of Human Malaria Parasite among Pregnant Women Attending Antenatal Care at Eldin Specialist Hospital Jos, Plateau State, Nigeria

Malaria infection is a severe parasitic infectious disease transmitted by the bite of an infected female Anopheles mosquito. The disease presents with severe consequences in pregnant women and children &lt;5 years, particularly in sub-Saharan Africa where the burden of the disease is highest. Malaria in pregnancy may present with severe symptoms such as miscarriages, premature delivery, low-birth-weight neonates, and neonatal death. This study therefore determined the prevalence of malaria infection among pregnant women in Jos Metropolis. Capillary blood samples were collected from 200 symptomatic pregnant women attending Eldin Specialist Hospital, in Jos, and thin and thick blood Giemsa-stained films were prepared and examined for the presence of malaria parasites. Our findings revealed that 70(35%) of the 200 pregnant women sampled were infected. Based on gravidity, incidence of malaria infection was highest in the primigravidae 45 (41.9%) as compared to multigravidae 25 (27.8%) although decreasing with gestational age, as subjects in their first trimester were more infected 44 (44.0%) with the least being those in their third trimester14 (21.5%). Two peaks of infection were encountered in respect to age; 46.2% in subjects aged 20-24 years and 42.9% in subjects aged 30-39 years with the least infection recorded in subjects aged &gt; 39 years, 17.9%. Malaria incidence is decreasing in the country due to scale up intervention and prevention measures, however, malaria still remains a major public health problem among pregnant women and deliberate efforts need to be put in place to forestall the negative impact of this disease.

Penyuluhan Pencegahan Penularan Penyakit Malaria kepada Masyarakat di Desa Kampung Baru Kelurahan Galang Baru Kota Batam

Malaria is an infectious disease caused by parasites of the genus Plasmodium. It targets erythrocytes and manifests with symptoms such as fever, chills, anemia, and splenomegaly, either acutely or chronically, transmitted to humans through the bite of infected female Anopheles mosquitoes. Malaria remains a public health problem in many areas, including Kampung Baru Village, Galang Baru Subdistrict, Batam City. In order to fulfill the mission of community service, we have designed a comprehensive and sustainable education program to increase awareness and knowledge of the community about malaria. This program not only focuses on educating about the prevention and management of the disease but also directly involves active participation of the community in the learning and implementation process. Through collaboration with various parties, such as community leaders, health workers, and local leaders, we aim to create an environment that supports sustainable behavioral change in malaria prevention efforts. Additionally, we integrate a multidisciplinary approach in this program by addressing specific challenges faced in the local context, such as limited access to healthcare services, environmental conditions conducive to the breeding of malaria-carrying mosquitoes, and socio-economic factors influencing prevention and treatment efforts. Thus, this education program aims not only to reduce the burden of malaria in Kampung Baru Village and its surroundings but also serves as a tangible contribution to fulfilling the tri dharma activities of higher education institutions in the field of community service.

Quantification of sporozoite expelling by Anopheles mosquitoes infected with laboratory and naturally circulating P. falciparum gametocytes

It is currently unknown whether all Plasmodium falciparum-infected mosquitoes are equally infectious. We assessed sporogonic development using cultured gametocytes in the Netherlands and naturally circulating strains in Burkina Faso. We quantified the number of sporozoites expelled into artificial skin in relation to intact oocysts, ruptured oocysts, and residual salivary gland sporozoites. In laboratory conditions, higher total sporozoite burden was associated with shorter duration of sporogony (p&lt;0.001). Overall, 53% (116/216) of infected Anopheles stephensi mosquitoes expelled sporozoites into artificial skin with a median of 136 expelled sporozoites (interquartile range [IQR], 34–501). There was a strong positive correlation between ruptured oocyst number and salivary gland sporozoite load (ρ = 0.8; p&lt;0.0001) and a weaker positive correlation between salivary gland sporozoite load and number of sporozoites expelled (ρ = 0.35; p=0.0002). In Burkina Faso, Anopheles coluzzii mosquitoes were infected by natural gametocyte carriers. Among salivary gland sporozoite positive mosquitoes, 89% (33/37) expelled sporozoites with a median of 1035 expelled sporozoites (IQR, 171–2969). Again, we observed a strong correlation between ruptured oocyst number and salivary gland sporozoite load (ρ = 0.9; p&lt;0.0001) and a positive correlation between salivary gland sporozoite load and the number of sporozoites expelled (ρ = 0.7; p&lt;0.0001). Several mosquitoes expelled multiple parasite clones during probing. Whilst sporozoite expelling was regularly observed from mosquitoes with low infection burdens, our findings indicate that mosquito infection burden is positively associated with the number of expelled sporozoites. Future work is required to determine the direct implications of these findings for transmission potential.

