Mosquitoes and mosquito-borne diseases remain one of the major public health burdens. In most cases, vector control is the main or the only intervention to mitigate these problems. We are facing the challenges of traditional, emerging, or resurging vectors and diseases, yet the availability and affordability of safe and effective mosquito control products are at a historical low. Development of new active ingredients (AI) and novel formulations based on currently available AI are demanded by mosquito control operations. This paper validated the bioactivity in the laboratory and evaluated the semi–field efficacy for 2 newly registered controlled-release products based on juvenile hormone analog S-methoprene: OmniPrene™ water-soluble pouch (WSP) and extended water-soluble pouch (XWSP). Along with technical S-methoprene, these 2 formulations showed high inhibition of adult emergence in laboratory bioassays against Aedes aegypti, Ae. albopictus, and Culex quinquefasciatus that are commonly found in catch basins. High initial and long-term residual efficacy were demonstrated in simulated catch basins against Cx. quinquefasciatus, where the OmniPrene WSP consistently provided over 90% control for 15 wk (105 days) and OmniPrene XWSP did the same for 38 wk (266 days). Considering the need for reliable mosquito control products, combined with commonly encountered product performance issues in catch basins, OmniPrene WSP and XWSP can be viable tools to combat mosquito species of public health concern that are associated with urban drainage systems.
Mosquitoes and mosquito-borne diseases remain one of the major threats to public health and well-being of humans and animals. In most cases, sustainable mosquito control is the main or only effective measure to mitigate mosquito-related illnesses. In mosquito control operations, larviciding and adulticiding are both important aspects in integrated mosquito management. When considering potential mosquito larvicides, biorational products based on natural materials or their mimics are desired because of strict regulatory issues and environmental concerns. During the past decades, limited numbers of products with microbial and insect growth regulator (IGR) origins have played crucial roles in controlling nuisance and vector mosquito species (Su 2016). Available mosquito larvicides are at a historic low from the standpoint of registered products or achievements in research and development. The strict regulations, narrow market niches, and development of resistance (Su 2022) further impact product availability. While diligent effort has been made to explore new active ingredient (AI), improvement in formulation technology by taking advantage of currently available AI is equally important.
Poorly designed and maintained urban stormwater catch basins in the USA and elsewhere are among the major mosquito production sites for Culex spp. and recently invasive species such as the yellow fever mosquito, Ae. aegypti (L.), and the Asian tiger mosquito, Ae. albopictus (Skuse) (Metzger et al. 2002; Su et al. 2003, 2014; Kwan et al. 2008; Harbison et al. 2011; Paploski et al. 2016; Rydzanicz et al. 2016; Gao et al. 2018; Wang et al. 2021). While many interventions can be considered based on the designs and functions of various stormwater devices (Metzger 2004; Metzger et al. 2008, 2018), strategic application of larvicides is one of the most common measures in mosquito population mitigation. Due to the nature of catch basin systems, such as unpredictable flooding, poor water quality, organic matter accumulation, etc., it is particularly difficult to achieve sustainable control effectiveness despite industrial effort to develop customized products for catch basin treatment. Quite often, complicated field conditions have made evaluation of effectiveness unfeasible and misleading (Siegel and Novak 1997, 1999; Stockwell et al. 2006; Su 2008; Anderson et al. 2011; Harbison et al. 2015, 2016, 2017, 2018; Nasci et al. 2017). Evaluation of juvenile hormone analog (JHA) S-methoprene–, or chitin synthesis inhibitor diflubenzuron-based products was even more challenging than microbial products due to their unique modes of action (Knepper et al. 1992, Stockwell et al. 2006, Bellini et al. 2009, Nasci et al. 2017, Harbison et al. 2018, Mian et al. 2020). Recently a new product line, OmniPrene™ based on S-methoprene, a commonly used synthetic biopesticide (Henrick 2007, Su 2018), has been developed and registered.
Among the numerous formulations of OmniPrene, the water-soluble pouch (WSP) and extended water-soluble pouch (XWSP) formulations were evaluated in the present study against the southern house mosquito, Culex quinquefasciatus Say, in the laboratory and simulated catch basins.
