Pesticide contamination is a threat to many aquatic habitats, and runoff from residential homes is a major contributor of these chemicals in urban surface streams and estuaries. Improved understanding of their fate and transport can help identify areas of concern for monitoring and management. In many urban areas, runoff water congregates in numerous underground catch basins before draining into the open environment; however, at present essentially no information is available on pesticide presence in these systems. In this study, we collected water samples from a large number of underground urban catch basins in different regions of California during the active pest management season to determine the occurrence and profile of the widely used pyrethroid in-secticides. Detectable levels of pyrethroids were found in 98% of the samples, and the detection frequency of individual pyrethroids ranged from no detection for fenpropathrin to 97% for bifenthrin. In the aqueous phase, total pyrethroid concentrations ranged from 3 to 726 ng/L, with a median value of 32 ng/L. Pyrethroids were found to be enriched on suspended solids, with total concentrations ranging from 42 to 93,600 ng/g and a median value of 2,350 ng/g. In approximately 89% of the samples, whole water concentrations of bifenthrin were predicted to have toxic units >1 for sensitive aquatic invertebrates. The high detection frequency of bifenthrin and overall pyrethroid concentrations, especially for particle-bound residues, suggest that under-ground urban catch basins constitute an important secondary source for extended and widespread contamination of downstream surface waters by pesticides such as pyrethroids in urban regions.

Pyrethroid insecticides are widely used to control mosquitoes that transmit pathogens such as West Nile virus (WNV) to people. Single nucleotide polymorphisms (SNP) in the knockdown resistance locus ( kdr ) of the voltage gated sodium channel ( Vgsc ) gene in Culex mosquitoes are associated with knockdown resistance to pyrethroids. RNAseq was used to sequence the coding region of Vgsc for Culex tarsalis Coquillett and Culex erythrothorax Dyar, two WNV vectors. The cDNA sequences were used to develop a quantitative reverse transcriptase PCR assay that detects the L1014F kdr mutation in the Vgsc . Because this locus is conserved, the assay was used successfully in six Culex spp . The resulting Culex RT kdr assay was validated using quantitative PCR and sequencing of PCR products. The accuracy of the Culex RT kdr assay was 99%. The L1014F kdr mutation associated with pyrethroid resistance was more common among Cx . pipiens than other Culex spp. and was more prevalent in mosquitoes collected near farmland. The Culex RT kdr assay takes advantage of the RNA that vector control agencies routinely isolate to assess arbovirus prevalence in mosquitoes. We anticipate that public health and vector control agencies may employ the Culex RT kdr assay to define the geographic distribution of the L1014F kdr mutation in Culex species and improve the monitoring of insecticide resistance that will ultimately contribute to effective control of Culex mosquitoes.

This synthesis is a short introduction to the Wetlands and Mosquito Control special issue of Wetlands Ecology and Management. The geographic extent of the articles in this Special Issue comprises four continents (America, Asia, Australia, and Europe) indicating a global interest in the issue of wetland ecology and mosquito control. The unifying theme across these diverse papers is the increasingly close collaboration between ecologists, mosquito control, and regulators. Continuing anthropogenic effects on the wetland habitat and demand for mosquito control call for close cooperation between environmental and mosquito control communities. The new generation of wetland managers and mosquito control practitioners understands and accepts the common and compatible goals of preserving the remaining wetlands while addressing the public health concerns. This special issue is dedicated to Bill Walton (1956–2020) who was the driving force behind this endeavor. His career and research illustrate the close relationship between mosquito control and wetland ecology showcasing on how integrating wetland restoration, sustainable mosquito control efforts, and the social and cultural values of the communities will be crucial for the success of wetland management and mosquito control in the twenty-first century.

