COMPARATIVE LABORATORY ACTIVITY AND SEMI-FIELD EFFICACY OF OMNIPRENE® G AND ALTOSID® PELLETS AGAINST AEDES AEGYPTI

In response to the emergence, reemergence, resurgence, or upsurgence of mosquitoes and mosquito-borne diseases worldwide (Gratz 1999, Chala and Hamde 2021), there is a high demand for mosquito control by environmentally compatible interventions. This scenario has intensified since the introduction of West Nile virus and invasive Aedes mosquitoes in the USA. In mosquito control operations, controlling habitat-concentrated aquatic stages, mainly larvae and sometimes pupae, is more cost-effective and feasible as compared with combating dispersed populations of the airborne adult stage (Antonio-Nkondjio et al. 2018, Derua et al. 2019). However, upon the gradual phasing out of conventional pesticides for larval control, available mosquito larvicides that meet the criteria of efficacy, safety, and quality are limited for reasons such as excessive cost in development and registration, narrow market margins, and strict regulations. Products based on microbial agents and insect growth regulators (IGRs) are the primary options for highly dynamic mosquito control and mosquito-borne disease management (Su 2022).

Among the IGRs, S-methoprene has been the most widely used juvenile hormone analog (JHA) in operational mosquito control (Su and Liu 2024). Considering this JHA’s advantages as a synthetic biopesticide as recognized by the US Environmental Protection Agency (EPA 2024), many products based on S-mehoprene have been developed against a wide variety of pests with economic significance (Henrick 2007, Su 2018). Along with the effort to develop novel active ingredients (AIs) (Su et al. 2023), innovations in formulation technologies to preserve and deliver the AIs in a tailor-made way are equally important. Altosid® Pellets and Altosid Pellets with DR-tech are among the well-recognized control release formulations of S-methoprene. The counterpart product, OmniPrene® G, with the same AI level, but different inert carrier and formulation technologies, has recently become available. To add more capacity to the mosquito control toolbox, the current paper is aimed at evaluating the comparative laboratory activity and semi-field efficacy of inhibition of adult emergence of the two aforementioned products against an important reference species, the yellow fever mosquito Aedes aegypti (L.).

The technical S-methoprene was provided by Synergetica International Inc. (Marlboro, NJ) with a purity of 98.06% (US EPA reg# 73487-1).

This granular formulation containing 4.25% S-methoprene (US EPA reg# 73487-2) was also provided by Synergetica International Inc. This formulation consists of 1.00–2.00 mm diam (10–18 mesh) diatomaceous earth (DE) granules with 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.

Altosid Pellets with DR-tech (US EPA reg# 2724-448) with 4.25% S-methoprene originated from the Central Life Sciences (Schaumburg, IL). This product uses plaster of Paris and activated carbon as carrier, has a diam of 4 mm, but varied length from 4 to 12 mm, with a bulk density 0.9–1.0 g/ml.

Aedes aegypti eggs were supplied by Benzon Research (Carlisle, PA) from its long-term susceptible colony. Eggs were hatched and larvae reared according to the procedures published previously (Su et al. 2019). The late 4^th instars, which were about to pupate, were used in laboratory bioassays.

Laboratory bioassay was conducted at the laboratory of EcoZone International (Riverside, CA) according to the previously published protocols (Su et al. 2018) with minor modifications along with the technical grade AI to validate the products to be evaluated. The technical S-methoprene was dissolved in and serially diluted by ACS pure acetone (Cole Parmer, Vernon Hills, IL). The OmniPrene G and Altosid Pellets were pulverized in a coffee grinder (Hamilton Beach Custom Grind; Southern Pines, NC) at the maximum speed in interruption mode until products were turned to fine powders, then suspended in deionized water by vortexing at 2,000 rpm for 3 min (Digital Vortex Genie 2; Scientific Industries, Inc., Bohemia, NY, USA). The 10x serial dilutions were then made in deionized water. For the bioassay, 5 concentrations (0.025, 0.5, 2.5, 10, and 25 ppb) within the dose range resulting in approximately 5–95% mortality, plus untreated control (UTC) were used, with 3 replicates at each concentration and UTC. Each aliquot, with appropriate dilution in the amount of 100–500 µl, was added to 100 ml deionized water, where the volume increase was negligible. In technical S-methoprene, such a small amount of acetone was used in the bioassay water that it evaporated quickly without observable impact on the Styrofoam polystyrene cup and test subjects as shown in UTC with solvent only. For each replicate, 25 late 4^th instars were placed in 100 ml deionized water in a 120-ml disposable cup. A small piece (approximately 100 mg) of rabbit pellets was added to each bioassay cup to have 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 or emerged as adults. Only the free pupal exuviae were considered as successful adult emergence.