MATERIALS AND METHODS
The technical S-methoprene (US Environmental Protection Agency [US EPA] 73487-1) was provided by Synergetica International Inc. (Marlboro, NJ). The sample had a Lot No. MT08-218 and purity 98.06% by high-performance liquid chromatography.
The test formulations OmniPrene WSP (Lot No. 122018SGWSP) and OmniPrene XWSP (Lot No. 122018SGXWSP) (US EPA 73487-2) were provided by the same supplier as the technical S-methoprene. Both formulations contain 1.00- to 2.00-mm-diam diatomaceous earth (DE) granules that contain 4.25% S-methoprene. The granules are wrapped in a polyvinyl alcohol water-soluble pouch with a net weight of 7.00 g for WSP and 27.83 g for XWSP, which is equivalent to 0.298 g and 1.1828 g AI, respectively (Fig. 1). These granules have a rough surface and high porosity. The overall bulk rate is 0.6 g/ml, but the net specific gravity is 4.0 g/ml when disregarding the extensive internal spaces.
Culex quinquefasciatus (EcoZone International, Riverside, CA), Ae. albopictus (Gainesville Mosquito Control, Gainesville, FL), and Ae. aegypti (Benzon Research Inc., Carlisle, PA) were used as test subjects. Considering the susceptible window of time for JHA, which is generally 12–24 h prior to pupation, the late-4th instars, sometimes called pupating larvae, were used in the bioassay (Su et al. 2021).
Bioassays and data analysis:
Laboratory bioassay was conducted according to previously published protocols (Su et al. 2018) to ensure the quality of the test materials. The technical S-methoprene was dissolved in and serially diluted by ACS-grade pure acetone (Cole Parmer, Vernon Hills, IL). The granules from the WSPs were pulverized in a coffee grinder (Hamilton Beach Fresh Grind; Glen Allen, VA) at the maximum speed in interruption mode until the granules were turned to fine powders, then suspended in tap water by vortexing for 3 min (Vortex Mixer VX200; Labnet International, Inc., Edison, NJ). In bioassay, 6 concentrations within the range resulting in approximately 5–95% mortality were used, with 3 replicates at each concentration. In each replicate, 25 pupating larvae were placed in 100 ml of tap water in a 120-ml disposable Styrofoam cup. A small piece (approximately 100 mg) of rabbit pellets (18% crude protein; Brookhurst Mill, Riverside, CA) was added to each bioassay cup to have a slow release of the nutrients to support larval growth to pupation. Bioassays were conducted at 27.0–29.0°C. The mortality was recorded when all exposed individuals died at larval or pupal stage, emerged incompletely, or emerged successfully as adults. Concentration–response data were analyzed using POLO (Robertson et al. 2006) to calculate the concentrations causing 10% (IE10), 50% (IE50), and 90% (IE90) inhibition of adult emergence and their 95% confidence intervals (95% CIs) (Su et al. 2018).