Mist sprayers (MS) are being rapidly adopted nationwide for applying larvicides to control peridomestic Aedes aegypti. Because MS can loft large quantities of larvicide over relatively long distances, we examined its efficacy in a tidal marsh habitat for controlling Ae. dorsalis. Liquid Vectobac 12AS larvicide, containing Bacillus thuringiensis israelensis, was applied at 1.16 liter/ha using a MS. Cards that change color when exposed to liquid were placed perpendicular to the path of the MS showed that the larvicide mist traveled up to 60 m from the MS and did not extend to 90 m. Use of the MS enabled a 4-fold increase in total hectares treated during 2020-21 relative to the prior 2 years without an increase in staff time. Notably, there was 83% reduction in the quantity of Ae. dorsalis larvae at 5 days posttreatment. Similarly, there was 63% reduction in adult female Ae. dorsalis that were collected in encephalitis virus surveillance traps from nearby communities relative to the prior 2 years. There were 2.3-fold fewer requests for service to address a mosquito problem from residents of communities that abut the tidal marshes, suggesting the applications had a positive impact on these communities. The MS offer an attractive alternative to hand treatments in tidal marshes where the use of unmanned aircraft or all-terrain vehicles is prohibited by national wildlife refuge managers.

Mosquitoes are major infectious disease-carrying vectors. Assessment of current and future risks associated with the mosquito population requires knowledge of the full repertoire of pathogens they carry, including novel viruses, as well as their blood meal sources. Unbiased metatranscriptomic sequencing of individual mosquitoes offers a straightforward, rapid, and quantitative means to acquire this information. Here, we profile 148 diverse wild-caught mosquitoes collected in California and detect sequences from eukaryotes, prokaryotes, 24 known and 46 novel viral species. Importantly, sequencing individuals greatly enhanced the value of the biological information obtained. It allowed us to (a) speciate host mosquito, (b) compute the prevalence of each microbe and recognize a high frequency of viral co-infections, (c) associate animal pathogens with specific blood meal sources, and (d) apply simple co-occurrence methods to recover previously undetected components of highly prevalent segmented viruses. In the context of emerging diseases, where knowledge about vectors, pathogens, and reservoirs is lacking, the approaches described here can provide actionable information for public health surveillance and intervention decisions.

The San Francisco Bay Area is a leader in environmental stewardship and home to numerous wetland restoration projects including the largest tidal wetland restoration project on the American West Coast. As tidal marsh wetlands are restored throughout the Bay Area many opportunities remain to reaffirm the importance of water management that reduces mosquito production and protects public health. Unlike the early 1900s when long term saltmarsh mosquito control was achieved with large scale surface water management projects, regulatory restrictions produce new hurdles that impact mosquito control and restoration projects alike. Work done in the wetlands surrounding the San Francisco Bay must comply with existing management plans, permit requirements, and government regulations. The same is true for emerging technologies. While unmanned airsystems employed for mosquito control improves efficiency and accuracy, regulations in this arena limit their broad use in wetlands that abut the San Francisco Bay. Mosquito abatement districts collect substantial scientific data that inform land management and mosquito control operations. This information is useful for evaluating wetland restoration progress in the Bay Area and fostering partnerships that keep a public health perspective at the forefront.

Organized mosquito control programs (MCP) in the United States have been protecting public health since the early 1900s. These programs utilize integrated mosquito management for surveillance and control measures to enhance quality of life and protect the public from mosquito-borne diseases. Because much of the equipment and insecticides are developed for agriculture, MCP are left to innovate and adapt what is available to accomplish their core missions. Unmanned aerial systems (UAS) are one such innovation that are quickly being adopted by MCP. The advantages of UAS are no longer conjectural. In addition to locating mosquito larval habitats, UAS affords MCP real-time imagery, improved accuracy of aerial insecticide applications, mosquito larval detection and sampling. UAS are also leveraged for applying larvicides to water in habitats that range in size from multi-acre wetlands to small containers in urban settings. Employing UAS can reduce staff exposure to hazards and the impact associated with the use of heavy equipment in sensitive habitats. UAS are utilized by MCP nationally and their use will continue to increase as technology advances and regulations change. Current impediments include a dearth of major UAS manufacturers of equipment that is tailor-made for mosquito control, pesticides that are optimized for application via UAS and regulations that limit the access of UAS to national airspace. This manuscript highlights the strengths and weaknesses of UAS within MCP, provides an update on systems and methods used, and charts the future direction of UAS technology within MCP tasked with public health protection.

