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).

Wide-Area Larviciding (WALS™) techniques for the control of “container-inhabiting” urban species of mosquitoes, such as Aedes aegypti (L.), Ae. albopictus (Skuse), and Culex quinquefasciatus Say, have become an integral part of many vector control programs across the USA and an indispensable method for the suppression or interruption of arboviral outbreaks (Tan et al. 2012, Setha et al. 2016, Stoddard 2018, Bohari et al. 2020, Harris et al. 2021). In general, WALS treatments are conducted using water-dispersible or aqueous suspensions of Bacillus thuringiensis israelensis de Barjac strain AM 65-52 (Bti) such as VectoBac® WDG (Valent Biosciences Corporation, Libertyville, IL) coupled with either ground-based or aerial dispersal equipment (Williams et al. 2014, Lucas et al. 2020, Williams et al. 2022). Using WALS treatments with suspensions of Bti require carefully calibrated hydraulic nozzles or rotary atomizers such as the Micronair AU-5000 (Micron Sprayers Limited, Bromyard, UK) to ensure the formation of an appropriate droplet spectrum as well as an adequate dispersal of the AI over peridomestic sources of larval mosquito habitat such as household containers, cisterns, gutters, tires, and other objects that may hold water (Jacups et al. 2013, Pruszynski et al. 2017, Murray et al. 2021, Burtis et al. 2022).

Most of the previous work on WALS methodologies has involved urban or suburban applications of Bti suspensions for the control of peridomestic container species of mosquitoes. Much less effort has been directed toward understanding the utility of a WALS-like larviciding approach for the control of mosquito species that inhabit floodwaters, forest pools, marshes, swamps, and other natural habitats. For instance, the utility of water-dispersible formulations of Lysinibacillus sphaericus (Ls) (Meyer and Neide) such as VectoLex® WDG (Valent Biosciences Corporation) dispersed with a WALS methodology has also been largely neglected except for only a couple of works. Floore et al. (2002) examined VectoLex WDG distributed via backpack sprayer to flooded areas ≤0.25 acres (0.10 ha) with positive results, and Burtis et al. (2022) examined VectoLex WDG in combination with VectoBac WDG using a bioassay methodology and colony-reared Ae. aegypti in urban residential areas. Since Ls can exhibit a longer interval of control than Bti in organically rich eutrophic environments (Lawler 2017) such as floodwaters and forest pools, the combination of a traditional WALS-like approach using equipment such as a Buffalo Turbine® Mist Sprayer (BTMS) (Buffalo Turbine Inc., Springville, NY) with aqueous suspensions of Ls represents a novel combination of material and equipment as well as a potentially effective treatment approach.

The North Shore Mosquito Abatement District (NSMAD) encompasses approximately 69 mi2 (179 km2) abutting the northern portions of the city of Chicago within Cook County, IL (USA). Bisecting the NSMAD from north to south is a portion of Cook County's “emerald necklace”: lines of nearly contiguous prairies, savanna, marshes, rivers, forests, and other natural or restored preserves embedded within the highly urbanized landscape. The North Branch Chicago River, the West Fork North Branch Chicago River, and the Skokie River each exhibit periodic seasonal inundation of surrounding floodplain, leaving behind large swaths of isolated standing water as the rivers recede. Other portions of preserved areas are underlain with a layer of hardpan that, when coupled with a lack of any significant topography, creates numerous areas where rainfall collects into large temporary pools and puddles. These ground pool habitats can be highly productive for the mosquitoes Ae. stimulans (Walker), Ae. vexans (Meigen), and Cx. restuans (Theobald), where adult populations can dramatically increase after significant rainfall events.

