Multiple Mechanisms Confer Fipronil Resistance in the German Cockroach: Enhanced Detoxification and Rdl Mutation

Abstract

Populations of Blattella germanica (L.) (German cockroach) have been documented worldwide to be resistant to a wide variety of insecticides with multiple modes of action. The phenylpyrazole insecticide fipronil has been used extensively to control German cockroach populations, exclusively in baits, yet the highest reported fipronil resistance is 38-fold in a single population. We evaluated five populations of German cockroaches, collected in 2018-2019 in apartments in North Carolina and assayed in 2019, to determine the status of fipronil resistance in the state. Resistance ratios in field-collected strains ranged from 22.4 to 37.2, indicating little change in fipronil resistance over the past 20 yr. In contrast, resistance to pyrethroids continues to escalate. We also assessed the roles of detoxification enzymes in fipronil resistance with four synergists previously shown to diminish metabolic resistance to various insecticides in German cockroaches-piperonyl butoxide, S,S,S-tributyl phosphorotrithioate, diethyl maleate, and triphenyl phosphate. These enzymes appear to play a variable role in fipronil resistance. We also sequenced a fragment of the Rdl (resistant to dieldrin) gene that encodes a subunit of the GABA receptor. Our findings showed that all field-collected strains are homozygous for a mutation that substitutes serine for an alanine (A302S) in Rdl, and confers low resistance to fipronil. Understanding why cockroaches rapidly evolve high levels of resistance to some insecticides and not others, despite intensive selection pressure, will contribute to more efficacious pest management.

Keywords: Blattella germanica; Rdl; German cockroach; fipronil; insecticide resistance; target site insensitivity; urban IPM.

Fig. 1.

Dose–response curves for fipronil-treated B. germanica adult males from a reference insecticide-susceptible strain (Orlando Normal) and five populations recently collected in apartments in Raleigh, NC. The lethal dose of fipronil that killed 50% of each population (LD₅₀) was determined by topical application. Mortality was assessed daily, and mortality at 4 d is reported. At least three replicates of 10 adult male cockroaches were performed per dose.

Fig. 2.

Effects of four detoxification enzyme inhibitors (i.e., insecticide synergists) on fipronil toxicity in B. germanica adult males. Each enzyme inhibitor (piperonyl butoxide [PBO], S,S,S-tributyl phosphorotrithioate [DEF], diethyl maleate [DEM], and triphenyl phosphate [TPP]) was topically applied in 1 µl acetone 1 h prior to application of a population-specific LD₅₀ (shown below the population name). Percent mortality was determined 4 d after treatment and mortality was corrected for control mortality (synergist only). The mean shown is of 3 replicates with 10 males each (n = 30 males per treatment). Significant differences between fipronil-only treatments and fipronil plus inhibitor treatments were determined using Chi-square analysis, with significance indicated by * (P < 0.05).

Fig. 3.

Nucleotide sequences of the TM2 region of the B. germanica Rdl gene, which includes the point mutation that results in the A302S substitution. (A) Representative sequences from the insecticide-susceptible Orlando Normal strain and five field-collected German cockroach populations were aligned against the reference sequence (MW267921.1), with the A302S region highlighted. (B) Representative direct sequencing chromatograms of homozygous susceptible (G/G) and homozygous resistant (T/T) cockroaches. The G-to-T point mutation site is shown within a box. MW267921.1 is the GenBank accession number for the B. germanica GABA-gated chloride channel complete cds (Jones et al. 2021).