Effects of different knock-down resistance polymorphisms on pyrethroid susceptibility in the stable fly Stomoxys calcitrans and deep amplicon sequencing to screen for resistance haplotypes on German dairy farms

Abstract text

Introduction

Stable flies (Stomoxys calcitrans) are biting flies that disturb the well-being of livestock and severely affect their productivity. Excessive, decades-long use of pyrethroids against insects has resulted in widespread resistance, and we previously reported the presence of pyrethroid resistance in stable flies on farms in Brandenburg (BB). Pyrethroid resistance in stable flies has been associated with kdr (L1014F) and kdr-his (L1014H) mutations but not super-kdr (M918T) in the voltage-sensitive sodium channel (vssc) gene.

Objectives

This study aimed to examine the correlation between pyrethroid resistance and the vssc genotype (kdr and kdr-his) in stable flies and to screen German stable fly populations for kdr and kdr-his mutations.

Materials & methods

In 2018, stable flies were collected from four dairy farms in BB and reared over two generations in the laboratory. The LD₅₀ values for deltamethrin were determined for each isolate by topical application and compared with a susceptible laboratory strain (MSD). Selected, phenotypically tested flies were genotyped using PCR amplification of specific alleles (PASA). Using pooled stable fly samples collected from 65 dairy farms in BB, Schleswig-Holstein, and Baden-Württemberg in 2019, deep amplicon sequencing of the vssc gene was used to investigate the frequency and diversity of resistance alleles.

Results

The four tested field populations were identified as being phenotypically resistant to deltamethrin, with LD₅₀ values of 18.0, 19.6, 40.5 and 47.0 ng deltamethrin/fly, 47.5 to 123.6-fold higher than for MSD strain. PASA showed the presence of kdr and kdr-his genotypes in all field populations, with significant (p < 0.05) higher odds to survive treatment already in heterozygous individuals compared to wild-type flies, and stronger effects of kdr than kdr-his on the resistance phenotype. Deep amplicon sequencing revealed the presence of kdr and kdr-his variants on 100% of the farms with up to 55% of kdr and 85% of kdr-his alleles on individual farms. Phylogenetic analysis suggested at least two independent origins of both, kdr and kdr-his variants. In 19/65 pools, the world-wide first evidence for super-kdr alleles in stable flies (frequency 0.6-9.5%) was observed.

Conclusion

Pyrethroid resistance genotypes are widespread in stable fly populations on German dairy farms. The deep amplicon sequencing assays will allow to screen for pyrethroid resistance markers on a nation-level scale.