Semi-field and surveillance data define the natural diapause timeline for Culex pipiens across the United States.

Reproductive diapause serves as biological mechanism for many insects, including the mosquito Culex pipiens, to overwinter in temperate climates. While Cx. pipiens diapause has been well-studied in the laboratory, the timing and environmental signals that promote diapause under natural conditions are less understood. In this study, we examine laboratory, semi-field, and mosquito surveillance data to define the approximate timeline and seasonal conditions that contribute to Cx. pipiens diapause across the United States. While confirming integral roles of temperature and photoperiod in diapause induction, we also demonstrate the influence of latitude, elevation, and mosquito population genetics in shaping Cx. pipiens diapause incidence across the country. Coinciding with the cessation of WNV activity, these data can have important implications for mosquito control, where targeted efforts prior to diapause induction can decrease mosquito populations and WNV overwintering to reduce mosquito-borne disease incidence the following season.

Wide-Area Larviciding with a Buffalo Turbine® Mist Sprayer and Vectolex® WDG.

Wide-Area Larviciding techniques (WALS™) using aqueous suspensions of Bacillus thuringiensis israelensis and various types of mist sprayers have typically been employed against container-inhabiting species of mosquitoes such as Aedes albopictus and Ae. aegypti in urban peridomestic environments. However, relatively little work has been done to examine the use of WALS-applied aqueous suspensions of Lysinibacillus sphaericus (Ls) against susceptible species of mosquitoes in larger natural habitats such as forest pools, floodwaters, and marshes. This study characterized the spray of an aqueous suspension of VectoLex® WDG (Ls) dispersed with a Buffalo Turbine® Mist Sprayer retrofitted with a Micronair rotary atomizer. The results indicate that when VectoLex WDG is applied similarly to traditional urban WALS methodologies, this material is highly effective at reducing larval mosquito populations in areas holding floodwater (an average 88.5% reduction over 6 wk).

Operational Basin Larvicide Evaluations in Northern Cook County, Illinois During 2019 and 2020.

Over the course of 2019 and 2020, 7 larvicide formulations for use in catch basins were evaluated using a standardized pass/fail protocol. A comparison between 1- and 2-pouch doses of VectoLex® water-soluble pouches (WSP; 10 g per pouch) and 20 g of VectoLex FG (loose granules) suggests that the WSP formulation may have a shorter duration than the FG formulation. Results also suggest that 20 g doses of each of 2 larvicides, Duplex™-G and Sumilarv® 0.5G, may have a minimum effective duration in basins for approximately 40 days. A 20 g dose of Altosid® XR-G and a single briquet Altosid XR each had an observed minimum effective duration of 20 days. Sustain MBG had an observed duration of only 7 wk posttreatment. These observations provide evidence that, in some locations, 20 g granular applications may have a longer effective duration than a single briquet. This work highlights the utility of applying a standardized protocol for routine quality control assessments of the thousands of catch basin larvicide applications performed seasonally by mosquito abatement districts.

Mosquito adaptations to hematophagia impact pathogen transmission.

Mosquito-borne diseases such as Dengue fever, Chikungunya, and Malaria are critical threats to public health in many parts of the world. Female mosquitoes have evolved multiple adaptive mechanisms to hematophagy, including the ability to efficiently draw and digest blood, as well as the ability to eliminate excess fluids and toxic by-products of blood digestion. Pathogenic agents enter the mosquito digestive tract with the blood meal and need to travel through the midgut and into the hemocele in order to reach the salivary glands and infect a new host. Pathogens need to adjust to these hostile gut, hemocele, and salivary gland environments, and when possible influence the physiology and behavior of their hosts to enhance transmission.

Gravid Culex pipiens Exhibit A Reduced Susceptibility to Ultra-Low Volume Adult Control Treatments Under Field Conditions.

In July and August of 2018, a field trial was conducted to examine the effectiveness of the North Shore Mosquito Abatement District's operational ultra-low volume (ULV) adulticide program. Two study sites were selected in Skokie, IL, and treated by truck-based ULV with d-phenothrin and prallethrin synergized with piperonyl butoxide over the course of a month. Natural mosquito populations were sampled via Biogents (BG)-counter baited with CO2 or Alfalfa infusion. The results from this study demonstrate that host-seeking mosquitoes were reduced by 65.3% after ULV treatment while gravid mosquitoes were reduced by only 29.2%. In addition, host-seeking mosquitoes rebounded dramatically (303.1%) 3 days posttreatment while gravid mosquitoes did not (5.7%). Based on the differential effect between gravid and host-seeking mosquitoes, we concluded that the gonotrophic cycle and timing of ULV adulticide operations are important factors affecting the resistance of West Nile virus vectors to pyrethroid exposures.

How micronutrients influence the physiology of mosquitoes.