Quantification of sporozoite expelling by Anopheles mosquitoes infected with laboratory and naturally circulating P. falciparum gametocytes.

It is currently unknown whether all Plasmodium falciparum-infected mosquitoes are equally infectious. We assessed sporogonic development using cultured gametocytes in the Netherlands and naturally circulating strains in Burkina Faso. We quantified the number of sporozoites expelled into artificial skin in relation to intact oocysts, ruptured oocysts, and residual salivary gland sporozoites. In laboratory conditions, higher total sporozoite burden was associated with shorter duration of sporogony (p<0.001). Overall, 53% (116/216) of infected Anopheles stephensi mosquitoes expelled sporozoites into artificial skin with a median of 136 expelled sporozoites (interquartile range [IQR], 34-501). There was a strong positive correlation between ruptured oocyst number and salivary gland sporozoite load (ρ = 0.8; p<0.0001) and a weaker positive correlation between salivary gland sporozoite load and number of sporozoites expelled (ρ = 0.35; p=0.0002). In Burkina Faso, Anopheles coluzzii mosquitoes were infected by natural gametocyte carriers. Among salivary gland sporozoite positive mosquitoes, 89% (33/37) expelled sporozoites with a median of 1035 expelled sporozoites (IQR, 171-2969). Again, we observed a strong correlation between ruptured oocyst number and salivary gland sporozoite load (ρ = 0.9; p<0.0001) and a positive correlation between salivary gland sporozoite load and the number of sporozoites expelled (ρ = 0.7; p<0.0001). Several mosquitoes expelled multiple parasite clones during probing. Whilst sporozoite expelling was regularly observed from mosquitoes with low infection burdens, our findings indicate that mosquito infection burden is positively associated with the number of expelled sporozoites. Future work is required to determine the direct implications of these findings for transmission potential.

Increased threat of urban arboviral diseases from Aedes aegypti mosquitoes in Colombia

ObjectivesOur study targets the potential of the local urban mosquito Aedes aegypti to experimentally transmit chikungunya virus (CHIKV), dengue virus (DENV), yellow fever virus (YFV), and Zika virus (ZIKV). MethodsWe collected eggs and adults of Ae. aegypti in Medellín, Colombia (from February to March 2020) for mosquito experimental infections with DENV, CHIKV, YFV and ZIKV and viral detection using the BioMark Dynamic arrays system. ResultsWe show that Ae. aegypti from Medellín was more prone to become infected, to disseminate and transmit CHIKV and ZIKV than DENV and YFV. ConclusionsThus, in Colombia, chikungunya is the most serious threat to public health based on our vector competence data.

Entomovirological investigation of mosquitoes (Diptera: culicidae) in areas with yellow fever epizootics in Goiânia, Goias, Brazil

The objective of this investigation was to analyze the occurrence and natural infection of mosquitoes in areas with epizootics of non-human primates (NHP) confirmed for yellow fever in the municipality of Goiânia, Goiás, Brazil from November 2020 to April 2021. This is an ecological, descriptive, retrospective study, with quantitative and qualitative data on the mosquito fauna collected in 10 areas. A total of 2,787 Culicidae were collected and allocated in 8 genera and 23 species. Of the total specimens captured, 2,341 (84%) were females and 446 males (16%). Haemagogus janthinomys occurred in 2.7% (74) of the cases and Haemagogus leucocelaenus in 4.5% (125); Sabethes chlropterus occurred in 0.1% (3) of the cases, Sabethes albiprivus in 1% (28), and Sabethes glaucodaemon in 0.3% (8); Aedes aegypti occurred in 19.7% (548) and Aedes albopictus in 3.9% (108) of the cases. The Sa. glaucodaemon species was recorded for the first time in Goiânia. Yellow fever infection was not detected in the collected specimens. We conclude that the presence of wild vectors, in addition to potential urban vectors, enabled the classification of receptive and vulnerable areas to the circulation of the yellow fever virus.&#x0D; KEYWORDS: Entomological surveillance; vector mosquitoes; epizootics; yellow fever.

Accelerating targeted mosquito control efforts through mobile West Nile virus detection.

