运用α-氰代酯荧光底物检测抗性棉蚜羧酸酯酶代谢活性

为了研究抗性和敏感棉蚜Aphis gossypii品系对菊酯类药剂代谢的差异, 本实验合成了溴氰菊酯和高效氯氰菊酯报告荧光底物, 应用这两种底物水解后生成具有荧光化合物的特性,测定了不同品系棉蚜羧酸酯酶的代谢活性。结果表明: 氧化乐果棉蚜抗性和敏感品系羧酸酯酶对溴氰菊酯报告荧光底物的代谢活性分别为10.0和3.4 pmol/min·mg; 对高效氯氰菊酯报告荧光底物的代谢活性分别为4.0和2.4 pmol/min·mg, 抗性品系羧酸酯酶对溴氰菊酯和高效氯氰菊酯报告荧光底物的代谢活性分别为敏感品系的2.9和1.7倍; 溴氰菊酯棉蚜抗性和敏感品系羧酸酯酶对溴氰菊酯报告荧光底物的代谢活性分别为7.6和6.2 pmol/min·mg; 对高效氯氰菊酯报告荧光底物的代谢活性分别为9.3和5.2 pmol/min·mg, 抗性品系羧酸酯酶对溴氰菊酯和高效氯氰菊酯报告荧光底物的代谢活性分别为敏感品系的1.2和1.8倍。这种衍生的报告荧光底物能够用来检测抗性棉蚜羧酸酯酶的水解活性, 表明羧酸酯酶可能参与棉蚜对溴氰菊酯和氧化乐果抗性的形成。     

Polymorphisms in a carboxylesterase gene between organophosphate-resistant and -susceptible Aphis gossypii (Homoptera: Aphididae)

Resistance to omethoate was suppressible by the hydrolytic enzyme inhibitor SSS-tributyl phosphorotrithioate in a laboratory-selected resistant cotton aphid, Aphis gossypii Glover, strain, suggesting the involvement of hydrolytic enzymes in the detoxification process. The kinetic properties of carboxylesterases from both resistant and susceptible cotton aphids were characterized by four acyl ester substrates: alpha-naphthyl acetate (alpha-NA), alpha-naphthyl butyrate (alpha-NB), alpha-naphthyl phosphate (alpha-NP), and beta-naphthyl phosphate (beta-NP). No significant differences of carboxylesterase activity were found between resistant and susceptible strains by using either alpha-NP or beta-NP as substrates. In contrast, the susceptible A. gossypii exhibited significantly higher activity compared with resistant aphids with either alpha-NA or alpha-NB as substrates. To understand the molecular basis of this esterase-mediated resistance, carboxylesterase genes from both strains were cloned. Two genes share 99.4% identity at the nucleic acid level and 99.2% identity at the amino acid level. The full length of the cDNA opening reading frame is 1581 bp, encoding 526 amino acids. Four amino acid substitutions, Thr210 --> Met210, Asn294 --> Lys294, Gly408 --> Asp408, and Ser441 --> Phe441, were identified in the resistant strain. Probing of Southern blots with the 0.5 kb esterase fragment showed the same banding patterns and intensities with genomic DNA extracts from both resistant and susceptible A. gossypii. Furthermore, the MspI and HpaII fragments are the same in both strains, indicating there is no methylation of sequences detected by the probe. The combined results suggest that the structural gene substitution is likely the molecular basis of the organophosphate resistance in this laboratory-selected cotton aphid strain.     