Aedes aegypti larvae used for the semi-field evaluation came from the susceptible colony of Manatee County Mosquito Control District (MCMCD) in Palmetto, FL. This colony consists of Orlando strain (ORL 1952) obtained from the United States Department of Agriculture-Agricultural Research Service (USDA-ARS) Center for Medical, Agricultural, and Veterinary Entomology (CMAVE), Gainesville, FL, and has been maintained at MCMCD since 2016 according to CMAVE colonization protocols.

Given the advantages of a previous semi-field test to accurately quantify larvicide efficacy under controlled conditions for new products based on S-methoprene (Su and Su 2022a, 2022b), the tests were carried out in semi-outdoor microcosms located in a protected screened enclosure room at the MCMCD facility in Palmetto, Florida (Fig. 1). Clear plastic tubs were selected based on qualitative observations that abandoned plastic containers are among the most common breeding grounds for Ae. aegypti in urban environment of Manatee County. The microcosms measured L0.60 × W0.37 × D0.39 m. Water depth was maintained at 11.5 cm for each microcosm (approximately 25.5 liters) with a surface area of 0.22 square meters. Relative shallow water was used to mimic habitats of Ae. aegypti: small containers intermittently flooded by rains or irrigation leakage from the landscape irrigation. A 3,800-ml emergence jar with a 7.5-cm diam screened top was installed in the middle of the lid at each microcosm to trap the emerged adults during observation. Larval food, which consisted of 20–40 ml of a 3:2 liver yeast slurry was added to each microcosm as larval food and organic enrichment, after flooding and once weekly on days that new larvae were introduced. The treatment was made on day 2 post-flooding, when the larval food sediment had settled down. The application doses for each of the two products were the lowest (2.8 kg/ha) and the highest (11.2 kg/ha) as recommended on product labels, equivalent to 61.59 mg and 246.40 mg per microcosm. Fifty late 2^nd to 3^rd instars of Ae. aegypti were introduced to each microcosm weekly for 9 weeks, after removing previously tested individuals before the new introduction. A submersible thermometer was put at bottom of one tub that was in the middle of the microcosm layout to monitor water temperature at time of observation, typically in the mid afternoon during the summer. Three replicates (tubs) were made of each treatment (2 products × 2 doses) and UTC, for a total of 15 tubs (Fig. 1).

Observations were made on a daily basis for larval growth, pupation, adult emergence, and stage-specific mortality until all introduced individuals died or emerged to adults. All individuals remained in the bins until the weekly observations were concluded. Mortalities were categorized: 1) “mortality before emergence” including dead larvae and dead pupae, 2) “mortality during emergence” indicating incomplete emergence with legs and/or wings attached to the pupal exuviae, and 3) “other mortality” - disintegrated or unrecoverable, meaning dead individuals were broken or unable to be recovered because of being consumed by the survivors. Successfully emerged adults were counted from pupal exuviae.

Concentration - response data in laboratory bioassays were corrected by Abbott formula to factor the mortality in UTC (Abbott 1925), then analyzed using POLO Plus (Robertson et al. 1980) for probit analysis to calculate the concentrations causing 10%, 50%, and 90% inhibition of emergence (IE) and their 95% confidence intervals (95% CIs). Significant differences in IE were indicated by separate 95% CIs (Su et al. 2018). In the semi-field test, the mean IE% for UTC and each treatment on each sampling day was calculated: IE% = 1- (number of successfully emerged adults/total number of larvae tested). The IE% between the products at the same dose on the same sampling intervals were analyzed by Chi square test after correction by Abbott formula (Abbott 1925) for the significance at χ² ≥ 3.84, P ≤ 0.05 and χ² ≥ 6.63, P ≤ 0.01 levels. Stage-specific mortality was charted by products, concentrations, and sampling intervals to reveal the general patterns.