Pyrethroid insecticides are widely used to control mosquitoes that transmit diseases such as West Nile virus (WNV) to humans. A single nucleotide polymorphism (SNP) in the knockdown resistance locus ( kdr ) of the voltage gated sodium channel ( Vgsc ) gene of Culex mosquitoes confers knockdown resistance to pyrethroids. PCR-based assays that detect these SNPs in Culex species are currently available for Culex pipiens Linnaeus and Culex quinquefasciatus Say. RNAseq was employed to sequence the coding region of Vgsc for Culex tarsalis Coquillett and Culex erythrothorax Dyar, two WNV vectors. We utilized the cDNA sequence to develop a quantitative reverse transcriptase PCR assay that detects the L1014F mutation in the kdr of V gsc . Because this locus is conserved, the assay successfully detected the SNPs in multiple Culex spp . vectors of WNV in the United States. The resulting Culex RT kdr assay was validated using quantitative PCR, CDC bottle bioassays, and sequencing of PCR products. Using sequencing, we determined the accuracy of the Culex RT kdr assay was 99%. Pyrethroid resistance was more common among Cx. pipiens than other Culex spp. and co-occured with agriculture. We anticipate that public health and vector control agencies may utilize the Culex RT kdr assay to map the distribution of pyrethroid resistance in Culex species to more efficiently control mosquitoes and the diseases they transmit.

The mosquito Culex erythrothorax Dyar is a West Nile virus (WNV) vector that breeds in wetlands with emergent vegetation. Urbanization and recreational activities near wetlands place humans, birds and mosquitoes in close proximity, increasing the risk of WNV transmission. Adult Cx. erythrothorax abundance peaked in a wetland bordering the San Francisco Bay of California (USA) during the first 3 hours after sunset (5527 ± 4070 mosquitoes / trap night) while peak adult Culex tarsalis Coquillett abundance occurred during the subsequent 3 h period (83 ± 30 Cx. tarsalis). When insecticide resistance was assessed using bottle bioassay, Cx. erythrothorax was highly sensitive to permethrin, naled, and etofenprox insecticides compared to a strain of Culex pipiens that is susceptible to insecticides (LC50 = 0.35, 0.71, and 4.1 μg/bottle, respectively). The Cx. erythrothorax were 2.8-fold more resistant to resmethrin, however, the LC50 value was low (0.68 μg/bottle). Piperonyl butoxide increased the toxicity of permethrin (0.5 μg/bottle) and reduced knock down time, but a higher permethrin concentration (2.0 μg/bottle) did not have similar effects. Bulk mixed-function oxidase, alpha-esterase, or beta-esterase activities in mosquito homogenates were higher in Cx. erythrothorax relative to the Cx. pipiens susceptible strain. There was no difference in the activity of glutathione S-transferase between the two mosquito species and insensitive acetylcholine esterase was not detected. Larvicides that were applied to the site had limited impact on reducing mosquito abundance. Subsequent removal of emergent vegetation in concert with larvicide applications and reduced daily environmental temperature substantially reduced mosquito abundance. To control Cx. erythrothorax in wetlands, land managers should consider vegetation removal so that larvicide can efficiently enter the water. Vector control agencies may more successfully control adult viremic Cx. erythrothorax that enter nearby neighborhoods by applying adulticides during the 3 h that follow sunset.

Mosquitoes are a disease vector with a complex ecology involving interactions between transmissible pathogens, endogenous microbiota, and human and animal blood meal sources. Unbiased metatranscriptomic sequencing of individual mosquitoes offers a straightforward and rapid way to characterize these dynamics. Here, we profile 148 diverse wild-caught mosquitoes collected in California, detecting sequences from eukaryotes, prokaryotes, and over 70 known and novel viral species. Because we sequenced singletons, it was possible to compute the prevalence of each microbe and recognize a high frequency of viral co-infection. By analyzing the pattern of co-occurrence of sequences across samples, we associated “dark matter” sequences with recognizable viral polymerases, and animal pathogens with specific blood meal sources. We were also able to detect frequent genetic reassortment events in a highly prevalent quaranjavirus undergoing a recent intercontinental sweep. In the context of an emerging disease, where knowledge about vectors, pathogens, and reservoirs is lacking, the approaches described here can provide actionable information for public health surveillance and intervention decisions.