Purification and characterization of a carboxylesterase involved in malathion-specific resistance from Tribolium castaneum (Coleoptera: Tenebrionidae)

Specific resistance to malathion in a strain of Tribolium castaneum is due to a 44-fold increase in malathion carboxylesterase (MCE) activity relative to a susceptible strain, whereas non-specific esterase levels are slightly lower. Unlike the overproduced esterase of some mosquito and aphid species, MCE in Tribolium castaneum accounts for only a small fraction (0.033-0.045%) of the total extractable protein respectively in resistant and susceptible strains. The enzyme was purified to apparent homogeneity from these two strains and has a similar molecular weight of 62,000. However, preparative isoelectricfocusing indicated that resistant insects possess one MCE with pI of 7.3, while susceptible insects possess a MCE with a pI of 6.6. Purified MCE from both populations had different K(m) and V(m) values for hydrolysis of malathion as well as for alpha-naphthyl acetate. The kinetic analysis suggests that MCE of resistant insects hydrolyses malathion faster than the purified carboxylesterase from susceptible beetles and that this enzyme has greater affinity for malathion than for naphthyl esters. Malathion-specific resistance is due to the presence of a qualitatively different esterase in the resistant strain.     

Enhanced esterase gene expression and activity in a malathion-resistant strain of the tarnished plant bug, Lygus lineolaris

Extensive use of insecticides on cotton in the mid-South has prompted resistance development in the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois). A field population of tarnished plant bugs in Mississippi with 11-fold higher resistance to malathion was used to examine how gene regulation conferred resistance to this organophosphate insecticide. In laboratory bioassays, synergism by the esterase inhibitors S,S,S,-tributylphosphorotrithioate (DEF) and triphenylphosphate (TPP) effectively abolished resistance and increased malathion toxicity by more than 80%. Esterase activities were compared in vitro between malathion susceptible and resistant (selected) strains. More than 6-, 3- and 10-fold higher activities were obtained with the resistant strain using alpha-naphthyl acetate, beta-naphthyl acetate, and p-nitrophenyl acetate, respectively. Up to 95% and 89% of the esterase activity in the susceptible and resistant strains, respectively, was inhibited by 1 mM DEF. Inhibition of esterase activity up to 75% and 85% in the susceptible and resistant strains, respectively, was obtained with 0.03 mM TPP. Esterase activities in field populations increased by up to 5.4-fold during the fall season. The increase was synchronized with movement of the insect into cotton where exposure to pesticides occurred. Esterase cDNA was cloned and sequenced from both malathion susceptible and resistant strains. The 1818-nucleotide cDNA contained a 1710-bp open reading frame coding a 570 amino acid protein which was similar to many insect esterases conferring organophosphate resistance. No amino acid substitution was observed between susceptible and resistant strains, indicating that esterase gene mutation was not involved in resistance development in the resistant strain in Mississippi. Further examination of esterase gene expression levels using quantitative RT-PCR revealed that the resistant strain had a 5.1-fold higher level of esterase mRNA than the susceptible strain. The results of this study indicated that up-regulation of the esterase gene appeared to be related to the development of resistance in the tarnished plant bug.     

应用酶标仪动力学方法监测棉蚜的抗药性

用酶标仪动力学测定法对3个抗性水平不同的棉蚜品系（R1、R2和R3）和1个敏感品系（S）的羧酸酯酶进行了研究,S、R1、R2和R3品系对α-乙酸萘酯（α-NA）的平均比活力分别为57．10、1171．69、1236．14和3293．00μmol·mgpro－1·min-1（分光光度计终点测定法）和38．24、85．27、198．14和762．25mOD·min－1·aphid-1（酶标仪动力学法）。终点测定法结果显示出不同品系间最大相差达60倍;酶标仪动力学测定法研究表明,4个棉蚜品系羧酸酶活性与其抗药性程度显著相关。通过对这两种方法的比较,酶动力学方法的测定结果更可靠。     

棉蚜抗氧化乐果品系及敏感品系羧酸酯酶性质的比较

在室内用氧化乐果逐代筛选的棉蚜抗性品系,相对于敏感品系的抗性倍数是17。用α-乙酸萘酯（α-NA）、α-丁酸萘酯（α-NB）、α-磷酸萘酯（α-NP）和β-磷酸萘酯（β-NP）作底物比较研究了氧化乐果抗性和敏感品系棉蚜Aphis gossypii羧酸酯酶的比活力、米氏常数（K_m）和最大反应速度V_max）等有关的动力学常数。以α-NA和α-NB作底物时,抗性品系棉蚜的比活力显著低于敏感品系的;以α-NP和β-NP作底物时,两个品系棉蚜的比活力、K_m和V_max没有明显差异。用α-NA、β-NA作底物染色做酯酶同工酶电泳,抗性品系棉蚜的酯酶同工酶染色比敏感品系棉蚜的浅。     

