Warfarin resistance test and polymorphism screening in the VKORC1 gene in Rattus flavipectus

Abstract

The buff-breasted rat (Rattus flavipectus) is a major agricultural pest across China. Warfarin-resistant animals have been found in several major provinces in China, and are hampering effective control. Molecular mechanisms underlying resistance to anticoagulant rodenticides have been determined for other species, but genetic information regarding resistance in R. flavipectus remains unknown. The vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1) encoded by VKORC1 gene is the molecular target of coumarin anticoagulants, and amino acid substitutions in VKORC1 coding-regions have been reported as one of the supposed mechanisms of warfarin resistance. Here, lethal feeding test in R. flavipectus (n = 36) was conducted in Zhanjiang, China. Four animals (11%) survived the test period of 25 days and were identified as warfarin resistance. Polymorphism across the whole genome DNA sequence of the VKORC1 gene was screened out and compared with resistant and non-resistant rats. A total of nine single nucleotide polymorphisms (SNPs) were identified including seven SNPs in introns and two SNPs in exons, and the SNP (2317A [ G) located in exon 3 led to the amino acid substitution (Tyr139Cys) in VKORC1 protein. Based on the characteristics of Tyr139Cys mutation of VKORC1 in humans or rats and its relationship with warfarin-resistance, Tyr139Cys mutation may be one mutation responsible for anticoagulant resistance in R. flavipectus. Given the low numbers of resistant rats in our feeding test, wider surveillance, tests of resistance development in a larger wild population and further researches on the genetic mechanisms of anticoagulant resistance in R. flavipectus are necessary.

Fertility control of Rattus nitidus using quinestrol: effects on reproductive organs and social behavior

“…Zeng et al (1999) report that the extremely flexible reproductive tactics of R. nitidus facilitate juvenile survival and enable this rodent species to maintain remarkably stable populations despite predictably periodic and frequent fluctuations in its agricultural environment. Current control strategies for rodent pests mainly rely on increasing death using anticoagulants; although these poisons have been useful in reducing immediate damage associated with rodents, the repeated and long-term application of anticoagulant rodenticides might result in environmental problems due to indirect and direct poisoning of non-target species and chemical residues, and might result in the rapid development of resistance to these compounds in wild rodent species in many countries (Zhang et al 2001;Liang 2005;Pelz et al 2005;Zhu 2005;Wu 2007;Wang et al 2008;Huang et al 2011). Investigation into new methods of rodent population control is urgently needed to deal with these problems in current pest rodent management .…”

Habitat evaluation for outbreak of Yangtze voles (Microtus fortis) and management implications

“…To control rodent pest animals, various methods from highly dangerous acute‐acting poisons to anticoagulant rodenticides have been used (Mushtaq et al ). However, these methods often lead to resistance in the population (Díaz et al ; Huang et al ). Considering the lack of strict adherence to rodent control in all regions, functional areas should be introduced into pest management science.…”

Low warfarin resistance frequency in Norway rats in two cities in China after 30 years of usage of anticoagulant rodenticides

“…Three Vkorc1 exons and their flanking intron regions were amplified using three pairs of primers from previous studies by Huang et al . The polymerase chain reaction (PCR) was conducted in a 25‐μL volume, including 1 μL of genomic DNA, 2 μL of 10 × buffer, 2 μL of 25 mM dNTP and 0.4 μ m of each primer.…”

“…Three Vkorc1 exons and their flanking intron regions were amplified using three pairs of primers from previous studies by Huang et al 35 The polymerase chain reaction (PCR) was conducted in a 25-L volume, including 1 L of genomic DNA, 2 L of 10 × buffer, 2 L of 25 mM dNTP and 0.4 M of each primer. The PCR recycling conditions were 95 ∘ C for 5 min, followed by 95 ∘ C for 30 s, 56 ∘ C for 30 s, 72 ∘ C for 1 min and a final extension at 72 ∘ C for 5 min.…”