Micronutrients or non-energetic nutrients (NEN) are needed in reduced amounts, but are essential for many mosquito physiological processes that influence biological traits from vector competence to reproductive capacity. The NEN include amino acids (AA), vitamins, salts, metals and sterols. Free AA plays critical roles controlling most physiological processes, from digestion to reproduction. Particularly proline connects metabolic pathways in energy production, flight physiology and ammonia detoxification. Metal, in particular iron and calcium, salts, sterol and vitamin homeostasis are critical for cell signaling, respiration, metabolism and reproduction. Micronutrient homeostasis influence the symbiotic relationships with microorganisms, having important implications in mosquitoes' nutrition, physiology and behavior, as well as in mosquito immunity and vector competence.

Male Aedes aegypti mosquitoes use JH III transferred during copulation to influence previtellogenic ovary physiology and affect the reproductive output of female mosquitoes.

The effect of male accessory gland substances on female reproductive physiology has been previously described as "activating" egg development. However, no mechanism has been described that can explain how male mosquitoes are able to influence egg development in female mosquitoes. To investigate how male mosquitoes are able to influence ovarian physiology and reproductive output we explored three main questions: (1) Do mating and male accessory gland substances affect ovarian physiology and alter markers of oocyte quality during the previtellogenic resting stage? (2) Does the male accessory gland contain JH III and is JH III transferred to the female during copulation? (3) Finally, does the nutritional history of the male affect the amount of JH III transferred to the female and alter reproductive output? By answering these questions it is clear that male mosquitoes are able to alter the female's resource allocation priorities towards reproduction by transferring JH III during copulation; reducing the rate of previtellogenic resorption and increasing the amount of stored ovarian lipids. These changes improve an individual follicle's likelihood of development after a blood meal. In addition, males maintained under better nutritional conditions make and transfer more JH III, prevent more follicular resorption and realize higher fecundities than other males. Together these results illustrate one mechanism behind the "activating" effect of mating described as well as the role sugar feeding plays in male mosquitoes.

Farnesyl phosphatase, a Corpora allata enzyme involved in juvenile hormone biosynthesis in Aedes aegypti.

BACKGROUND: The juvenile hormones (JHs) are sesquiterpenoid compounds that play a central role in insect reproduction, development and behavior. The late steps of JH III biosynthesis in the mosquito Aedes aegypti involve the hydrolysis of farnesyl pyrophosphate (FPP) to farnesol (FOL), which is then successively oxidized to farnesal and farnesoic acid, methylated to form methyl farnesoate and finally transformed to JH III by a P450 epoxidase. The only recognized FPP phosphatase (FPPase) expressed in the corpora allata (CA) of an insect was recently described in Drosophila melanogaster (DmFPPase). In the present study we sought to molecularly and biochemically characterize the FPP phosphatase responsible for the transformation of FPP into FOL in the CA of A. aegypti. METHODS: A search for orthologs of the DmFPPase in Aedes aegypti led to the identification of 3 putative FPPase paralogs expressed in the CA of the mosquito (AaFPPases-1, -2, and -3). The activities of recombinant AaFPPases were tested against general phosphatase substrates and isoprenoid pyrophosphates. Using a newly developed assay utilizing fluorescent tags, we analyzed AaFPPase activities in CA of sugar and blood-fed females. Double-stranded RNA (dsRNA) was used to evaluate the effect of reduction of AaFPPase mRNAs on JH biosynthesis. CONCLUSIONS: AaFPPase-1 and AaFPPase-2 are members of the NagD family of the Class IIA C2 cap-containing haloalkanoic acid dehalogenase (HAD) super family and efficiently hydrolyzed FPP into FOL. AaFPPase activities were different in CA of sugar and blood-fed females. Injection of dsRNAs resulted in a significant reduction of AaFPPase-1 and AaFPPase-2 mRNAs, but only reduction of AaFPPase-1 caused a significant decrease of JH biosynthesis. These results suggest that AaFPPase-1 is predominantly involved in the catalysis of FPP into FOL in the CA of A. aegypti.

Aldehyde dehydrogenase 3 converts farnesal into farnesoic acid in the corpora allata of mosquitoes.