棉蚜抗氧化乐果品系的羧酸酯酶基因突变

用氧化乐果对室内敏感品系棉蚜Aphis gossypii (Glover)进行抗性选育,经24代筛选,抗性指数达到124.7倍。以α-乙酸萘酯（α-NA）为底物,比较了氧化乐果敏感和抗性品系棉蚜羧酸酯酶的比活力,发现抗性品系羧酸酯酶比活力明显小于敏感品系。对这两个品系的羧酸酯酶基因进行了克隆,通过对抗性和敏感品系羧酸酯酶基因核苷酸序列及推导的氨基酸序列比较,发现抗性品系有4个氨基酸残基发生了替代 (His¹⁰⁴→Arg, Ala¹²⁸→Val, Thr³³³→Asp, Lys⁴⁸⁴→Arg)。对其蛋白质三维结构分析推测只有His¹⁰⁴→Arg的替代是位于其活性中心。棉蚜氧化乐果敏感和抗性品系羧酸酯酶基因cDNA全长的GenBank登录号分别为AY485216和AY485214。     

棉蚜啶虫脒抗性种群交互抗性和增效剂增效作用的研究

【目的】明确棉蚜Aphis gossypii Glover啶虫脒抗性品系与其它杀虫剂的交互抗性现状以及增效剂的增效作用,为延缓和治理棉蚜对啶虫脒的抗性提供依据。【方法】采用单头反选育和群体汰选的方式,获得了棉蚜啶虫脒敏感和抗性品系;采用叶片药膜法测定了13种杀虫剂对啶虫脒的交互抗性以及增效剂对啶虫脒的增效作用。【结果】经过室内棉蚜敏感和抗性品系的筛选,获得了相对抗性倍数为82.33倍的棉蚜啶虫脒抗性品系。棉蚜啶虫脒抗性品系的交互抗性谱的研究表明,交互抗性倍数小于5的药剂为:吡蚜酮,甲基阿维菌素;交互抗性倍数在5~10倍的药剂为:噻虫嗪,联苯菊酯,毒死蜱,马拉硫磷,丙溴磷,辛硫磷;交互抗性倍数在10~15倍的药剂为:硫丹,阿维菌素,高效氯氰菊酯,三唑磷,氧化乐果;交互抗性倍数大于1 5倍的药剂为:吡虫啉。增效剂实验表明,TPP和PBO在啶虫脒敏感品系中增效作用不明显,但在抗性品系中增效作用显著。在啶虫脒抗性品系中的增效比为1.77、1.61,在啶虫脒敏感品系中的增效比为1.02、1.03。DEM在啶虫脒抗性、敏感品系中的增效作用均不明显,增效比为1.04、1.02。TPP和PBO对啶虫脒有很好的增效作用。以室内棉蚜敏感品系(LC_(50)为0.180 mg/L)为基础,对新疆各主要棉区的棉蚜种群进行了啶虫脒药剂的抗性调查,结果表明新疆各主要棉区棉蚜对啶虫脒的相对抗性倍数为6.1~22.0倍。【结论】由此说明新疆主要棉区棉蚜对啶虫脒具有一定的抗性风险,生产中可以利用无交互抗性的吡蚜酮和甲基阿维菌素来治理抗性棉蚜种群。     