Concentration - response was established in the concurrent bioassays using technical S-methoprene, OmniPrene G, and Altosid Pellets against the test species, with low mortality in UTC (3.6–8.5%). High activity of inhibition of adult emergence was indicated after data correction by Abbott formula. By comparing the IE₁₀, IE₅₀, and IE₉₀, there were no significant differences among the technical S-methoprene and the formulated products at three IE levels as shown by overlapping 95% CIs (Table 1). Mortality occurred in an overall pattern of incomplete adult emergence, dead pupae, dead “puparvae” (larval-pupal intermediates) (Su et al. 2020), or dead larvae, upon the increases of S-methoprene concentrations.

The overall mortality was low in UTC, ranging from 0.7% to 6.0% during the 9-week evaluation. At the low dose of 2.8 kg/ha, Altosid Pellets showed lower efficacy than OmniPrene G during the initial 6 weeks (χ² = 5.28–34.57, P < 0.05–0.01), this difference became negligible on week 7, followed by higher efficacy in Altosid Pellets on weeks 8 and 9 (χ² = 7.05–8.66, P < 0.01). More uniform efficacy was observed at the high dose of 11.2 kg/ha. Equal performance between two products was revealed during weeks 2 to 6, with the OmniPrene G outperforming the Altosid Pellets on week 1 (χ² = 7.17, P < 0.01), but opposite during weeks 7 to 9 (χ² = 19.15–27.27, P < 0.01) (Fig. 2).

Generally, stage-specific mortality patterns were similar in both products, i.e., a majority of the mortality occurred before emergence, primarily dead pupae and dead larvae. Mortality during emergence, i.e., incomplete emergence was more prevalent at the lower doses where some adults attempted to emerge from pupae. However, the proportions of “other mortality” - disintegrated or unrecoverable individuals were higher at the high doses. At the low dose, overall higher incomplete emergence was noted in Altosid Pellets than in OmniPrene G during the 9-week test period. At the high dose, on the other hand, noticeable incomplete emergence only prevailed at Altosid Pellets on week 1, and became moderately common in OmniPrene G during weeks 7–9 (Fig. 3).

Water temperatures at the time of observation ranged 25.7°–29.5°C (average 27.8°C) during the 9-week evaluation.

Upon the further understanding of insect JHAs for their safety and mode of action, methoprene earned the revision of registration status with US EPA from conventional chemical pesticide to biopesticides during 1970–1980. Along with the advancement in synthetic chemistry, original R,S-methoprene (CAS 40596-69-8) was replaced by S-methoprene (CAS 65733-16-6) with enhanced bioactivity (Su et al., unpublished data) during 1980–1990, followed by the addition of newly innovated S-methobutene (CAS 2699134-88-6) with significantly improved bioactivity and stability with the same mode of action as S-methoprene (Su et al. 2023). At the same time, innovation in methoprene-based formulations has been relatively limited, early formulations with accepted field performance have dominated the marketplace for decades with minimal additions of new products, most notably MetaLarv® S-PT and XRP (Valent BioSciences 2024) in recent years. To enhance product diversity and meet customized needs in mosquito control operations, it is advantageous to have newly developed products available such as OmniPrene G, that are formulated utilizing innovative carriers and cutting-edge control release technologies.

Bioassays on technical grade pesticide can be straightforward, although tests on JHAs do need more detailed involvements (Su et al. 2021). Testing of control release formulations such as OmniPrene G and Altosid Pellets is more challenging than those for technical grade materials. In current studies, a full concentration - response range was achieved for both test materials and IE₁₀, IE₅₀ and IE₉₀ and their 95% CIs were successfully obtained without needs for projections. In mosquito species, late instar larvae are more susceptible than the early instar larvae to external JHAs such as methoprene (Noguchi and Ohtaki 1974). Young larvae have low susceptibility to JHAs because of high internal concentrations of juvenile hormone. The bioassay using young larvae would underestimate the activity of JHAs, take longer to complete or even end with inconclusive results because the peak concentration of JHAs in bioassay cups and susceptibility window of larvae miss each other upon degradation of AI and larval growth. Therefore, late instars, sometimes called pupating larvae, were used in current studies to ensure the maximized and synchronized inhibition of emergence in the laboratory bioassays. The observed low mortality (3.6–8.5%) in UTC and conclusion from the week-long bioassay were in support of the test protocols mentioned above. The similar IE levels between the technical grade and both products with the same AI levels further endorsed the analysis process as previously published (Su et al. 2018).