The mosquito Culex erythrothorax Dyar is a West Nile virus (WNV) vector that breeds in wetlands with emergent vegetation. Urbanization and recreational activities near wetlands place humans, birds and mosquitoes in close proximity, increasing the risk of WNV transmission. Adult Cx. erythrothorax abundance peaked in a wetland bordering the San Francisco Bay of California (USA) during the first 3 hours after sunset (5527 ± 4070 mosquitoes / trap night) while peak adult Culex tarsalis Coquillett abundance occurred during the subsequent 3 h period (83 ± 30 Cx. tarsalis). When insecticide resistance was assessed using bottle bioassay, Cx. erythrothorax was highly sensitive to permethrin, naled, and etofenprox insecticides compared to a strain of Culex pipiens that is susceptible to insecticides (LC50 = 0.35, 0.71, and 4.1 μg/bottle, respectively). The Cx. erythrothorax were 2.8-fold more resistant to resmethrin, however, the LC50 value was low (0.68 μg/bottle). Piperonyl butoxide increased the toxicity of permethrin (0.5 μg/bottle) and reduced knock down time, but a higher permethrin concentration (2.0 μg/bottle) did not have similar effects. Bulk mixed-function oxidase, alpha-esterase, or beta-esterase activities in mosquito homogenates were higher in Cx. erythrothorax relative to the Cx. pipiens susceptible strain. There was no difference in the activity of glutathione S-transferase between the two mosquito species and insensitive acetylcholine esterase was not detected. Larvicides that were applied to the site had limited impact on reducing mosquito abundance. Subsequent removal of emergent vegetation in concert with larvicide applications and reduced daily environmental temperature substantially reduced mosquito abundance. To control Cx. erythrothorax in wetlands, land managers should consider vegetation removal so that larvicide can efficiently enter the water. Vector control agencies may more successfully control adult viremic Cx. erythrothorax that enter nearby neighborhoods by applying adulticides during the 3 h that follow sunset.

An unmanned aircraft system (UAS; i.e., drone) with an attached multispectral camera was used to quantify accumulated surface water on a 0.54-km(2) tidal marsh that abuts San Francisco Bay, CA, USA. The results of the survey showed unequal accumulation of surface water and provided information for focused inspections of potential mosquito breeding areas and identified areas where existing ditches needed improvement for increasing water circulation in the marsh to reduce mosquito breeding. The UAS was also outfitted with a high-magnification zoom video camera and piloted at varying heights to measure the video camera's ability to visualize immature mosquitoes in 2 small containers of contrasting colors during simulation tests in a marsh habitat. Immature mosquitoes could be seen clearly in white or black containers at heights up to 14 and 8 m, respectively. An artificial intelligence algorithm identified mosquito larvae and pupae in videos of the white tray with 94.1% and 52.8% accuracy, respectively. Together, our studies show that an UAS equipped with multispectral and zoom cameras provides a means for vector control agencies to rapidly and quantitatively assess the landscape for the presence of surface water and mosquito larvae.

Oviposition cup traps (OCT) are commonly used to detect gravid invasive Aedes mosquitoes. Employing OCT during hot summer months or over broad geographic areas is labor intensive because the water in these small-volume traps must be frequently replenished to maintain their attractiveness to mosquitoes. We developed low-cost and simple-to-build oviposition bucket traps (OBT) that attract mosquitoes for more than 1 wk. Comparison of adjacently placed OCT and OBT in the city of Madera, CA, showed OBT captured significantly more Ae. aegypti eggs per trap-night relative to the OCT (8.8 ± 2.6 and 4.1 ± 1.1, respectively; paired t-test, P = 0.0076), and a significantly greater proportion of OBT contained Ae. aegypti eggs relative to OCT (83% of OBT and 65% of OCT; Fisher's exact test, P = 0.0214). The results suggest that OBT can collect larger quantities of Ae. aegypti eggs relative to OCT while potentially offering greater flexibility in scheduling trap inspections.