Field test facility and treatment:
Field evaluation was carried out in suburban Riverside, in inland southern California, where applications of S-methoprene products in general and in catch basins are negligible (local mosquito and vector control agency, personal communication). A simulated catch basin test system was designed to mimic those employed in earlier catch basin studies (Su 2008). To achieve this design, plastic totes (Rubbermaid; 43.2–61.03 × 43.2–61.0 × 71.1 cm, tilted sides, 151.4 liters in volume) were fitted with an inverted 2-in. polyvinyl chloride overflow pipe (5.1 cm long horizontally and 10.2 cm long vertically) at the 50.8-cm level of the tub. Basin lids were cut to have 5 slots to make catch basin cover. Basins were installed with 19 liters of sludge (90% topsoil and 10% sandy loam soil). The topsoil was Miracle-Gro brand (The Scotts Miracle-Gro Co., Maryville, OH) with organic label—no pesticides, herbicides, or other additives added. The sandy loam soil was collected from the neighboring orchard where no pesticides or herbicides were applied during the past decade. Each basin was further enriched with 100 g of rabbit pellets (18% crude protein; Brookhurst Mill). The basins were then filled with tap water to the exit level (about 125 liters) and allowed to ferment outdoors for 16 days prior to testing. During this time, basins were covered with window screen to prevent oviposition by mosquitoes of local wild populations. The screen was then removed after initial fermentation, and natural oviposition was allowed to occur thereafter. Four replicates (basins) were made for each treatment and untreated control. A minimum–maximum thermometer was placed in one of the basins located in the middle of the basin layout to monitor the water temperature during study period. Treatment was made on day 28 postflooding when larval 1st–3rd instars presented in large numbers (occasional 4th instars, but no pupae) by tossing 1 WSP or XWSP to each replicate basin. Nineteen liters of water, about 15% of original volume, were gently added through the top grids to each basin weekly posttreatment and allowed to exit out of the overflow to simulate rain events. After each flushing, 50 g of rabbit pellets were added to each basin to maintain the organic level and oviposition attractancy.
Sampling, observation, and data analysis:
Basins were monitored for presence of pupae and exuviae, and live pupae were collected for evaluation of adult emergence weekly for up to 64 wk (448 days), when observation and sampling were concluded. Twenty-five (25) pupae collected from each basin were held in 200 ml of water from the same basin in a Styrofoam cup. Cups with pupae were placed in a 61 × 61 × 61-cm mosquito cage; emerged adults were confined in this cage and died later due to lack of water and carbohydrate. Only free exuviae were counted and considered as successful adult emergence when all pupae died or emerged. Mortality mostly presented as dead pupae and sometimes as incomplete emergence of adults with wings and/or legs attached to the exuviae. Observation on inhibition of emergence was conducted at 27–29°C. The inhibition of emergence was calculated: IE% = 100 × [1 − (number of successfully emerged adults/total number of pupae collected)]. The significance in IE% was analyzed by chi square test at χ2 = 3.83, P = 0.05 level (Social Science Statistics 2022).
Approximately 50 late-stage larvae (3rd and 4th instars) were collected from randomly selected basins in weeks 15, 30, and 60 posttreatment. Larvae were killed by 95% ethanol, and species was identified morphologically under a dissecting microscope.
Observation on WSPs and granules during application and retrieval of granules upon evaluation completion:
Qualitative observations were made during application of the WSPs for pouch dissolving and granule releasing. Upon completion, best effort was made to retrieve the granules from the sediment, rinsed with tap water, dried at room temperature, and compared with the granules prior to treatment from the WSPs for their physical appearance and integrity.
Laboratory bioassays on the granules from the WSPs indicated typical S-methoprene mode of action, i.e., inhibition of adult emergence against test species. The typical mortality pattern of incomplete emergence (legs and/or wings attached to pupal exuviae), dead pupae, and dead larvae was observed from the low to high concentrations. The JHA activity was expressed as IE10, IE50, and IE90 and their 95% CIs against 3 test species. The mortality in the untreated control was all less than 10%. The susceptibility to S-methoprene was significantly higher (P < 0.05) in Aedes spp. than in Culex as indicated by separate 95% CIs across 3 test materials. The difference in susceptibility to S-methoprene between 2 Aedes species was negligible (Table 1).
Efficacy of OmniPrene WSP:
During the 18-wk evaluation period, the untreated control basins showed a negligible inhibition of adult emergence ranging from 0% to 6.0% with an average of 2.9%. At the same time, a consistently greater than 90% efficacy was achieved with an inhibition of emergence fluctuating from 94.0% to 100% (average 97.9%) for the first 15 wk (105 days) against the wild Cx. quinquefasciatus populations. During each of these 15 wk, the inhibition of emergence in treatment was significantly higher than that in untreated control (χ2 = 154.9–196, P < 0.0001). A gradual decline of efficacy to 76.0–88.0% (average 81.3%) ensued for the remaining weeks 16–18, when treatment still provided significant control (χ2 = 111.5–142.0, P < 0.0001) (Fig. 2).