During the 9-week semi-field evaluation, late 2^nd to 3^rd stage larvae of susceptible Ae. aegypti were used in consideration of the control release formulations, with the belief that the susceptible window of the growing larvae would synchronize with the concentration of persistently released AI. Here introduced larvae did have longer exposure to the AI as compared with the late instars in the previously described bioassays where controlled release mechanism in formulations tested was mostly negated by pulverization of the granules or pellets. As a benchmark product, Altosid Pellets showed lower and more varying efficacy than OmniPrene G at the low dose. One of the three replicates, in particular, provided lower efficacy, which is believed to be attributable to the possibly uneven AI loading in the bulky Altosid Pellets with highly varying sizes (4 × 4–12 mm). Moreover, the role of inner carriers in controlled release of the AI in Altosid Pellets remained unknown in current evaluation. OmniPrene G consisting of 1–2 mm diam granules did show the advantages of even coverage and timely release. Its efficacy, however, declined in week 7, when it was beyond the product label’s recommended duration of 6 weeks (EPA 2022). The initial AI release by OmniPrene G remained beneficial at the high dose, as shown by the higher efficacy than Altosid Pellets in week 1 post-treatment. On the other hand, Altosid Pellets did show the advantages of longer residual efficacy than OmniPrene G beyond week 7, as the larger matrix of the carrier in the pellets probably had provided better protection and allowed longer residual for the AI.

Based on the mode of action, S-methoprene as a true JHA that is categorized in Group 7A by Insect Resistance Action Committee (IRAC) (IRAC 2024), the stage-specific mortality pattern uniformly showed major lethal activity in pupae and larvae (mortality before emergence). It was the incomplete emergence (mortality during emergence) that showed product- and dose-dependent differences. Incomplete emergence, considered as a marginal success of survivorship, was associated with the scenarios of low efficacy such as the bulky formulation having a slower release characteristic, lower doses or later time of evaluation period when free methoprene concentrations in the water had declined. The higher proportions of “other mortality” - disintegrated or unrecoverable individuals at the high doses indicated that some larvae might have died earlier and been consumed by the surviving ones.

Regarding the OmniPrene G, the inert carrier DE granules consist of natural fossilized remains of unicellular algae called diatoms. The properties of DE such as low density, high porosity, large surface area per given weight or volume, and high adsorption capacity allow its unique industrial value and extensive uses in pesticide formulation (Tsai et al. 2006, Jia et al. 2007, Rose 2010). Additionally, the DE granules have a specific gravity of 4.0 g/ml if disregarding the internal spaces, which helps to anchor the granules down to the bottom of the habitats. However, the bulk rate of the DE granules is 0.6 g/ml if the internal spaces are considered. It is this “low density” feature that prevents them from further sinking and being buried into the sediment, thus allowing the granules to move around close to the top of the sediment in the habitats. Moreover, the proprietary formulation process and binding agents also play a crucial role to ensure the protection and proper release of the S-methoprene within and beyond labeled longevity (Su and Su 2022a, 2022b). The Altosid Pellets use a composite carrier consisting of plaster of Paris (CAS 26499-65-0, 69.16%), activated carbon (CAS 7440-44-0, 9.26%), silica sand (CAS 14808-60-7, 0.7%) and others (balanced to 100% by materials that are non-hazardous and/or do not meet criteria for classification), which render protection and controlled release of the S-methoprene. This composite carrier results in a bulk density 0.9–1.0 g/ml, which is believed to prevent the pellets from washing away or being embedded into the sediment in mosquito larval habitats. When considering their dust-free hardy carriers, both products are expected to withstand the stress of handling and application under real world conditions. Further evaluations are warranted in the future to compare with other similar formulations such as MetaLarv S-PT, as well as against other mosquito species.

When considering the increasing demand for mosquito control to face the nuisance species and mosquito-borne diseases, more viable intervention tools are highly desired. It is expected that S-methoprene-based OmniPrene G, along with the long recognized Altosid Pellets, and very few other products such as MetaLarv® S-PT will continue to play important roles in combating mosquito species of public health and well-being significance now and in the future.

The authors are grateful to Synergetica International Inc. (Marlboro, NJ) and Central Life Science (Schaumburg, IL) for provision of test samples. Our special thanks are due to the intern Megan Walder for her assistance in microcosm test.