Pyrethroid Resistance in Culex tarsalis in Regions of Northern California
Presenter: Tara Thiemann PhD, Associate Professor, University of the Pacific with research contributions by graduate students Sumiko De La Vega and Bonnie Ryan
Culex tarsalis is an important vector of West Nile Virus and other arboviruses in the Western US. Once more common in rural areas, Culex tarsalis is now often found in residential areas and faces insecticide pressure both from vector control efforts and agricultural spraying. Until recently, few reports of insecticide resistance in Cx. tarsalis had been reported, but over the last few years that has changed.
To investigate this, Thiemann launched a research project with two main objectives. The first was to determine the prevalence of pyrethroid resistance among mosquitos in select study sites across five counties in Northern California. Once the onset of resistance could be verified, Thiemann wanted to better understand the mechanisms behind it, in particular, the presence of enzymatic resistance and target-site mutations.
Pyrethroids target the nervous system of the mosquito by binding to, and opening, voltage-gated sodium channels (VGSC) in synaptic cells. This binding activity holds those channels open, which in turn increases the influx of sodium into the neurons and starts a chain reaction that overexcites mosquito’s neurons. As a result, the mosquito uses all of its energy contracting muscles and sending action potentials, ultimately leading to death.
One way insecticide resistance develops is when mosquitos adapt and become able to produce more enzymes (esterases and oxidases in the case of pyrethroids) that are able to break down or inhibit the insecticide. A second mechanism is target site mutation. In the latter, a mutation in the insect’s DNA leads to an amino acid substitution that reduces the ability of the insecticide to bind to its target site. For Thiemann and her team, the mutation of interest was Knockdown Resistance (kdr) – mutations that prevent the insecticide from binding to the target site. Two common mutations in mosquitoes relate to changes in the gene that codes for voltage-gated sodium channels, substitutions involving the amino acid Leucine changing to Phenylalanine or Serine.
To first determine the prevalence of resistance, mixed age females were collected using CO2-baited traps. Between three and five populations were collected in each of the collaborating county districts in California: San Joaquin, Sac-Yolo, Placer, and Lake, and were evaluated with the CDC bottle bioassays. Mortality rates were recorded every 15 minutes for a two-hour period.
Among the susceptible (control) populations, results showed high and rapid mortality rates in bottles containing the pyrethroid Permethrin alone as well as in bottles containing Permethrin + PBO (a synergist that inhibits oxidases). While recorded mortality was also high for wild mosquitos in the Permethrin + PBO bottles, mortality rates in the Permethrin only bottles were typically quite low. The results not only confirmed the existence of resistant populations, but also, as Thiemann put it, that the challenge was “a bit more than we expected.” (see Summary of 17 Study Sites).
For the second objective of the study, researchers used enzyme (microplate) assays and molecular testing. In the microplate assays, mosquitoes were ground in a buffer and then added to the plates. A substrate and other reactants were then added to the plates to start a chemical reaction. The resulting product assumed a color varying in intensity depending on how much enzyme activity was in the sample. An instrument was then used to analyze the color to measure the amount of enzyme present.
The team measured for levels for a variety of detoxifying enzymes including oxidases, alpha-esterases, beta-esterases, glutathione-S-transferases (GSTs) and acetylcholinesterase (ACE), but the ensuing data proved difficult to analyze. While levels were elevated among the wild population samples, researchers learned that the control group also showed higher levels of the enzymes than they had anticipated. This made comparative analysis challenging. Thiemann states she is working with a statistician to determine if a different approach might be used to analyze the results.
Testing for target site mutations, however, proved far more definitive.
Thiemann and her team employed SYBR green qPCR molecular assays using three different primers that matched up to the three alleles they were looking for: Leucine, Phenylalanine, and Serine. The results showed a high prevalence of the Leucine to Phenylalanine kdr mutation as well as a lesser amount of the Leucine to Serine mutation (which are linked to insecticide resistance in these mosquito populations).
Intrigued by the results, Thiemann brought some Culex tarsalis samples “out of the freezer” from her grad school days (2006-2008) and was able to closely match them to a few capture sites from the 2018 study. Not only was the comparison conclusive, it was dramatic. Her analysis showed high prevalence of the kdr alleles, particularly the Leucine to Phenylalanine mutation. Moreover, the ten-year comparison showed that samples of kdr alleles had completely reversed from susceptible to a current high prevalence of resistance alleles. (see kdr Allele Prevalent Change Over Time).