Efficacy of OmniPrene XWSP:
In this extended 64-wk evaluation, the untreated control exhibited a low-level inhibition of emergence of 1.0–7.0% with an average of 4.2%. Under the same conditions, the XWSP provided high initial and long-term residual efficacy with inhibition of emergence against the same test species from the wild populations. During the first 38 wk (266 days), efficacy was consistently above 90%, ranging from 94.0% to 100% with an average of 98.9% (χ2 = 176.7–196.0, P < 0.0001). Control level fluctuated between 82.0% and 99.0% during the remaining weeks 39–64, when the average efficacy was still as high as 90.5% (χ2 = 117.2–184.4, P < 0.0001) (Fig. 3).
Water temperature experienced the full range of seasonal changes during the test period. During the evaluation period, the minimum water temperatures ranged from 8.3 to 20.6°C with an average of 14.9°C, while the maximum water temperatures fluctuated between 20.0 and 34.4°C with an average of 27.3°C (Fig. 4).
Species identification on 3rd to 4th instars indicated that Cx. quinquefasciatus was the predominant species that inhabited the simulated catch basins; other species such as the foul water mosquito, Cx. stigmatosoma Dyar, and the winter mosquito, Culiseta incidens Thompson, were present at much lower levels (0–6.0%) (Table 2).
Observation on WSPs and granules during application and retrieval of the granules upon test completion:
The WSPs stayed afloat on water surface immediately after application, the polyvinyl alcohol film gradually dissolved within a couple of minutes, then the granules inside the pouch were released, remaining afloat for some seconds, and then sank to the bottom. Upon completion of the test, the granules retrieved from the basins were still mostly intact, except their color changed somewhat after collecting some organic matter while sitting at the basin bottom for 15–64 wk (105–448 days) (Fig. 1).
The extensive catch basins in urban environments are important mosquito production sites due to poor design, installation, and maintenance (Metzger et al. 2002, 2018; Su et al. 2003; Metzger 2004; Rydzanicz et al. 2016), especially in the areas that are topographically flat. Urbanization and demographic growth further worsened this situation due to the addition of more stormwater drainage systems. Quite often, prevention of mosquito production at stormwater devices are not considered during design and installation phases of such systems. Traditional and typical mosquito species, namely, Cx. pipiens L. complex, Cx. restuans Theobald, Cx. stigmatosoma etc., are commonly found in storm drains. Floodwater mosquitoes, such as invasive Ae. albopictus and Ae. aegypti, also use urban drainage systems as their habitats for immature stages and as resting places for adults. The high diversity of mosquito species that dwell in the stormwater drainage systems has made mosquito management even more difficult and demanding to mitigate the risk of public nuisance and disease transmission.
Some of the challenges in controlling mosquitoes in the underground storm drain systems, including catch basins and lateral convey pipelines, are due to unexpected and variable flooding, extensive edges and pockets when water flow is interrupted, accumulation of organic matter, poor water quality, existence of sediment, and others. These challenges are greater in southwestern USA where rainfall is scarce during warm mosquito season and habitats are mostly created by spillage and leakage of urban water consumption (Su et al. 2003). The aforementioned challenges compromise the field effectiveness of mosquito larvicides, despite the design of customized formulations for such habitats. Well-designed controlled-release formulations based on limited microbial and IGR larvicides, such as water-soluble pouches and briquets, can perform below the product label claims when facing the challenges associated with catch basins. The most common issues with product performance are low initial effectiveness, shorter longevity, inconsistent effectiveness during labeled longevity, or total failure to perform (Anderson et al. 2011; Su et al. 2014; Harbison et al. 2015, 2016, 2017, 2018; Nasci et al. 2017). Under certain field conditions, effectiveness evaluation can be quite difficult due to high variability of data among the replicates, which makes field evaluation inconclusive, and leading to even more questions about product quality, performance claims, and impact of storage and handling (Su et al. 2018).
