MicroRNA-275 targets sarco/endoplasmic reticulum Ca2+ adenosine triphosphatase (SERCA) to control key functions in the mosquito gut.

The yellow fever mosquito Aedes aegypti is the major vector of arboviruses, causing numerous devastating human diseases, such as dengue and yellow fevers, Chikungunya and Zika. Female mosquitoes need vertebrate blood for egg development, and repeated cycles of blood feeding are tightly linked to pathogen transmission. The mosquito's posterior midgut (gut) is involved in blood digestion and also serves as an entry point for pathogens. Thus, the mosquito gut is an important tissue to investigate. The miRNA aae-miR-275 (miR-275) has been shown to be required for normal blood digestion in the female mosquito; however, the mechanism of its action has remained unknown. Here, we demonstrate that miR-275 directly targets and positively regulates sarco/endoplasmic reticulum Ca2+ adenosine triphosphatase, which is implicated in active transport of Ca2+ from the cytosol to the sarco/endoplasmic reticulum. We utilized a combination of the gut-specific yeast transcription activator protein Gal4/upstream activating sequence (Gal4/UAS) system and miRNA Tough Decoy technology to deplete the endogenous level of miR-275 in guts of transgenic mosquitoes. This gut-specific reduction of miR-275 post blood meal decreased SERCA mRNA and protein levels of the digestive enzyme late trypsin. It also resulted in a significant reduction of gut microbiota. Moreover, the decrease of miR-275 and SERCA correlated with defects in the Notch signaling pathway and assembly of the gut actin cytoskeleton. The adverse phenotypes caused by miR-275 silencing were rescued by injections of miR-275 mimic. Thus, we have discovered that miR-275 directly targets SERCA, and the maintenance of its level is critical for multiple gut functions in mosquitoes.

MicroRNA-8 targets the Wingless signaling pathway in the female mosquito fat body to regulate reproductive processes.

Female mosquitoes require a blood meal for reproduction, and this blood meal provides the underlying mechanism for the spread of many important vector-borne diseases in humans. A deeper understanding of the molecular mechanisms linked to mosquito blood meal processes and reproductive events is of particular importance for devising innovative vector control strategies. We found that the conserved microRNA miR-8 is an essential regulator of mosquito reproductive events. Two strategies to inhibit miR-8 function in vivo were used for functional characterization: systemic antagomir depletion and spatiotemporal inhibition using the miRNA sponge transgenic method in combination with the yeast transcriptional activator gal4 protein/upstream activating sequence system. Depletion of miR-8 in the female mosquito results in defects related to egg development and deposition. We used a multialgorithm approach for miRNA target prediction in mosquito 3' UTRs and experimentally verified secreted wingless-interacting molecule (swim) as an authentic target of miR-8. Our findings demonstrate that miR-8 controls the activity of the long-range Wingless (Wg) signaling by regulating Swim expression in the female fat body. We discovered that the miR-8/Wg axis is critical for the proper secretion of lipophorin and vitellogenin by the fat body and subsequent accumulation of these yolk protein precursors by developing oocytes.

Mosquito-specific microRNA-1174 targets serine hydroxymethyltransferase to control key functions in the gut.

Lineage-specific microRNAs (miRNAs) may contribute to functions specific to hematophagous mosquitoes and, as such, have potential for contributing to the development of future mosquito control approaches. Here we report that the mosquito- and gut-specific miRNA, miR-1174, is required for proper sugar absorption, fluid excretion, blood intake, and, consequently, egg maturation and survival in female mosquitoes. miR-1174 is highly expressed and localized in the posterior midgut, the blood-digesting portion of the mosquito alimentary canal. Depletion of miR-1174 results in severe defects in sugar absorption and blood intake. We identified serine hydroxymethyltransferase (SHMT) is a direct miR-1174 target. The adverse phenotypes caused by miR-1174 silencing were rescued by SHMT RNA interference. Our results suggest that miR-1174 is essential for fine-tuning the SHMT transcript to levels necessary for normal mosquito gut functions.

Mosquito-specific microRNA-1890 targets the juvenile hormone-regulated serine protease JHA15 in the female mosquito gut.

