Discussion

This study reveals significant variation in susceptibility and resistance to fipronil among German cockroach (Blattella germanica) populations. Using probit analysis across a concentration range of 0.001 to 25 μl in 1000 μl acetone, the susceptible strain exhibited an LD₅₀ of approximately 0.68 ppm, while resistant field populations showed LD₅₀ values more than 20-fold higher, indicating substantial resistance ratios (Scharf & Gondhalekar) (Table 4). These findings align with previous reports documenting highly variable mortality rates (8.5–98.4%) in field populations exposed to fipronil baits after 7 days (Khoobdel et al., 2022a; H Nasirian, 2007; H Nasirian et al., 2006b). Probit analysis effectively described the dose-response relationships, providing reliable mortality predictions based on the cumulative normal distribution a standard approach for evaluating insecticide efficacy in pest species (González-Morales et al., 2022) (Table 4).

Time period | Location | RRa range | Type of treatment | Assay used | Time of mortality assessment (Castilhos et al.) | References | Notes
1997–2010 | United States | 1.0–1.8 (1.8–7.7) b | Topical | Dose-response | 4 | Scott and Wen (1997) | Baseline susceptibility
 | United States | 1.0–1.3 | Topical | Dose-response | 1 | Valles et al. (1997) | Low resistance
 | United States | 1.2–>17c, d, e | Topical | Dose-response | 3 | Holbrook et al. (2003) | Emerging high resistance
 | United States | 8.7–9.3 | Topical | Dose-response | 3 | Wang et al. (2004) | Moderate resistance
 | Denmark | 1–15 | Topical | Dose-response | 3 | Kristensen et al. (2005) | Variable resistance
 | Iran | 1–2.6 | Topical | Dose-response | 3 | Nasirian et al. (2006a) | Low resistance; permethrin cross-check
 | Singapore | 1.0–10.0 | Topical | Dose-response | 2 | Chai and Lee (2010a) | Moderate resistance
2011–2020 | United States | 37.9 | Topical | Dose-response | 3 | Gondhalekar and Scharf (2012) | High resistance
 | Singapore | 1.2–3.0 (10.8–25.8) b | Topical | Dose-response | 2 | Ang et al. (2013) | Increasing secondary kill resistance
 | Puerto Rico | 5.6 (15.9) | Topical | Dose-response | 2 | Ko et al., (2016) | Moderate resistance
 | United States | 0.9–1.4 (2.5–25.0) b | Topical | Dose-response | 5 | Liang et al., (2017) | Variable, high secondary
 | United States | 2.0–8.7 | Topical | Dose-response | 3 | Wu and Appel (2017) | Moderate resistance
 | United States | 6–23 | Topical | Dose-response | 2 | DeVries et al. (2019) | High resistance
 | Taiwan | 1.5–3.8 | Surface contact | Time-course (LT50) | 7 | Hu et al. (2020) | Low-moderate; LT50 focus
2021–2025 | United States | ∼27.7f | Ingestion | Discriminating doses | 3 | Lee et al. (2022) | Stable high resistance
 | United States | 22.4–37.2 | Topical | Dose-response | 4 | González-Morales et al. (2022) | Linked to A302S mutation
 | Indonesia | 12.21 | Topical | Dose-response | 2 | )Van Dini et al., (2023) | Moderate-high resistance
 | Iran | N/A (76.5% decline at 7 days) | Bait (gel/powder) | Field trial (sticky traps) | 7 | Khoobdel et al. (2022a) | Efficacy study; no RRa, high mortality with 0.02% fipronil; potential A302S link
 | Present study | 1.77–13.35 | Topical | Dose-response | 4 | – | Low-moderate; LD50 focus

A global review indicates a consistent upward trend in fipronil resistance in B. germanica across multiple continents (Table 4). Studies from North America (González-Morales et al., 2022), Asia (R.-Y. Chai and Lee, 2010b; H Nasirian, 2007), and Europe (Kristensen et al., 2005) have documented resistance factors reaching up to 100-fold in some populations. This pattern reflects strong selection pressure imposed by repeated fipronil applications in urban environments, which favors the survival and spread of resistant genotypes (Fig. 5). In Iran, clear geographic variation in fipronil resistance was observed among field populations. Resistance ratios (RR), calculated relative to the susceptible strain (LD₅₀ = 0.68 ppm), ranged from low to moderate in Dormitory and Apartment populations (RR = 1.77–3.36). In these settings, fipronil may still provide effective control when integrated with optimized application strategies and regular resistance monitoring (Kristensen et al., 2005; Scott and Wen, 1997; Valles et al., 1997). In contrast, Fast-food restaurant and Hospital populations exhibited high resistance levels (RR = 12.48–13.35), suggesting that standard fipronil treatments may no longer be reliable in these environments (Gondhalekar and Scharf, 2012; González-Morales et al., 2022; Holbrook et al., 2003; S.-H. Lee et al., 2022). The relatively low probit slope values (0.49–1.07) further indicate substantial heterogeneity in susceptibility within populations, likely reflecting underlying genetic and physiological diversity and the possible coexistence of multiple resistance mechanisms (Abbott, 1925; Finney, 1971; Robertson et al., 2017).

Fig. 5: Temporal trends in fipronil resistance development in german cockroach (Blattella germanica) populations.

The present study also provides new insights into the molecular basis of resistance by identifying two previously unreported amino acid substitutions in the Rdl gene of B. germanica: A299S and A301S. While the well-known A302S mutation was not detected, substitutions at nearby positions (including A296S, A299S, and A301S) have been linked to altered GABA receptor function and insecticide resistance in other insects such as Anopheles spp., Musca domestica, Nilaparvata lugens, and Drosophila melanogaster (Garrood et al., 2017; Liu et al., 2020; Ozoe et al., 2015; Remnant et al., 2014). The presence of these variants in a highly conserved region of the GABA-gated chloride channel suggests they may represent novel adaptive changes under insecticide selection pressure.

Haplotype analysis of a 245-bp fragment of the Rdl gene revealed exceptionally high genetic diversity, with 17 unique haplotypes among 21 individuals (Hd = 1.000 ± 0.020; π = 0.0405 ± 0.007), 24 polymorphic sites, and 27 mutations. The haplotype network showed a star-shaped pattern with Hap_18 (accession MW267921.1, USA) at the center, likely representing an ancestral haplotype. Hospital and fast-food restaurant populations displayed greater mutational distances, while the laboratory strain exhibited lower diversity and clustered near the central haplotype. This high level of standing genetic variation is consistent with the known genetic plasticity of urban cockroach populations under recurrent insecticide pressure (Wada-Katsumata and Schal, 2024; Fardisi et al., 2019) and may be maintained by balancing selection or demographic processes (Nei, 1987). Overall, these findings underscore the genetic complexity of fipronil resistance evolution in B. germanica and highlight the need for location-specific resistance management. Integrating phenotypic bioassays with molecular surveillance of Rdl variants will be essential for tracking resistance dynamics. Future studies combining functional validation of novel mutations, broader geographic sampling across Iran, and population genetic analyses will help develop predictive tools for resistance emergence. Such integrated approaches are critical for implementing effective Integrated Pest Management (IPM) strategies and preserving the long-term efficacy of fipronil and other insecticides in urban cockroach control.