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Technical Challenges and Solutions for Low-Rate Applications
Presenter: Banu Kesavaraju PhD, Global Technical Manager, Valent BioSciences
From a high level, low-rate applications have easy-to-understand safety, economic, and environmental impact benefits. However, the benefits of low-rate applications can only be fully realized with the right product in the right environment, and when equipment has been adequately characterized to ensure application consistency.
According to Kesavaraju, the main benefit for districts performing low-rate aerial applications of granular larvicides for floodwater mosquito control is lower operational costs. Single brood efficiency, application flexibility, and high potency can also be achieved. In addition to potentially lower product costs, a lower use rate means reduced ferry time for aerial applicators: fewer trips back and forth to refill the aircraft. This delivers savings on fuel and means less wear and tear on the equipment. For districts that contract their applications, additional savings are realized through less time in the air. With the introduction of drones, low-rate applications have the additional benefit of maximizing area of application relative to the battery life of the vehicle.
While the benefits can be significant, Kesavaraju cautions that effective low-rate applications require thorough characterization of equipment. He recommends that technicians depend on a variable called coefficient of variation (COV) when conducting their characterization activities. He mentioned that Valent BioSciences recommends a COV of less than 0.3 (or 30%).
Kesavaraju explained that a traditional way of setting up a characterization is to place buckets or tote containers at regular intervals in a field setting, then fly the aircraft over the containers. As the product is applied, granules will drop into each of the containers. By collecting and weighing the amount of material captured in each bucket or tote, technicians can compare samples to evaluate variability of the application in a live setting. COV provides an excellent way to measure variability of application across the target site and is particularly important when characterizing for low-rate applications. According to Kesavaraju, technicians should never rely solely on “mean” or “average” granule weights across containers as they can be quite misleading.
He stressed that the effectiveness of low rate applications is dependent on evenness rather than on average distribution. Since these kinds of applications are already positioned at the low end of the recommended range, product delivered at less than that rate will likely be ineffective. In the same vein, short bursts of overapplication (streaking) may deliver an inordinate amount of material into just a few spots across the swath. This can dramatically change the result, as Kesavaraju demonstrated in one example.
For a characterization run where the targeted calibration was 4 lbs/acre and the minimum preferred rate was 2 lbs/acre, the spreader had an average application rate of 2.6 lbs/acre with containers spaced 5 feet apart. Although the average rate of application was comfortably within the 2-4 lbs/acre range, data showed that two of the containers received approximately 10 lbs/acre each because of improperly calibrated equipment. Furthermore, only one of the containers actually received an amount of product within that desired range. This variation in distribution was captured by the COV value which was greater than 1.
Tote or Bucket
Kesavaraju also explained that while helping districts in achieving even distribution (low COV) of product, his team learned that the kind of collection container being used can dramatically impact results. He said many districts default to buckets for materials collection, then demonstrated how tote containers typically deliver more reliable data especially lower COV.
Kesavaraju showed an example of a characterization run using a line of tote boxes on one side and a line of buckets on the other. (see Tote vs Bucket). Evaluating the resulting data, he showed how the totes produce a lower COV value of 0.3 (optimal) compared to a COV of 0.7 (too high) from the buckets. Despite the fact that all of the conditions were the same, the same aircraft, product, wind speed, temperature, and distance interval, he explained that mouth of the tote opening is simply larger than the area of the bucket opening. As a result, tote measurements provide COV values that better represent the consistency of application across the entire surface being treated.
Other Low-Rate Challenges
Kesavaraju then went on to explore specific challenges associated with fixed-wing and rotary aircraft. For fixed-wing applications, he noted that streaking can often be overcome by installing a custom roller in the system to help deliver a more consistent rate of product to the boom. This helps applicators improve both the consistency of their swath width and more even distribution across the swath.
According to Kesavaraju, COV challenges in rotary aircraft are often attributable to inconsistencies related to hopper openings and booms. He advised technicians to thoroughly test their hopper openings and booms for even distribution, which will result in more consistent swath widths and distribution.
More new products with lower label rates will come into the market in response to modern needs like drone based mosquito control. Districts can reduce their costs by taking advantage of these products by having a pragmatic approach to characterization and lower COV.
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Managed Wetlands Mosquito Control Strategies
Presented by: Marty Scholl, Ecological Management Supervisor; Sacramento-Yolo Mosquito & Vector Control District, CA
California’s Sac-Yolo Mosquito and Vector Control district spans the bottom end of the Sacramento Valley and the top end of the San Joaquin valley. The district is home to a series of rivers that converge to make up what’s called the California Delta, which contains numerous public and private managed wetlands. A movement to restore wetlands is being experienced not only in California, but across the country. If left unchecked, however, wetlands can become prime breeding grounds for floodwater mosquitoes that pose a risk to public health.