Females of the hematophagous mosquito species require a vertebrate blood meal to supply amino acids and other nutrients necessary for egg development, serving as the driving force for the spread of many vector-borne diseases in humans. Blood digestion utilizes both early and late phase serine proteases (SPs) that are differentially regulated at the transcriptional and post-transcriptional level. To uncover the regulatory complexity of SPs in the female mosquito midgut, we investigated involvement of miRNAs in regulating the juvenile hormone (JH)-controlled chymotrypsin-like SP, JHA15. We identified regulatory regions complementary to the mosquito-specific miRNA, miR-1890, within the 3' UTR of JHA15 mRNA. The level of the JHA15 transcript is highest post eclosion and drastically declines post blood meal (PBM), exhibiting an opposite trend to miR-1890 that peaks at 24 h PBM. Depletion of miR-1890 results in defects in blood digestion, ovary development and egg deposition. JHA15 mRNA and protein levels are elevated in female mosquitoes with miR-1890 inhibition. JHA15 RNA interference in the miR-1890 depletion background alleviates miR-1890 depletion phenotypes. The miR-1890 gene is activated by the 20-hydroxyecdysone pathway that involves the ecdysone receptor and the early genes, E74B and Broad Z2. Our study suggests that miR-1890 controls JHA15 mRNA stability in a stage- and tissue- specific manner.

Evaluation of a novel triple-action adulticide containing a pyrethroid, macrocyclic lactone, and fatty acid against pyrethroid-resistant Aedes aegypti and Culex quinquefasciatus (Diptera: Culicidae).

Insecticide resistance in mosquito populations has long been recognized as a significant global public health challenge, motivating the development of new control chemistries. ReMoa Tri is a novel triple-action space spray that employs a different mode of action than traditional adult mosquito control formulations. It combines 3 components: fenpropathrin, a mixed-type I/II pyrethroid; abamectin, a macrocyclic lactone; and C8910, a patented fatty acid chain. As an ultra-low volume adulticide, ReMoa Tri has the potential to target mosquito species that are resistant to pyrethroid and organophosphate-based control materials. To determine whether ReMoa Tri effectively targets resistant mosquito species in Florida's Collier County, United States, we conducted ground-based field cage trials using field-caught pyrethroid-resistant Culex quinquefasciatus (Say) and Aedes aegypti (L.), of which the latter also displayed developing resistance to organophosphates. Trials were also conducted against the same mosquito populations with Merus 3.0, a pyrethrin-based adulticide used by the Collier Mosquito Control District. ReMoa Tri was effective against Collier's pyrethroid-resistant Cx. quinquefasciatus, resulting in more than 95% mortality in semifield cage trials by 24 h postapplication. Similarly, ReMoa Tri applications against Collier's pyrethroid-resistant Ae. aegypti resulted in 72%-89% mortality at 24 h postapplication and 74%-97% mortality at 48 h postapplication. This study represents the first field data on this novel space spray, and its findings shed light on the performance of ReMoa Tri against local mosquito populations that have developed resistance to currently available adulticides.

Aedes Tortilis, Culex Declarator, and Culex Tarsalis: New County Records for Mosquito Species in Collier County, Florida.

Understanding the distribution of mosquito species is an important element of surveillance. This is especially true in Florida, where detections of nonnative mosquitoes have been increasing. Collier Mosquito Control District performs routine adult mosquito surveillance for operational purposes throughout the year. Here, we report records for 3 species collected in 2021 that had not been documented previously in Collier County, FL: Aedes tortilis, Culex declarator, and Cx. tarsalis. Specimens were initially identified based on morphology, then each species was confirmed by comparing the cytochrome c oxidase subunit I gene sequences to those of other related mosquito species. Although Ae. tortilis and Cx. declarator were collected at multiple sites, Cx. tarsalis was collected only once, making it unclear whether this species has established a permanent population within the county.

Baseline Susceptibility and Effectiveness of Adulticides to Local Aedes Taeniorhynchus From Collier County, Florida.

The black salt marsh mosquito, Aedes taeniorhynchus, is the primary nuisance mosquito in the coastal regions of Florida. This study aimed to establish the baseline susceptibility of Ae. taeniorhynchus to adulticide products used for mosquito control by the Collier Mosquito Control District (CMCD). The Centers for Disease Control and Prevention bottle bioassay was used to test technical-grade and formulated products, and Merus 3.0® was evaluated in semifield cage trials through aerial applications. The results revealed the baseline susceptibility of Ae. taeniorhynchus to the tested materials and the effectiveness of Merus 3.0 to effectively control the Ae. taeniorhynchus. The study provides important information for the development of an integrated mosquito management strategy for controlling Ae. taeniorhynchus mosquito populations in southwest Florida.

Detection of Aedes Scapularis In Southwestern Florida.

Aedes scapularis has recently been detected for the first time in southwestern Florida. During the course of research and surveillance activities by local mosquito control districts in 2020 and 2021, a total of 190 adult females were collected from 14 separate locations in Collier and Lee Counties. To date, Ae. scapularis has been found in 5 counties since its rediscovery in Florida in 2006. Its detection and likely northward expansion into Collier and Lee Counties from the southern Florida Peninsula is in line with ecological niche model predictions that found the environment of the Gulf Coast of southwestern Florida to be highly suitable for the species. Due to its potential ability to transmit both exotic and endemic pathogens such as Everglades virus and Dirofilaria immitis, understanding the range and distribution of Ae. scapularis should be a priority for Florida mosquito control and public health agencies.