A strain of Adoxophyes honmai resistant to Adoxophyes honmai nucleopolyhedrovirus (AdhoNPV) was established from a field-collected colony by repeated selection. Fifth-instar larvae of this resistant strain (R-strain) had over 66 666-fold greater resistance in terms of 50 % lethal concentration values to oral infection of AdhoNPV than non-selected strain larvae (susceptible for AdhoNPV; S2-strain). In this study, the mechanism of resistance to AdhoNPV was determined in R-strain larvae. An assessment of viral genome replication in AdhoNPV-infected S2- and R-strain larvae by quantitative PCR showed no viral genome replication occurring in R-strain larvae. Transcription of AdhoNPV ie-1, vp39 and polyhedrin genes was also not detected in R-strain midgut cells. Besides, a fluorescent brightener had no effect on AdhoNPV infection in either S2- or R-strain. However, binding and fusion of occlusion-derived virus with R-strain were significantly lower than those of S2-strain. These findings suggest that R-strain Adoxophyeshonmai larvae possess a midgut-based resistance to oral infection by AdhoNPV in which midgut epithelial cells are infected less efficiently.

Saccharomyces cerevisiae produces prostaglandin E2 (PGE2) in the presence of arachidonic acid (AA). S. cerevisiae and its metabolites may be consumed in products manufactured using the yeast (e.g. beer) and yeast PGE2 can potentially modify human immune cell functions to exacerbate pre‐existing inflammation. The biochemical pathway utilized by S. cerevisiae to produce PGE2 is unknown. The identification of genes involved in AA metabolism may direct approaches to reduce off‐target effects of yeast PGE2 on immune cells. As S. cerevisiae does not have genes homologous to those involved in mammalian AA metabolism, identification of such genes will reveal a novel pathway for lipid biosynthesis. RNAseq transcriptome sequencing was conducted and we observed 2044 genes upregulated in yeast cultured with AA relative to the control yeast cultured without AA. Notably, genes encoding mitochondrial proteins that direct fatty acid beta‐oxidation (CRC1, YAT1 and CEM1) were upregulated 1.1‐3.0‐fold and genes encoding peroxisomal proteins that mediate beta‐oxidation of fatty acids (POT1, POX1, FAA1 and FAA2) were upregulated 1‐2.3‐fold. Upregulation of fatty acid metabolizing genes points to enzymes that may metabolize AA to produce PGE2 in S. cerevisiae. Currently, we are testing knock‐out strains of S. cerevisiae that lack the genes identified in the RNA‐seq studies for reduced PGE2 production.Grant Funding Source: Supported by a grant from the National Institutes of Health to E.J.H‐S (SC3GM092298).

Virulence and pathogenesis of the baculovirus Autographa californica M nucleopolyhedrosis virus were quantified in penultimate instar Pseudoplusia includens (soybean looper) larvae using a recombinant encoding the lacZ reporter gene (AcMNPV-hsp70/lacZ). Larvae inoculated orally with AcMNPV-hsp70/lacZ occlusion bodies (OB) or intrahemocoelically with budded virus (BV) were susceptible to fatal infection (LD50 = 40.8 OB; 13.8 BV plaque forming units). Pseudoplusia includens displayed increased developmental resistance as larvae were orally inoculated with OB at later times during the penultimate instar. The optical brightener M2R, an inhibitor of the apoptotic processes that drive developmental resistance, did not significantly affect the virulence of AcMNPV-hsp70/lacZ OB. To study pathogenesis, newly molted penultimate instar P. includens were orally inoculated with 60 OB and examined from 0.5 to 96 h post inoculation (h.p.i). Infection in the midgut was first detected at 4 h.p.i in 32% of the larvae and was apparent in cells of the tracheal system at 8 h.p.i. LacZ-positive (LacZ+) hemocytes were first observed 2 h later. At 18 h.p.i, a low proportion of the hemocytes were LacZ+ (5.6%). However, flow cytometry analysis of cell surface expression of the viral protein GP64 showed that 84.5% of the hemocytes collected at 18 h.p.i were infected with AcMNPV, suggesting that flow cytometry may be a more sensitive method for identifying AcMNPV-infected cells. Because P. includens display no physiological barriers to AcMNPV OB and is permissive to fatal infection, this species could be controlled in organic cropping systems using naturally occurring strains of AcMNPV.