家蚕羧酸酯酶基因克隆及差异表达

家蚕浓核病毒 (Bombyx mori densonucleosis virus,BmDNV)是蚕业生产上危害比较严重的一类病毒。用完全抗浓核病中国镇江株(BmDNV-Z)的家蚕品系秋丰、感性品系华八及以华八为轮回亲本回交8代和自交8代构建的近等基因系BC₈为材料,采用mRNA荧光差显技术首次分离克隆了家蚕羧酸酯酶（B. mori carboxylesterase,BmCarE）基因全长cDNA,并用实时荧光定量PCR检测了添毒后12 h、36 h、72 h BmCarE在感、抗BmDNV-Z家蚕品系中肠内的表达差异。结果表明： （1）添毒后12 h不同品系家蚕中肠BmCarE表达差异最大,抗性品系BC₈和秋丰分别是感性品系华八的17.714倍和3.602倍,三者彼此间的差异达到极显著水平;（2）同一品系添毒后12 h与添清水后12 h BmCarE表达也有较大差异, BC₈添毒是BC₈添清水的15.08倍, 秋丰添毒是秋丰添清水的3.39倍, 差异达到极显著水平,而华八添毒和添清水的BmCarE表达量均低,二者差异不显著;（3）同一品系添毒后不同时间BmCarE表达也有较大差异, BC₈和秋丰添毒后12 h BmCarE表达量最高,显著高于各自添毒后36 h和72 h表达水平,而添毒后36 h与72 h表达无显著差异;华八添毒后12 h、36 h和72 h,BmCarE表达无显著差异。上述结果提示羧酸酯酶基因可能与家蚕抗浓核病毒有一定关系。     

Differential mRNA expression levels and gene sequences of a putative carboxylesterase-like enzyme from two strains of the parasitoid Anisopteromalus calandrae (Hymenoptera: Pteromalidae).

Carboxylesterase-like enzyme cDNAs have been cloned and sequenced from malathion-resistant and susceptible strains of the parasitoid Anisopteromalus calandrae (Howard) (Hymenoptera: Pteromalidae). The cDNAs consist of 1963 nucleotides including a 35 bp untranslated 5'-end, a 1596 bp open reading frame, and a 332 bp untranslated 3'-end. The open reading frame encodes 532 amino acid residues. The predicted protein sequence from these cDNAs includes 2 potential N-glycosylation sites, a carboxylesterase type-B serine active site FGGDSENVTIFGESAG, and conserved residues Ser187, Glu317, and His432 to function as the catalytic triad. The predicted carboxylesterase-like enzyme sequence is most similar to that of the carboxylesterase from the peach-potato aphid, Myzus persicae with 45% sequence identity. Alignment of the parasitoid carboxylesterase-like enzyme cDNAs revealed that there are two nucleotide differences in the open reading frame between the parasitoid strains, including a silent mutation and a point mutation that presumably causes a gene product difference. A nucleotide thymine at position 658 in the susceptible strain cDNA is replaced by a guanine in the resistant strain cDNA. This substitution leads to an amino acid change from tryptophan (Trp220) in the susceptible strain to glycine (Gly220) in the resistant strain. This substitution is genetically linked to resistance but it is not known how or if this amino acid substitution affects detoxification of malathion. Northern blot analyses demonstrated that expression level of the carboxylesterase-like enzyme mRNA in adult A. calandrae is approximately 30-fold higher in the resistant strain relative to that in the susceptible strain. Southern analysis indicated that Pst I or Eco RI restriction sites are different in the two strains. Both a modified gene structure and an increase in expression of carboxylesterase may be responsible for the high level of resistance found in this beneficial wasp.     

