Effect of Cup Material on S-Methoprene Bioavailability and Susceptibility Estimates in Larval Mosquito Bioassays

Mosquito control programs rely on larvicides such as S-methoprene to suppress vector populations and prevent mosquito-borne diseases including West Nile virus (WNV). Resistance surveillance is essential for maintaining the long-term effectiveness of larvicide active ingredients and is typically conducted using cup bioassays to evaluate larval susceptibility. Disposable Styrofoam cups are commonly used for these assays because they minimize adsorption of S-methoprene under standard testing conditions. However, recent restrictions on expanded polystyrene products in California necessitated the identification and evaluation of suitable replacement cup materials for larval resistance bioassays. Four alternative cup materials, high-density polyethylene (HDPE), polyethylene terephthalate (PET), polypropylene (PP), and paper cups, were compared to Styrofoam cups. Cup bioassays were conducted using a laboratory colony of Culex quinquefasciatus exposed to S-methoprene concentrations of 1-40 ppb. Emergence inhibition data were analyzed using probit regression to estimate the concentrations causing 50% and 90% emergence inhibition (LC₅₀ and LC₉₀, respectively) for each cup type. Cup material significantly affected concentration-response relationships. HDPE and paper cups produced significantly higher LC₅₀ and LC₉₀ estimates than Styrofoam, whereas PET and PP produced responses more comparable to the Styrofoam reference. Concentration-response slopes were similar among cup materials, indicating that cup material primarily shifted the concentration required to achieve a given level of emergence inhibition. These findings demonstrate that cup material can significantly influence estimated S-methoprene LC values. Replacement assay cups should therefore be validated before implementation in larval resistance monitoring programs.