Human apolipoprotein A–I (apoA–I) is a 28kDa protein and a major component of high-density lipoproteins, mediating several essential metabolic functions related to heart disease. In the present study the potential protective role against bacterial pathogens was explored. ApoA–I suppressed bacterial growth of Escherichia coli and Klebsiella pneumoniae. The protein was able to bind lipopolysaccharides and showed a strong preference for bilayer vesicles made of phosphatidylglycerol over phosphatidylcholine. Lysine side chains of apoA–I were acetylated to evaluate the importance of electrostatic forces in the binding interaction with both membrane components. Electrophoresis properties, dot blot analysis, circular dichroism, and fluorescence spectroscopy to probe for changes in protein structure indicated that the acetylated protein displayed a strongly reduced lipopolysaccharide and phosphatidylglycerol binding. A mutant containing only the N-terminal domain of apoA–I also showed a reduced ability to interact with the membrane components, although to a lesser extent. These results indicate the potential for apoA–I to function as an antimicrobial protein and exerts this function through lysine residues.

Antimicrobial peptides are generated in insects exposed to pathogens for combating infection. Gloverin is a small cationic antibacterial protein whose expression is induced in the hemocytes and fat body cells of Trichoplusia ni larvae exposed to bacteria. The purpose of this study was to determine the role of gloverin during baculovirus infection. We found that gloverin expression is induced in T. ni systemically infected with the baculovirus Autographa californica M nucleopolyhedrovirus (AcMNPV). Two gloverin genes were cloned using RNA isolated from the hemocytes of T. ni larvae that were systemically infected with AcMNPV budded virus (BV) and C-terminal 6x-His and V5 epitope tags were incorporated to facilitate gloverin isolation, detection and functional studies. The supernatants of Sf9 cells stably transfected with the two gloverin expression plasmids and affinity purified gloverin proteins reduced the quantity of infectious AcMNPV BV as measured in vitro by plaque assay with untransfected Sf9 cells. Nanomolar concentrations of affinity column purified gloverin protein caused calcein to be rapidly released from unilamellar vesicles comprised of phosphatidylglycerol, but not from vesicles made up of phosphatidylcholine, suggesting that gloverin interaction with membranes is rapid and affected by membrane charge. Both the BV inactivation and calcein release activities of gloverin increased with higher concentrations of gloverin. These results demonstrate that gloverin is an antiviral protein that interacts with vesicle membranes to cause the contents to be released.

Human apolipoprotein A‐I (apoA‐I) is a 28 kDa protein and a major component of high‐density lipoproteins, mediating several essential metabolic functions related to heart disease. Recently, a novel protective role against bacterial pathogens has emerged. ApoA‐I suppressed bacterial growth of log phase Escherichia coli and Klebsiella pneumoniae, resulting in a reduced colony count. We suggest that apoA‐I interacts with gram‐negative bacteria in two ways: 1) by associating with lipopolysaccharides (LPS) which are the major constituent of the outer bacterial membrane, thereby reducing endotoxic effects, and 2) destabilizing the negatively charged phospholipid bilayer of the inner bacterial membrane, resulting in cell lysis. To analyze the role of lysine in LPS and phospholipid binding, lysine side chains were acetylated using acetic anhydride in saturated sodium acetate. Electrophoresis analysis and 1‐anilino‐naphthalene‐8‐sulfonate fluorescence of apoA‐I in the absence or presence of LPS indicated decreased binding upon acetylation. ApoA‐I was able to lyse phosphatidylglycerol vesicles; however, acetylated apoA‐I showed a marked decrease in activity. These results indicate the potential for apoA‐I to function as an antimicrobial protein and the importance of lysine residues.