The complex Delta landscape provides numerous challenges for the Sac-Yolo district, challenges which require thoughtful and creative management. A key presenter at Valent BioSciences’ January 19, Virtual Floodwater Mosquito Control Summit for the Western US, Sac-Yolo’s Ecological Management Supervisor Marty Scholl shared the district’s Managed Wetlands approach with an audience totaling 275 mosquito abatement professionals and other stakeholders.
In the Sac-Yolo district, wetlands ownership runs the gamut of federal, state, and county agencies as well as non-agency entities such as conservancies, wildlife organizations, and private estates and hunting grounds. Some of the wetlands are natural, but a significant portion of the acreage is manually flooded at various points throughout the year. (insert image from slide 11 in this general vicinity)
The close proximity of these locations to urban areas in and around Sacramento, and the corresponding risk of vector-borne disease to public health, makes mosquito control in wetlands a priority. Federal agency owners such as the US Fish and Wildlife Service and the Bureau of Land Management, along with State agencies such as the California Department of Fish and Wildlife, the Department of Water Resources, and the Department of Parks and Recreation may oversee management of some or all of their own wetlands. For many non-agency wetlands, however, the Sac-Yolo district is called upon to help owners to actively manage mosquito populations. Scholl indicated this is where his team spends much of its time.
While the district treats a small amount of permanent wetlands, more of its resources are required for wetlands that are dry for most of the year, then flooded. Scholl said these wetlands range from private hunting grounds to agricultural land (mostly rice), totaling more than 60,000 acres. He said that a typical scenario is wetlands that hold their water through the winter months, then drain it sometime between March and May to promote vegetation growth. Irrigation is then used for 30 to 90 days to drown weeds and germinate desirable plants, then perimeters are mown down and the ground is disked under toward the end of summer. At some point in the late summer or fall, the lands are then flash flooded to moisten the soil, and the cycle repeats.
Communication and Collaboration
Scholl made it clear that effective control in their managed wetlands starts with proactive communication and collaboration with property owners. To assist in these efforts, the district created a Mosquito Reduction Best Management Practices Manual (BMP)s, available on its website. First published in 2008, the BMP Manual was compiled from several sources including scientific literature, collaborative inter–agency documents, and experienced vector control professionals. The Manual lays out the guidelines for program development and execution in managed wetlands and other source areas.
Plan development starts from scratch for a new wetland area or involves an annual review process for existing programs. Regardless, Scholl’s first step is to understand what the property manager is trying to accomplish so they can determine how the district can help and where it fits in. Potential treatment areas can range from very small to more than ten thousand acres.
Together with ownership, Scholl facilitates a pre-season meeting to review how the Sac-Yolo district approaches different types of mosquitoes and the different habitats that can be found on the property. Since it promotes an Integrated Vector Management strategy, the district helps property owners understand mosquito control technology and the possibilities and limitations of each. Beyond the discussion of annual goals, the pre-season meeting includes important topics such as irrigation timing, fall flooding timing, and timing of maintenance. Central to all of these discussions, of course, are costs, and how BMPs can be utilized to keep costs down both for the district and property owner. In addition to application services, the district offers imaging services, mowing and backhoe services, and heavy equipment rentals.
Since the district is responsible for managing such a wide area that includes both urban and rural areas, demands on district resources during the summer months are high. As a result, Sac-Yolo has implemented an innovative cost share incentive program to encourage managed wetland property owners to delay flooding as late into the fall as possible. By agreeing to delay application of floodwater larvicides into the latter part of the year, property owners can reduce their portion of material costs by as much as 100%. (See Fall Flooding Cost Share)
Sac-Yolo uses technology to its advantage. Unmanned aerial system imaging helps the team understand the dynamics of the changing landscape and what application technologies will make the most sense, and when. In addition to fixed wing aircraft, the team also makes extensive use of drones in areas where it doesn’t make sense to treat with a plane.
From a product standpoint, Scholl says he tries to use single brood Bacillus thuringiensis israelensis (Bti) when possible, especially in habitats with open water and less vegetation. In the fall, he looks at using residual products, initially, depending on flood timing. If the property will be flooded in early September, he starts with a residual followed by a cleanup, and often uses two to three Bti or single brood products to help manage resistance.
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Collier MCD Use of UAS Platforms in Areas of Salt Marsh Mosquitoes
Presenter: Peter Brake, Director of Technical Development, Collier Mosquito Control District, Naples FL
Just a few years ago, use of unmanned aircraft systems (UAS), or drones in mosquito control programs was in its infancy. Today, numerous districts have fully integrated drones into their programs with a host of important safety and economic benefits.