When considering the challenges in mosquito control in general but in particular the ones associated with catch basins, screening new AI and development of new formulations based on existing AI are equally important. Laboratory bioassays on both OmniPrene products along with the technical S-methoprene indicated the high inhibition of emergence activity of Cx. quinquefasciatus, the common species that is associated with catch basins (Su et al. 2003). The other 2 Aedes spp. that have been found in storm drains in recent years (Paploski et al. 2016, Gao et al. 2018) were also tested, and their susceptibility was higher than Culex, which agrees with earlier findings (Su et al. 2019a, 2019b). The higher susceptibility of Ae. aegypti and Ae. albopictus made OmniPrene WSPs more meaningful to control operations as these species can be less susceptible to the commonly used microbial larvicides based on Bacillus sphaericus Neide (Su et al. 2019a).
The OmniPrene WSP and XWSP have their product label claims for 30 days and 150 days of residual efficacy, respectively (EPA 2022), against Aedes, Anopheles, and Culex spp. The performance to control the wild Culex species from the study area was well beyond their product label claims under challenging conditions such as high organic content, weekly 10% flush to simulate rain events, exposure to aboveground environment with ultraviolet irradiation, and wide temperature fluctuations as compared with underground catch basins. The OmniPrene WSP provided greater than 90% efficacy for 15 wk (105 days), while the OmniPrene XWSP provided the same level of control for 38 wk (266 days). Due to the higher susceptibility of Aedes spp. to S-methoprene than Culex spp. (Su et al. 2019a, 2019b), higher efficacy can be reasonably expected under the same conditions when these products are used to control Aedes spp. Higher dose is expected for the same levels of efficacy in the catch basins with greater volumes of water when one considers the high diversity in size and configurations of catch basins in general. It is believed that the extended efficacy by these novel formulations is attributable to the following factors. The inert carrier DE granules consist of natural fossilized remains of unicellular algae called diatoms. These granules are manufactured from sedimentary silica rocks that are typically composed of 87–91% silicon dioxide (SiO2), plus significant amounts of alumina (Al2O3) and ferric oxide (Fe2O3) (Tsai et al. 2006). The unique properties of DE, such as a low conductivity, low density, high porosity, large surface area per given weight or volume, high adsorption capacity, and excellent thermal resistance, allow its unique industrial value and extensive uses in household, farming, gardening, pesticides, pharmaceutics, pets, etc. (Jia et al. 2007, Rose 2010). The chemical and physical properties of DE granules conferred excellent binding capacity of methoprene molecules due to their rough surfaces and porosity. Additionally, the DE granules have a specific gravity of 4.0 g/m if disregarding the internal spaces, which helped to anchor them down to the bottom of the simulated catch basins. However, the bulk rate of the DE granules is 0.6 g/ml if the internal spaces are considered; this feature prevented them from further sinking and being buried into the sediment and allowed the granules to move around close to the top of the sediment in the basins. Finally, the proprietary formulation process and binding agents also played crucial role in ensuring the protection and proper release of the S-methoprene along and beyond labeled longevity.
The dry amorphous DE powder can be used as a pesticide, which impacts the target species by dehydration and being abrasive against their cuticle (Rose 2010). However, this is not the case in the current studies where the DE is in 1.0- to 2.0-mm granules and impregnated in water all the time. The lack of direct impact of the DE granules on mosquito larvae and pupae was therefore not applicable, which was clarified in another separate study on blank DE granules against the immature mosquitoes of Cx. quinquefasciatus (Su and Su, unpublished data). The retrieved DE granules at the completion of product evaluation confirmed that the DE granules only function as a carrier; these fossilized remains of diatoms stayed intact in the aquatic habitats throughout the extended test period.
When considering the demand for mosquito control in urban catch basins against indigenous and invasive species, combined with the inconsistent performance of currently available products based on microbials and IGRs, it is expected that the new products OmniPrene WSP and XWSP will serve as another viable tool to combat urban pestiferous mosquito species.
EcoZone International LLC, 7237 Boice Lane, Riverside, CA 92506.
Synergetica International Inc., 9 Inverness Drive, Marlboro, NJ 07746.