Different mosquito control strategies have been implemented to mitigate or prevent mosquito-related public health situations. Modern mosquito control largely relies on multiple approaches, including targeted, specific treatments. Given this, it is becoming increasingly important to supplement these activities with rapid and mobile diagnostic capacities for mosquito-borne diseases. We aimed to create and test the applicability of a rapid diagnostic system for West Nile virus that can be used under field conditions. In this pilot study, various types of adult mosquito traps were applied within the regular mosquito monitoring activity framework for mosquito control. Then, the captured specimens were used for the detection of West Nile virus RNA under field conditions with a portable qRT-PCR approach within 3-4h. Then, positive samples were subjected to confirmatory RT-PCR or NGS sequencing in the laboratory to obtain genome information of the virus. We implemented phylogenetic analysis to characterize circulating strains. A total of 356 mosquito individuals representing 7 species were processed in 54 pools, each containing up to 20 individuals. These pools were tested for the presence of West Nile virus, and two pools tested positive, containing specimens from the Culex pipiens and Anopheles atroparvus mosquito species. As a result of subsequent sequencing, we present the complete genome of West Nile virus and Bagaza virus. The rapid identification of infected mosquitoes is the most important component of quick response adulticide or larvicide treatments to prevent human cases. The conceptual framework of real-time surveillance can be optimized for other pathogens and situations not only in relation to West Nile virus. We present an early warning system for mosquito-borne diseases and demonstrate its application to aid rapid-response mosquito control actions.

Comparison of different trapping methods to collect malaria vectors indoors and outdoors in western Kenya

BackgroundVector surveillance is among the World Health Organization global vector control response (2017–2030) pillars. Human landing catches are a gold standard but difficult to implement and potentially expose collectors to malaria infection. Other methods like light traps, pyrethrum spray catches and aspiration are less expensive and less risky to collectors.MethodsThree mosquito sampling methods (UV light traps, CDC light traps and Prokopack aspiration) were evaluated against human landing catches (HLC) in two villages of Rarieda sub-county, Siaya County, Kenya. UV-LTs, CDC-LTs and HLCs were conducted hourly between 17:00 and 07:00. Aspiration was done indoors and outdoors between 07:00 and 11:00 a.m. Analyses of mosquito densities, species abundance and sporozoite infectivity were performed across all sampling methods. Species identification PCR and ELISAs were done for Anopheles gambiae and Anopheles funestus complexes and data analysis was done in R.ResultsAnopheles mosquitoes sampled from 608 trapping efforts were 5,370 constituting 70.3% Anopheles funestus sensu lato (s.l.), 19.7% Anopheles coustani and 7.2% An. gambiae s.l. 93.8% of An. funestus s.l. were An. funestus sensu stricto (s.s.) and 97.8% of An. gambiae s.l. were Anopheles arabiensis. Only An. funestus were sporozoite positive with 3.1% infection prevalence. Indoors, aspiration captured higher An. funestus (mean = 6.74; RR = 8.83, P < 0.001) then UV-LT (mean = 3.70; RR = 3.97, P < 0.001) and CDC-LT (mean = 1.74; RR = 1.89, P = 0.03) compared to HLC. UV-LT and CDC-LT indoors captured averagely 0.18 An. arabiensis RR = 5.75, P = 0.028 and RR = 5.87, P = 0.028 respectively. Outdoors, UV-LT collected significantly higher Anopheles mosquitoes compared to HLC (An. funestus: RR = 5.18, P < 0.001; An. arabiensis: RR = 15.64, P = 0.009; An. coustani: RR = 11.65, P < 0.001). Anopheles funestus hourly biting indoors in UV-LT and CDC-LT indicated different peaks compared to HLC.ConclusionsAnopheles funestus remains the predominant mosquito species. More mosquitoes were collected using aspiration, CDC-LTs and UV-LTs indoors and UV-LTs and CD-LTs outdoors compared to HLCs. UV-LTs collected more mosquitoes than CDC-LTs. The varied trends observed at different times of the night suggest that these methods collect mosquitoes with diverse activities and care must be taken when interpreting the results.

Assessment of Mayaro virus vector competence of the mosquito Aedes aegypti (Linnaeus, 1762) populations in Argentine using dose-response assays.

