Material and methods

Collection and Rearing

Between 2023 and 2024, four field populations of B. germanica were collected from public and residential settings across two Iranian cities: a university dormitory in Shiraz (29°38′35.44″N, 52°30′44.57″E), a residential apartment in Shiraz (29°37′19.19″N, 52°32′27.14″E), a fast-food restaurant in Tehran (35°42′46.83″N, 51°24′24.06″E), and Namazi Hospital in Shiraz (29°35′28.38″N, 52°35′01.06″E). All four field populations were sourced from locations with documented histories of insecticide application. A laboratory-susceptible strain, maintained under insecticide-free conditions for over 30 years at the insectary of the Department of Vector Biology and Control of Disease, School of Public Health, Tehran University of Medical Sciences, was used as the untreated control throughout all bioassays (Ladonni, 2001).

The cockroaches were reared separately in standard containers under controlled environmental conditions (24 ± 2 °C, 30–50% relative humidity, and a 12:12 L:D photoperiod). They were provided with food (dog food), water, and cardboard harborages as shelter (Gemeno Marín et al., 2011).

Insecticide and bioassay

Technical grade of fipronil, with a purity of 95%, was obtained from Moshkfam-Fars™ Chemical Company, Shiraz, Iran. The 50%. 90%,95% and 99% lethal dose (LD50, LD90, LD95 and LD99) of each population was determined using topical application bioassay (Nasirian et al., 2006b). Groups of 20 adult male cockroaches were briefly anesthetized with CO2 in a plastic Petri dish. Fipronil was topically applied in acetone using a micro-applicator (Hamilton, Reno, NV) equipped with a 50 μl glass syringe (Hamilton Co.), delivering 1 μl onto the ventral thorax of each cockroach (Scott et al., 1990). Serial dilutions of fipronil were prepared in acetone to achieve final doses ranging from 0.0002 to 5.685 ng/insect, corresponding to concentrations of 0.001, 0.01, 1, 2.5, 5, 7, 10, 15, and 25 ppm. For each of the five strains, bioassays were conducted using nine concentrations, with three independent replicates per concentration (540 individuals per population). Mortality was assessed at 24-, 48-, 72-, and 96-h post-treatment by gently probing each individual with forceps. Cockroaches that failed to exhibit coordinated locomotion in response to mechanical stimulation were recorded as dead. Final mortality rates were determined at 96 h post-treatment (González-Morales et al., 2022).

DNA extraction and Rdl gene sequencing

To investigate target-site resistance mechanisms, five adult males from each of the four field populations of B. germanica and one individual from the laboratory-susceptible strain (21 samples) were screened for Rdl gene mutations associated with the A302S amino acid substitution (Ang et al., 2013). The heads of individual cockroaches were homogenized for 30 s, and DNA was extracted using the Smbio™ DNA extraction Kit according to manufacturer's instructions. A 245-bp genomic fragment of the GABA receptor gene that includes the Rdl mutation site was amplified with the primers BG-Rdl-F (5′-GTGCGGTCCATGGGATACTA-3′) and BG-Rdl-R (5′-AACGACGCGAAGACCATAAC-3′) (M. Kristensen et al., 2005). A negative control with no template DNA was included in every PCR run. The following thermal cycle program was used for amplification: Initial activation at 95 °C for 5 min followed by 40 cycles of 94 °C for 30 s, 60.0 °C for 30 s, and 72 °C for 30 s and a final extension at 72 °C for 5 min (Gondhalekar and Scharf, 2012). Two microliters of each PCR product were electrophoresed on a 1.2% agarose gel to confirm the presence of bands with sized estimated. The remaining PCR product was sequenced by Sanger sequencing method. Each sequence was manually inspected for the GCC to TCC, TCC to AGC and GCA to TCA mutation that results in the A302S, A301S and A299S substitutions**.**

Haplotype reconstruction and confirmation

Genotype data quality was evaluated using Chromas v2.6.6 within BioEdit version 7.2.5 and MEGA 11 (Hall, 1999; Kumar et al., 2018). Rdl gene sequences obtained from 21 specimens, comprising 20 field-collected individuals and one susceptible strain from four populations, were aligned and analyzed in DnaSP v6 for haplotype identification and reconstruction (Librado and Rozas, 2009). Genetic relationships among haplotypes were visualized by constructing a median-joining haplotype network in PopART v1.7 (Leigh, Bryant, Nakagawa, and Evolution, 2015). Haplotype assignments were further confirmed through manual examination of sequence alignments and validation of polymorphic nucleotide sites.

Mutation analysis

For molecular analysis, DNA sequences were aligned and analyzed using MEGA11 and BioEdit version 7.2.5 software to identify point mutations, focusing on the A302S/N substitution associated with fipronil resistance (Ang et al., 2013). Sequence comparisons between field-collected and susceptible B. germanica strain were conducted to evaluate mutation frequencies and patterns. The reference sequence used was the complete coding sequence of the B. germanica GABA-gated chloride channel, deposited in GenBank under accession number MW267921.1 (Jones et al., 2021).

Statistical analysis of bioassay data

The LD50, LD90, LD95, and LD99 values for each cockroach population were determined using log-dose probit-mortality analysis. Probit analysis was done using R software version 4.3.2 with the STATS and ggplot2 package. When necessary, Abbott's formula correction was applied for data of mortality (Abbott, 1925). The lethal dose ratio at LD50 was then used to calculate resistance ratios (Valles et al., 1997). To compare mortality at each fipronil concentration with the acetone control within each population, a chi-square test of independence was performed. For each concentration, a 2 × 2 contingency table was constructed (control vs. treatment, dead vs. alive). The resulting p-values were adjusted using the Bonferroni correction. A corrected p-value <0.05 was considered statistically significant.

In addition, to confirm the dose-dependent relationship independently of the probit model, Spearman correlation analyses were conducted between log-transformed dose and mortality proportion for each population (using only concentrations >0 ppm).

Goodness-of-fit of the probit models was assessed using the Pearson chi-square statistic. A non-significant p-value (p > 0.05) indicates that the model adequately describes the observed dose-mortality relationship.

Resistance ratios (RRs) and resistance-level classifications were adopted from previous studies on German cockroach insecticide resistance and other urban insect pests. Populations with RR ≤ 1 were considered susceptible, those with RR > 2 as exhibiting low resistance, RR > 5 as moderate resistance, and R_R_ > 10 as high resistance (R. Y. Chai and Lee, 2010a; González-Morales et al., 2022; K.-M. Lee and Lee, 2002; Öz et al., 2021).