Common methods for treating ground pool sources of mosquitoes within the NSMAD consist of applications of granular larvicides by manual broadcast spreader, Stihl backpack blower (Stihl Inc., Virginia Beach, VA), or truck-based Vortex granular spreader (Vortex Granular Systems LLC, Lighthouse Point, FL). While these methods are effective in reducing larval populations of mosquitoes, they are also extremely time consuming and laborious. These methods require a field technician to physically traverse each of the treatment areas to ensure adequate and homogenous coverage of the granular larvicide. This equipment is also limited by a very narrow treatment swath width. The goal of this study was to determine if an aqueous suspension of VectoLex WDG applied using a ground-based methodology identical to WALS can reduce larval mosquito populations in natural and artificial ground pool habitats within the NSMAD as well as to determine the length of any residual control effect from Ls applications. In urban WALS treatments for container species of mosquitoes using VectoBac WDG, effective control can be observed at a swath width of up to 300 ft (91.4 m) (Williams et al. 2014) and for a period greater than 6 wk (Jacups et al. 2013, Williams et al. 2014). Many of the previously described ground pool larval habitats within the NSMAD occur within 300 ft (91.4 m) of a paved road, bicycle or walking path, or other access road. Therefore, a ground-based WALS-like application of Ls has the potential to greatly decrease the labor and time necessary to apply a larval control treatment to floodwater habitats while extending the duration of control. A WALS-based methodology coupled with a BTMS could also potentially improve the distribution of larvicide and efficacy within a target area when compared with other mister/sprayer systems (Williams et al. 2014), backpack blowers, or vortex-based applications of granular larvicides.

The output nozzle of a BTMS (Model CSM2) was modified with a Micronair Buffalo Turbine Conversion Kit (Part Number PC1131/324; Micron Sprayers Limited) and an AU-5000 rotary atomizer (Micron Sprayers Limited). This Micronair kit replaces the factory-equipped hydraulic nozzles with a metal collar and rotary atomizer centered in the output air flow. The AU-5000 atomizer was fitted with size 20 mesh screen as well as 2.75-in. (6.985-cm) blades (Part Number EX6353; Micron Sprayers Limited). Blades were set at an angle of 55° to produce approximately 5,000 revolutions per minute (RPMs) of the atomizer blades at full throttle (Micron Sprayers Limited 2013). This initial combination of blade angle, screen mesh size, and RPMs was selected to produce a droplet spectrum with an estimated median droplet size (DV0.5) of approximately 175 μm (Micron Sprayers Limited 2013, Williams et al. 2014). In addition to the conversion from factory hydraulic nozzles to rotary atomizer, a brass check valve/regulator (Part Number 10742A; Spray Systems Company, Glendale, IL) was installed, without a filter and restrictor orifice plate, between the output of the BTMS liquid pump and the AU-5000 rotary atomizer. The bypass valve of the BTMS liquid pump was set to create an operating pressure of 50 psi at full throttle. The combination of bypass pressure, check valve, and throttle position delivered 3.2 gal/min (12.11 liter/min) to the AU-5000 rotary atomizer.

To characterize the droplet distribution and dispersion of the BTMS for WALS applications, 25 lb (11.34 kg) of VectoLex WDG and 1.5 lb (0.68 kg) of granular FD&C Red #40 food dye (Sensient Colors LLC, St. Louis, MO) was thoroughly mixed with 22 gal (83.28 liters) of water to create a suspension at a final weight-to-volume ratio of 1 lb (454 g) of VectoLex WDG to 1 gal (4.55 liters) of final mixture (i.e., a 12% W:W suspension). At a vehicle speed of 10 mph (16.1 km/h), an assumed 300-ft (91.4-m) swath width, and with the BTMS set to full throttle, this setup was calculated to deliver a VectoLex WDG suspension to the target area at a rate of 0.5 lb/acre (560.44 g/ha). One-sided 9-cm × 7-cm glossy Kromekote® cards (CTI Paper USA Inc., Sun Prairie, WI) were affixed to compact disc jewel cases with a binder clip and laid horizontally on the ground parallel to the prevailing wind and perpendicular to the vehicle's spray route at 30-ft (9.14-m) intervals starting at 0 ft and ending at 300 ft (91.44 m) for a total of 11 cards per trial. A total of 3 trial runs were conducted. Trial applications were made by angling the output nozzle of the BTMS approximately 5° from vertical and driving the vehicle at 10 mph (16.1 km/h) perpendicular to the line of Kromekote card collection stations. The ground and air temperatures were recorded at 29.5°F (−1.38°C) and 40.8°F (4.88°C), respectively, indicating a lack of convective currents during the study period. Wind speed was recorded before each trial at 4.4 mph (7.08 km/h), 4.8 mph (7.72 km/h), and 5.2 mph (8.37 km/h). The RH was similarly stable with values recorded at 89.3%, 89.9%, and 90.3% before each trial. The BTMS was permitted to distribute the red-dyed material for 200 ft (60.96 m) before and 200 ft (60.96 m) after the line of collection stations. Kromekote cards stained with red droplets from each trial application were allowed to dry on the ground for 10 min before being collected and analyzed by BacDrop™ software (Valent BioSciences Corporation) to determine spray characteristics such as droplet density and droplet distribution within the 300-ft (91.44-m) swath.