Modelling distributions of Aedes aegypti and Aedes albopictus using climate, host density and interspecies competition.

Florida faces the challenge of repeated introduction and autochthonous transmission of arboviruses transmitted by Aedes aegypti and Aedes albopictus. Empirically-based predictive models of the spatial distribution of these species would aid surveillance and vector control efforts. To predict the occurrence and abundance of these species, we fit a mixed-effects zero-inflated negative binomial regression to a mosquito surveillance dataset with records from more than 200,000 trap days, representative of 53% of the land area and ranging from 2004 to 2018 in Florida. We found an asymmetrical competitive interaction between adult populations of Aedes aegypti and Aedes albopictus for the sampled sites. Wind speed was negatively associated with the occurrence and abundance of both vectors. Our model predictions show high accuracy (72.9% to 94.5%) in validation tests leaving out a random 10% subset of sites and data since 2017, suggesting a potential for predicting the distribution of the two Aedes vectors.

The Impact of Hurricane Irma on Our Community and the Collier Mosquito Control District's Mission.

The Collier Mosquito Control District, located in southwest Florida, is uniquely positioned in a subtropical environment between the Gulf of Mexico and Everglades National Park. The District's mission is focused on the control of disease vector and nuisance mosquitoes in Collier County, which is accomplished through integrated mosquito management. Hurricane Irma made landfall in the county on September 10, 2017, leaving in its wake tremendous property and infrastructure damage, and it also disrupted communications and airport operations. These factors greatly affected the District's operations and its ability to meet its mission. In addition, the lengthy loss of electrical power forced most residents outdoors, increasing their exposure to mosquitoes. From challenges in completing poststorm treatments to outdated policies that caught us off-guard, the event prompted a new hurricane policy and plan to ensure improved preparedness for the next natural disaster. The poststorm environment also provided a rich foundation for research into mosquito populations after tropical disturbances of this scale. Here we report the impact on the District's aerial mosquito control operations, changes to internal policies, and mosquito population abundance following Hurricane Irma.

Oxidase, Esterase, and KDR-Associated Pyrethroid Resistance in Culex quinquefasciatus Field Collections of Collier County, Florida.

In several insect species, resistance to pyrethroids and DDT (dichlorodiphenyltrichloroethane) is linked to point mutations in the voltage-gated sodium channel (VGSC) gene. Pyrethroid-based insecticides prolong the opening of sodium channels, causing paralysis known as a "knockdown" effect before mortality occurs. Point mutations in the VGSC gene result in decreased pyrethroid binding and reduced sensitivity to the insecticide-this resistance mechanism is known as knockdown resistance (kdr) as insects do not die but recover from paralysis with time. In Culex mosquito species loss of target site sensitivity to pyrethroids is linked to a number of substitutions, one of which is leucine (L) to phenylalanine (F) at residue 1014 (L1014F) in the VGSC gene. Here we report the identification of kdr-associated pyrethroid resistance and developing resistance in Cx. quinquefasciatus field collections from Collier County, FL. Evaluation of position 1014 of the VGSC in Cx. quinquefasciatus collections from 7 locations in Collier County, FL, revealed a wide range of genotypes from one part of the district to the other. Centers for Disease Control and Prevention bottle bioassay, linear regression analysis, and cage trial evaluations suggest that the L1014F mutation plays a role, at least in part, to the pyrethroid resistance status of Cx. quinquefasciatus collected in Collier County, FL. Furthermore, we identified resistance attributed to both oxidase and esterase activity, indicating that multiple mechanisms are responsible for pyrethroid resistance in Collier County Cx. quinquefasciatus.

Do socioeconomic factors drive Aedes mosquito vectors and their arboviral diseases? A systematic review of dengue, chikungunya, yellow fever, and Zika Virus.

As the threat of arboviral diseases continues to escalate worldwide, the question of, "What types of human communities are at the greatest risk of infection?" persists as a key gap in the existing knowledge of arboviral diseases transmission dynamics. Here, we comprehensively review the existing literature on the socioeconomic drivers of the most common Aedes mosquito-borne diseases and Aedes mosquito presence/abundance. We reviewed a total of 182 studies on dengue viruses (DENV), chikungunya virus (CHIKV), yellow fever virus (YFVV), Zika virus (ZIKV), and presence of Aedes mosquito vectors. In general, associations between socioeconomic conditions and both Aedes-borne diseases and Aedes mosquitoes are highly variable and often location-specific. Although 50% to 60% of studies found greater presence or prevalence of disease or vectors in areas with lower socioeconomic status, approximately half of the remaining studies found either positive or null associations. We discuss the possible causes of this lack of conclusiveness as well as the implications it holds for future research and prevention efforts.