Mayaro virus (MAYV; Alphavirus: Togaviridae) is an emerging pathogen in Latin America, causing fever and polyarthritis. Sporadic outbreaks of MAYV have occurred in the region, with reported human cases being imported to Europe and North America. Although primarily a risk for those residing in the Amazon basin's tropical forests, recent reports highlight that urbanization would increase the risk of MAYV transmission in Latin America. Urban emergence depends on human susceptibility and the ability of mosquitos like Aedes aegypti (Linnaeus, 1762) (Diptera: Culicidae) to transmit MAYV. Despite the absence of active MAYV transmission in Argentine, the risk of introduction is substantial due to human movement and the presence of Ae. aegypti in the region. This study aimed to evaluate the susceptibility of different Argentine Ae. aegypti populations to MAYV genotype L (MAYV-L) using dose-response assays and determine barriers to virus infection, dissemination and transmission. Immature mosquito stages were collected in Buenos Aires, Córdoba and Rosario cities. Female Ae. aegypti (F2) were orally infected by feeding on five concentrations of MAYV-L, ranging from 1.0 to 6.0 log10 PFU/mL. Abdomens, legs and saliva were analysed using viral plaque assays. Results revealed that MAYV-L between infection and dissemination were associated with viral doses rather than the population origin. Infection rates varied between 3% and 65%, with a 50% infectious dose >5.5 log10 PFU/mL. Dissemination occurred at 39%, with a 50% dissemination dose of ~6.0 log10 PFU/mL. Dissemination among infected mosquitoes ranged from 60% to 86%, and transmission from disseminated mosquitoes ranged from 11% to 20%. Argentine Ae. aegypti populations exhibited a need for higher viral doses of MAYV-L than those typically found in humans to become infected. In addition, only a small proportion of infected mosquitoes were capable of transmitting the virus. Understanding MAYV transmission in urban areas is crucial for public health interventions.

Alpha-mannosidase-2 modulates arbovirus infection in a pathogen- and Wolbachia-specific manner in Aedes aegypti mosquitoes.

Multiple Wolbachia strains can block pathogen infection, replication and/or transmission in Aedes aegypti mosquitoes under both laboratory and field conditions. However, Wolbachia effects on pathogens can be highly variable across systems and the factors governing this variability are not well understood. It is increasingly clear that the mosquito host is not a passive player in which Wolbachia governs pathogen transmission phenotypes; rather, the genetics of the host can significantly modulate Wolbachia-mediated pathogen blocking. Specifically, previous work linked variation in Wolbachia pathogen blocking to polymorphisms in the mosquito alpha-mannosidase-2 (αMan2) gene. Here we use CRISPR-Cas9 mutagenesis to functionally test this association. We developed αMan2 knockouts and examined effects on both Wolbachia and virus levels, using dengue virus (DENV; Flaviviridae) and Mayaro virus (MAYV; Togaviridae). Wolbachia titres were significantly elevated in αMan2 knockout (KO) mosquitoes, but there were complex interactions with virus infection and replication. In Wolbachia-uninfected mosquitoes, the αMan2 KO mutation was associated with decreased DENV titres, but in a Wolbachia-infected background, the αMan2 KO mutation significantly increased virus titres. In contrast, the αMan2 KO mutation significantly increased MAYV replication in Wolbachia-uninfected mosquitoes and did not affect Wolbachia-mediated virus blocking. These results demonstrate that αMan2 modulates arbovirus infection in A. aegypti mosquitoes in a pathogen- and Wolbachia-specific manner, and that Wolbachia-mediated pathogen blocking is a complex phenotype dependent on the mosquito host genotype and the pathogen. These results have a significant impact for the design and use of Wolbachia-based strategies to control vector-borne pathogens.

Interkingdom interactions shape the fungal microbiome of mosquitoes

BackgroundThe mosquito microbiome is an important modulator of vector competence and vectoral capacity. Unlike the extensively studied bacterial microbiome, fungal communities in the mosquito microbiome (the mycobiome) remain largely unexplored. To work towards getting an improved understanding of the fungi associated with mosquitoes, we sequenced the mycobiome of three field-collected and laboratory-reared mosquito species (Aedes albopictus, Aedes aegypti, and Culex quinquefasciatus).ResultsOur analysis showed both environment and host species were contributing to the diversity of the fungal microbiome of mosquitoes. When comparing species, Ae. albopictus possessed a higher number of diverse fungal taxa than Cx. quinquefasciatus, while strikingly less than 1% of reads from Ae. aegypti samples were fungal. Fungal reads from Ae. aegypti were < 1% even after inhibiting host amplification using a PNA blocker, indicating that this species lacked a significant fungal microbiome that was amplified using this sequencing approach. Using a mono-association mosquito infection model, we confirmed that mosquito-derived fungal isolates colonize Aedes mosquitoes and support growth and development at comparable rates to their bacterial counterparts. Strikingly, native bacterial taxa isolated from mosquitoes impeded the colonization of symbiotic fungi in Ae. aegypti suggesting interkingdom interactions shape fungal microbiome communities.ConclusionCollectively, this study adds to our understanding of the fungal microbiome of different mosquito species, that these fungal microbes support growth and development, and highlights that microbial interactions underpin fungal colonization of these medically relevent species.