The DV0.5 of the Vectolex WDG suspension declined steadily across the width of the swath. As expected, larger droplets (those ≥200 μm in diam) were the first to settle onto the Kromekote cards within 30 ft (9.14 m) of the vehicle path. The DV0.5 of the application continued to decline slightly at each collection station until the end of the swath, where the DV0.5 of the application reached an average of 122 μm. The average number of drops/cm2 across the swath was more variable across the 3 trials and ranged from <1 drop/cm2 at 0 ft to nearly 40 drops/cm2 at 120 ft (36.58 m). By the end of the swath, the average number of drops/cm2 declined to 5.54. The average DV0.5 of all 3 trial runs and all 11 stations combined (n = 33) was 175 μm with a standard deviation of ±6.02 μm. The average relative span (relative span is a ratio that describes the width of the droplet distribution) of the 3 trials was 0.83, which suggests a relatively narrow droplet spectrum characteristic of rotary atomizers. Together, these attributes (droplet density, DV0.5, and relative span) demonstrate a droplet distribution and droplet spectrum within the 300-ft (91.44-m) swath width similar to other works that have examined the use of AU5000 rotary atomizers for this purpose (Williams et al. 2014, Burtis et al. 2022, Wieland et al. 2022).

To evaluate the field efficacy of VectoLex WDG applied by a WALS methodology, a 1.25-acre (0.506-ha) flooded area was identified along the Skokie River and North Branch Bike trail within Northfield, IL (42°05′34.2″N, 87°45′36.3″W), for an early-spring treatment (Fig. 1). A similarly sized (0.94-acre/0.38-ha) untreated control parcel (42°05′25.5″N, 87°45′34.2″W) was also identified approximately 640 ft (approximately 195.07 m) to the south-southeast (Fig. 1). During April and May, both parcels are typically flooded with up to approximately 1 ft (approximately 30 cm) of pooled rainwater covered by a dense understory of invasive common buckthorn, Rhamnus cathartica (L.), in the early stages of spring leaf out. These locations are typically highly productive habitats for early Ae. stimulans and later Ae. vexans and Cx. restuans. Prior to application, QGIS software (QGISA 2022) was used to generate 10 random latitude–longitude locations within each parcel for a pretreatment inspection and 6 weekly posttreatment inspections (Fig. 1). Random point locations identified by QGIS were found in each parcel by handheld Global Positioning System unit (Mobile Demand xTablet T1400; Mobile Demand LLC, Hiawatha, IA) and sampled with a single dip from a standard mosquito larvae dipper (Part Number 1132BQH; BioQuip Products, Rancho Dominguez, CA). Therefore, each parcel received 10 random pretreatment sample dips as well 10 random posttreatment sample dips per week for a total of 70 samples per parcel. The use of random preassigned sampling locations ensured that statistically independent samples free from sampling bias were collected (Fig. 1). On April 25, 2019, VectoLex WDG was applied to the treatment site at a rate of 0.5 lb/acre (560.44 g/ha) under the equipment calibration conditions previously used to characterize the droplet distribution and swath width of the BTMS. At the time of treatment, the temperature was 53.6°F (12°C) and the wind speed <3 mph (4.83 km/h) from the southwest (Visual Crossing Corporation 2019). The total number of immature mosquitoes per dip was averaged for each weekly sampling event and location. Mulla's formula (Mulla et al. 1971) was used to determine a weekly posttreatment percentage reduction based on week 1 as the pretreatment observation.

Fig. 1.

Diagram of study area in Northfield, IL, detailing control (gray shaded) and treatment parcels, QGIS random sampling locations, and other attributes of the treatment. Diamonds within treatment and control parcels indicate randomly selected sampling locations.

