| The anticoagulant rodenticides have been widely used for rodent control all over the world.Anticoagulant resistance has evolved in different rodent species and arisen as a big challenge in practical usage of anticoagulants.The main mechanism of warfarin resistance involved the mutations in the vitamin K epoxide reductase complex,subunit 1 gene(Vkorc1).Previous investigation of warfarin resistance in the Norway rats in China had revealed resistance rats in different areas after 30 years usage of anticoagulants and the highest resistance frequency of 21.6% was found in Zhanjiang City.However,the polymorphisms of Vkorc1 in Norway rats in China are rarely studied.In addition,some rats were found to be resistant but did not carry any Vkorc1 resistance mutation,however,the associated mechanism was not very clear.In this study,we investigated the Vkorc1 polymorphisms in a number of Norway rat populations in China,and explored other resistance mechanisms in induced warfarin resistance rats without carrying Vkorc1 resistance mutations.The main results were as follows:1.We analyzed Vkorc1 polymorphisms and its correlation with resistance in 641 Norway rats in Zhanjiang and Harbin district for 8 consecutive years from 2008 to 2015.Totally,3 new mutations were found,including 1 amino acid mutation Ala140Thr(frequencies 0~4.0%)and 2 synonymous mutations His68 His and Leu105 Leu.The physiological resistance frequenceiss of the Norway rat populations in Harbin and Zhanjiang were 0% and 5~17%,respectively.Mutations His68 His and Ile105 Ile were not associated with resistance.Both the Vkorc1 polymorphisms and the laboratory resistance tests suggested low resistance frequencies in the Norway rat populations in two regions.2.We further analyzed Vkorc1 polymorphisms in 491 Norway rats from 24 provinces in China and found 7 mutations,including 2 amino acid mutations Ala140 Thr and Cys96 Tyrthat were detected in Leizhou and Baoding population,respectively.Analysis of the frequencies of Vkorc1 polymorphismssuggested that the resistance frequencies of Norway rat populations in most areas of China were low.In contrast,at least at least 9 different resistance mutations in Norway rats in European countries.Despite the gene flow analysis suggested the ancient Norway rat population spread from northern China to Southeast Asia,and then to Europe,the Vkorc1 resistance mutaions in Norway rats in European countries was likely de novo mutations,rather than historical spread of ancestral Vkorc1 polymorphisms.3.In order to explore other possible resistance mechanisms,we screened sensitive Norway rats without carrying Vkorc1 resistance mutations(sF0 generation)by sublethal doses of warfarin for 6 consecutive generations(sF0~sF5).we found the survival rate had been greatly increased over generations,and 5.7~7.1% of sF3~sF5 rats showed resistance phenotype,indicating that warfarin resistance can be induced and inheritable in the Norway rats.4.In order to explore the molecular regulation mechanism of the plastic resistance phenotype,we analyzed the liver transcriptomes of sensitive rats(uF5),tolerant rats(sF5),and warfarin-treated tolerant rats(sF5R).The differential expression of innate-immune-related genes was found in sF5 ratsas compared to the sensitive uF5 rats.After warfarin administration,sF5 female rats showed significant gene expression changes in multiple physiological processes such as medicine metabolism,detoxification,blood coagulation and wound healing,while sF5 male rats mainly changed the gene expression of multiple metabolic pathways such as proteins and lipids.In summary,the frequency of warfarin resistance was very low in most area in China based on the analysis of Vkorc1 polymorphisms and some low-toxic first-generation anticoagulant rodenticides are still effective for rat control in China.We also found that warfarin resistance could be induced independent of Vkorc1 mutations,which involved many innate-immune-related genes,as well as some metabolic pathways. |
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