The juvenile hormones (JHs) play a central role in insect reproduction, development and behavior. Interrupting JH biosynthesis has long been considered a promising strategy for the development of target-specific insecticides. Using a combination of RNAi, in vivo and in vitro studies we characterized the last unknown biosynthetic enzyme of the JH pathway, a fatty aldehyde dehydrogenase (AaALDH3) that oxidizes farnesal into farnesoic acid (FA) in the corpora allata (CA) of mosquitoes. The AaALDH3 is structurally and functionally a NAD(+)-dependent class 3 ALDH showing tissue- and developmental-stage-specific splice variants. Members of the ALDH3 family play critical roles in the development of cancer and Sjögren-Larsson syndrome in humans, but have not been studies in groups other than mammals. Using a newly developed assay utilizing fluorescent tags, we demonstrated that AaALDH3 activity, as well as the concentrations of farnesol, farnesal and FA were different in CA of sugar and blood-fed females. In CA of blood-fed females the low catalytic activity of AaALDH3 limited the flux of precursors and caused a remarkable increase in the pool of farnesal with a decrease in FA and JH synthesis. The accumulation of the potentially toxic farnesal stimulated the activity of a reductase that converted farnesal back into farnesol, resulting in farnesol leaking out of the CA. Our studies indicated AaALDH3 plays a key role in the regulation of JH synthesis in blood-fed females and mosquitoes seem to have developed a "trade-off" system to balance the key role of farnesal as a JH precursor with its potential toxicity.

The fate of follicles after a blood meal is dependent on previtellogenic nutrition and juvenile hormone in Aedes aegypti.

Juvenile hormone (JH) mediates the relationship between fecundity and nutrition during the gonotrophic cycle of the mosquito in three ways: (1) by regulating initial previtellogenic development, (2) by mediating previtellogenic resorption of follicles and (3) by altering intrinsic previtellogenic follicle "quality", physiology, and competitiveness thereby predetermining the fate of follicles after a blood meal. To support a role for JH in mediating the response of ovarian follicles after a blood meal, we explored three main questions: (1) Do changes in nutrition during the previtellogenic resting stage lead to relevant biochemical and molecular changes in the previtellogenic ovary? (2) Do hormonal manipulations during the previtellogenic resting stage lead to the same biochemical and molecular changes? (3) Does nutrition and hormones during the previtellogenic resting stage affect vitellogenic resorption and reproductive output? We examined the accumulation of neutral lipids in the previtellogenic ovary as well as the previtellogenic expression of genes integral to endocytosis and oocyte development such as the: vitellogenin receptor (AaVgR), lipophorin receptor (AaLpRov), heavy-chain clathrin (AaCHC), and ribosomal protein L32 (rpL32) under various previtellogenic nutritional and hormonal conditions. mRNA abundance and neutral lipid content increased within the previtellogenic ovary as previtellogenic mosquitoes were offered increasing sucrose concentrations. Methoprene application mimicked the effect of offering the highest sucrose concentrations on mRNA abundance and lipid accumulation in the previtellogenic ovary. These same nutritional and hormonal manipulations altered the extent of vitellogenic resorption. Mosquitoes offered 20% sucrose during the previtellogenic resting stage had nearly 3 times less vitellogenic resorption than mosquitoes offered 3% sucrose despite taking smaller blood meals and developed ∼10% more eggs during the first gonotrophic cycle. Mosquitoes treated with JH III during the previtellogenic resting stage and then offered a blood meal had a ∼40% reduction in the amount of vitellogenic resorption and developed ∼12% more eggs. Taken together, these results suggest that previtellogenic nutrition alters the extent and pattern of resorption after a blood meal through the effect of JH on mRNA abundance and lipid accumulation in previtellogenic follicles.

Nutrient limitation results in juvenile hormone-mediated resorption of previtellogenic ovarian follicles in mosquitoes.

Juvenile hormone (JH) is a central hormonal regulator of previtellogenic development in female Aedes aegypti mosquitoes. JH levels are low at eclosion and increase during the first day after adult emergence. This initial rise in JH is essential for female reproductive maturation. After previtellogenic maturation is complete, the mosquito enters a 'state-of-arrest' during which JH synthesis continues at a slower pace and further ovary development is repressed until a blood meal is taken. By examining the relationships between juvenile hormone, follicular resorption and nutrition in A. aegypti, we were able to define a critical role of JH during the previtellogenic resting stage. The rate of follicular resorption in resting stage mosquitoes is dependent on nutritional quality. Feeding water alone caused the rate of follicular resorption to reach over 20% by day 7 after emergence. Conversely, feeding a 20% sucrose solution caused resorption to remain below 5% during the entire experimental period. Mosquitoes fed 3% sucrose show rates of resorption intermediate between water and 20% sucrose and only reached 10% by day 7 after emergence. Follicular resorption is related to JH levels. Ligated abdomens separated from a source of JH (the corpora allata) showed an increase in resorption comparable to similarly aged starved mosquitoes (16%). Resorption in ligated abdomens was reduced to 6% by application of methoprene. The application of methoprene was also sufficient to prevent resorption in intact mosquitoes starved for 48 h (14% starved vs. 4% starved with methoprene). Additionally, active caspases were localized to resorbing follicles indicating that an apoptotic cell-death mechanism is responsible for follicular resorption during the previtellogenic resting stage. Taken together, these results indicate that JH mediates reproductive trade-offs in resting stage mosquitoes in response to nutrition.