Fig. 1.

Diagram of study area in Northfield, IL, detailing control (gray shaded) and treatment parcels, QGIS random sampling locations, and other attributes of the treatment. Diamonds within treatment and control parcels indicate randomly selected sampling locations.

Close modal

As Fig. 2A demonstrates, treatment with 0.5 lb/acre (560.44 g/ha) VectoLex WDG using a WALS approach was highly effective in reducing larval mosquito populations in the treatment parcel. Treatment efficacy was sustained for the duration of the study (6 wk) while percent reductions in immature mosquitoes remained above 70% with half of the week observed (3 out of 6) exhibiting 100% control as measured with our sampling methodology (Fig. 2A). Statistical testing of each week's immature mosquito count using a Mann–Whitney U-test revealed that during week 1 (pretreatment) the treatment and control parcel contained the same median abundance of mosquitoes (Fig. 2B). Analysis of posttreatment week revealed that the treatment parcel during weeks 2, 3, 4, 6, and 7 were all statistically significantly different from the control parcel at an α = 0.05 (Fig. 2B). Although the week 5 treatment area remained at 0 (zero) mosquitoes, the Mann–Whitney U-test returned a P-value of 0.0867 (Fig. 2C) due to the lower overall number of mosquitoes sampled in the control site as well as high variability between sample dips obscuring the treatment effect and limiting the ability of the statistical test to resolve a significant difference at such a low α. The level and duration of control at the minimum application rate range (0.5 lb/acre [560.44 g/ha]) exceeded the maximum treatment interval of 4 wk suggested on the product label (Valent BioSciences Corporation). In this study, we observed excellent control for at least 6 wk at the minimum label rate. This result agrees well with Floore et al. (2002), who found that 1 application of VectoLex WDG (applied by backpack) was effective for up to 35 days (5 wk) at a similar application rate (0.5 lb/acre [560.44 g/ha]). Similar work by Wieland et al. (2022) with VectoBac 12AS (Bti) applied using a WALS-like methodology against Ae. dorsalis (Meigen) in a salt-marsh environment showed a similar 84% decrease of immature mosquitoes at 5 days posttreatment. Taken together, these results suggest an outstanding level of control as well as a dramatic reduction in the labor required for application. For example, the actual time required (exclusive of mixing and loading) to cover the 1.25-acre (0.506-ha) treated parcel at a vehicle speed of 10 mph (16.093 km/h) with a BTMS was less than 30 sec. The high effectiveness, long duration of control, and reduced labor requirements of a WALS methodology for VectoLex WDG demonstrate that this methodology has the potential to be widely useable and effective for mosquito control operations nationwide.

Fig. 2.

Results from the operational field trial. (A) In weeks 2 and 3 posttreatment there was 100% control of mosquitoes. The control percentage decreased to 79% by week 7 (mean reduction; Mulla's formula). (B) Average number of mosquitoes per dip in control and treatment areas. The treatment area exhibited sustained reductions in immature mosquito numbers when compared with the untreated control. The arrow indicates the week of treatment (mean ± SD of 10 dips; * P ≤ 0.05, ** P ≤ 0.01; Mann–Whitney U-test). (C) Summary of Mann–Whitney test statistics and percent reductions.

Fig. 2.

Results from the operational field trial. (A) In weeks 2 and 3 posttreatment there was 100% control of mosquitoes. The control percentage decreased to 79% by week 7 (mean reduction; Mulla's formula). (B) Average number of mosquitoes per dip in control and treatment areas. The treatment area exhibited sustained reductions in immature mosquito numbers when compared with the untreated control. The arrow indicates the week of treatment (mean ± SD of 10 dips; * P ≤ 0.05, ** P ≤ 0.01; Mann–Whitney U-test). (C) Summary of Mann–Whitney test statistics and percent reductions.

Close modal

We thank Banugopan Kesavaraju, Leanne Lake, and Peter DeChant of Valent Biosciences LLC, for the technical assistance in setting up the BTMS as well as helping to characterize and calibrate the BTMS for VectoLex WDG. We also thank the North Shore Mosquito Abatement District's Board of Trustees for their continued support of applied research efforts at the District.

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