The Impact of Hurricane Irma on Population Density of the Black Salt-Marsh Mosquito, Aedes taeniorhynchus, in Collier County, Florida.

In 2017, the southeastern USA and Caribbean endured an extremely active year for tropical storm and hurricane activity. On September 10, 2017, Hurricane Irma made a 2nd landfall in Florida, striking Collier County-including Naples and Marco Island, FL. Areas affected by Hurricane Irma are abundant in salt-marsh and mangrove swamps, prime habitats for the black salt-marsh mosquito, Aedes taeniorhynchus. Using both human landing rate and Biogents BG-Counter trap data, here we report the year-to-year fluctuations in population density of Ae. taeniorhynchus. In 2017, Collier County experienced a population explosion of Ae. taeniorhynchus, with daily landing rates reaching as high as 150 adult females/2-min period. The following year a dramatic population crash of Ae. taeniorhynchus was observed, representing some of the lowest Ae. taeniorhynchus numbers ever recorded in Collier County. Analysis of human landing rate and trap data following major hurricane strikes, including Hurricane Irma (2017) and Hurricane Wilma (2005), have revealed severe reductions in Ae. taeniorhynchus populations. Because Ae. taeniorhynchus accounts for a large majority of adulticiding missions in the state of Florida, an enhanced understanding of the factors contributing to Ae. taeniorhynchus population dynamics may allow for improved operational planning and decision-making.

Quantification of permethrin resistance and kdr alleles in Florida strains of Aedes aegypti (L.) and Aedes albopictus (Skuse).

Recent outbreaks of locally transmitted dengue and Zika viruses in Florida have placed more emphasis on integrated vector management plans for Aedes aegypti (L.) and Aedes albopictus Skuse. Adulticiding, primarily with pyrethroids, is often employed for the immediate control of potentially arbovirus-infected mosquitoes during outbreak situations. While pyrethroid resistance is common in Ae. aegypti worldwide and testing is recommended by CDC and WHO, resistance to this class of products has not been widely examined or quantified in Florida. To address this information gap, we performed the first study to quantify both pyrethroid resistance and genetic markers of pyrethroid resistance in Ae. aegypti and Ae. albopictus strains in Florida. Using direct topical application to measure intrinsic toxicity, we examined 21 Ae. aegypti strains from 9 counties and found permethrin resistance (resistance ratio (RR) = 6-61-fold) in all strains when compared to the susceptible ORL1952 control strain. Permethrin resistance in five strains of Ae. albopictus was very low (RR<1.6) even when collected from the same containers producing resistant Ae. aegypti. Characterization of two sodium channel kdr alleles associated with pyrethroid-resistance showed widespread distribution in 62 strains of Ae. aegypti. The 1534 phenylalanine to cysteine (F1534C) single nucleotide polymorphism SNP was fixed or nearly fixed in all strains regardless of RR. We observed much more variation in the 1016 valine to isoleucine (V1016I) allele and observed that an increasing frequency of the homozygous V1016I allele correlates strongly with increased RR (Pearson corr = 0.905). In agreement with previous studies, we observed a very low frequency of three kdr genotypes, IIFF, VIFF, and IIFC. In this study, we provide a statewide examination of pyrethroid resistance, and demonstrate that permethrin resistance and the genetic markers for resistance are widely present in FL Ae. aegypti. Resistance testing should be included in an effective management program.

Regulation of physiological processes by microRNAs in insects.

MicroRNAs (miRNAs) are small non-coding RNAs that function in gene regulatory processes in plants and animals by targeting sites within messenger RNA. In insects, miRNAs have been shown to regulate a variety of physiological processes throughout insect development, including molting, metamorphosis, oogenesis, embryogenesis, behavior and host-pathogen interactions. The roles of miRNAs in the model organism, Drosophila melanogaster, have been studied extensively due to the conserved nature of miRNA function among highly divergent species. However, seeking to understand miRNA function in non-drosophilid insect species has become a growing trend in insect science. Here, we highlight the recent discoveries regarding miRNA function in insect physiology and development.

Small RNAs: a new frontier in mosquito biology.

The discovery of small non-coding RNAs has revolutionized our understanding of regulatory networks governing multiple functions in animals and plants. However, our knowledge of mosquito small RNAs is limited. We discuss here the state of current knowledge regarding the roles of small RNAs and their targets in mosquitoes, and describe the ongoing efforts to understand the role of the RNA interference (RNAi) pathway in mosquito antiviral immunity and transposon silencing. Providing a clear picture into the role of small RNAs in mosquito vectors will pave the way to the utilization of these small molecules in developing novel control approaches that target mosquito immunity and/or reproductive events. Elucidation of the functions of small RNAs represents a new frontier in mosquito biology.