On January 21, as part of Valent BioSciences’ Virtual Floodwater Mosquito Control Summit for the Eastern US, Peter Brake, Director of Technical Development with Florida’s Collier Mosquito Control District (CMCD) provided insights on how drones have transformed the way the district conducts operations.
Located in near the southwestern tip of the state, Collier is the second largest county in Florida. It is surrounded by five neighboring counties and the Gulf of Mexico to the west. Collier spans more than 2300 square miles of land mass, 1500 square miles of which are protected state and federal lands. The county is home to 383,000 full-time residents plus another 80,000 in the winter, meaning tourism is a big part of its industry. In 2018, Southwest Florida hosted more than two million visitors who brought an estimated $1.5 billion to the local economy. Mosquito control is essential.
The district employs eight full-time pilots and seven field technicians. Its conventional aerial fleet consists of three fixed wing Skyvans (used strictly for adulticiding over large areas), plus one Bell 407 and three McDonnell Douglas helicopters used for larvidicing and applications to smaller areas.
The newest member of the aerial team, however, is CMCD’s diverse fleet of drones – each of which performs a unique task within the district’s program. Brake reported CMCD was among the first of the state’s mosquito control districts to make wide use of drone technology and was the first organization in the state of Florida to receive authorization to dispense material from drones.
The Drone Fleet
DJI Mavic Pro Platinum: The district owns three of these drones, which Brake calls their “workhorses.” Used for wetland areas inspection, these drones give the team quick and easy visual access to hard to reach areas while offering improved safety (think spiders and snakes). Operated by a control interface and tablet, these drones allow operators to see what the drone sees, in real time. They also record weather data and images that can be downloaded post-flight. For precision mapping, the Mavic Pro uses Visible Atmospherically Resistant Index (VARI) imaging data, which emphasizes vegetation similar to NDVI (see Phantom 4) and provides significantly higher resolution than Google Earth. Brake said these operations do have some limitations, namely with control tablets overheating in mid-day sun.
Matrice 660P (PV13): The fleet includes one of these application drones, which can carry 13 pounds of material. The PV13 travels at a speed of between eight and 16 mph treating between 0.4 and 1 acre/minute. For liquid (WDG) application, the PV13 applies in a 40-foot swath and can cover about 200 acres on a single tank for an adulticide application. For granular applications, the unit treats in a 30-foot swath with a 1-10 acre per tank capacity and a battery life of 3-4 loads. The drone is especially useful in urban area applications.
Phantom 4 NDVI: This mapping drone is equipped with Normalized Difference Vegetation Index (NDVI), a graphical indicator that can be used to analyze whether or not the target being observed contains live, green vegetation. It measures the level of chlorophyll in the trees and has a red filter that shows transitional areas of dead or dying habitat where mosquitoes often lay their eggs.
SwellPro Splash Drone: This specialized drone is used for observation below the surface of water. It is waterproof and has a camera mounted underneath for inspection of fish and larvae.
Quantum-Systems Trinity: This drone is mainly used for larger surface areas, typically over 100 acres. The flight software is installed on a laptop or PC and uploaded to the aircraft. Brake said that since this drone operates using a different controller, it can fly longer without heat being a factor – making the mapping process more efficient. With a 7.5-foot wingspan, The Quantum Trinity takes off vertically. Once it has reached its assigned altitude, propellers on its wings shut down and another propeller on the tail propels the aircraft forward. Flight times can last as long as 90 minutes. This drone has NDVI and a 20-megapixel camera, with a max range of three to five miles.
To process imaging data captured by the drones for map creation, CMCD uses advanced drone mapping software called DroneDeploy. At a cost of $3600 per year, the software allows technicians to import and export in various file formats and generate an unlimited number of maps.
Brake said CMCD’s drone fleet and systems cost around $86,000. The payback comes in safety, precision, efficiency, and reduced environmental impact.
Safety improvements for urban area application settings are considerable. With helicopter applications, pilots have to fly over and around buildings in highly populated areas, often generating noise and safety complaints. With drones, the team can limit flights to the space directly over the area of treatment, and with far less noise.
The drones’ enhanced imaging capabilities also reduce environmental impact and provide direct cost savings . Originally, CMCD manned aircraft had to rely on Google Earth images, which can be 12 to 18 months out of date. Brake showed an example of a treatment area from 2018 that spanned 326 acres. Re-imaging the site in 2019 using drones, the team was able to reduce the treated area within the block by 87 acres, or 27%. Figuring in an application cost of $125/acre, the savings amounted to $10,875 per mission. With the area receiving three treatments per year, the single-year savings was more than $32,000.
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