Deltamethrin‐induced feeding plasticity in pyrethroid‐susceptible and ‐resistant strains of the maize weevil,Sitophilus zeamais

Phenotypic plasticity contributes to the adaptative evolution of populations exposed to new or altered environments. Feeding plasticity is a component of phenotypic plasticity not usually considered in insect strains adapted to insecticide‐altered environments, but which may either accentuate or mitigate insecticide resistance. This is a concern in the pyrethroid‐resistant strains of the maize weevil Sitophilus zeamais Motsch. (Col., Curculionidae), and the reason for this study. A pyrethroid‐susceptible and two pyrethroid‐resistant strains of maize weevil were subjected to free‐choice and no‐choice tests with maize grains sprayed with increasing doses of the pyrethroid, deltamethrin. The insects from the pyrethroid‐resistant strains exhibited higher feeding avoidance with increased deltamethrin doses than insects from the susceptible strain when subjected to free‐choice tests. The strains of maize weevil physiologically resistant to pyrethroids were also behaviourally resistant to deltamethrin – an additional management concern. The resistant strains avoid deltamethrin‐sprayed grains and are less nutritionally affected by this compound, with divergent responses from the susceptible strain with increased doses of deltamethrin. Furthermore, the higher relative growth rate and consequently higher efficiency of food conversion observed in the insecticide‐resistant strains were significant even without insecticide exposure, indicating that these traits are stimulus‐independent and may persist even without further insecticide selection, potentially limiting the options available for their management.     

不同地区小菜蛾种群羧酸酯酶的毒理学性质研究

在1995～1997年对湖北武汉、河北张家口地区小菜蛾Plutella xylostella(L.)种群的抗药性进行了研究。结果表明对阿维菌素的抗性和台湾敏感种群相比,武汉种群抗性为4.3倍,张家口种群抗性为1.8倍;对马拉硫磷的抗性武汉和张家口种群分别为2.2和2.9倍;对氟铃脲的抗性分别为3.2和0.5倍;对溴氰菊酯的抗性分别为2.4和1.7倍。对羧酸酯酶(Care)的研究结果表明,三个种群幼虫CarE对a-乙酸萘酯或β-乙酸萘酯(a或β-NA)水解活性差异显著,但成虫Care活性没有明显差异。武汉和张家口种群幼虫CarE对a-NA和β-NA的亲和力没有明显差异,但是武汉种群幼虫Care对底物的亲和力高于张家口种群。敏感品系Care对a—NA的亲和力明显高于对β-NA,相差约3倍。不同类型的抑制剂对小菜蛾幼虫CarE的抑制能力不同。增效磷和对氧磷对敏感品系CarE水解a-NA具有明显的抑制作用,分别比对武汉种群Care的抑制作用大4.577倍(SVl)和2.576倍(对氧磷)。     

棉蚜抗杀灭菊酯品系的某些生物学特性

1908-1990年抗性监测表明,河南棉区棉蚜Aphis gossypii Glover对杀灭菊酯、氧化乐果和 久效磷的抗性水平明显增长,特别是对杀灭菊酯的抗性已达较高水平。使用杀灭菊酯对敏感棉蚜连续选择8次,棉蚜对杀灭菊酯抗性上升1392.67倍(浸溃法测定),并表现对久效磷和氧化乐果有3-4倍的正交互抗性。对4个不同抗性品系棉蚜的实验种群生命表分析表明,杀灭菊酯抗性棉蚜的适合度并不降低,抗性棉 蚜的繁殖力不表现降低趋势。利用棉田常用8种有机磷、菊酯类和脒类农药的单剂及混剂测定了对高抗棉蚜的毒力水平,DDVP表现对高抗杀灭菊酯棉蚜有较强毒力;脒类和菊酯类农药混配有显著的增效作用, 但与DDVP混用则有拮抗作用。     

Isolation and characterization of a ferulic acid esterase (Fae1A) from the rumen fungus Anaeromyces mucronatus

Aims: A novel ferulic acid esterase gene from rumen fungus Anaeromyces mucronatus was cloned, heteroexpressed in Escherichia coli and characterized. Methods and Results: A total of 30 clones exhibiting activity on α‐naphthyl acetate (α‐NA) were isolated from an A. mucronatus YE505 cDNA library. Sequence analysis revealed that these clones represented two esterase‐coding sequences. The gene, fae1A, showed highest amino acid sequence identity to CE family 1 esterases from anaerobic micro‐organisms such as Orpinomyces sp., Ruminococcus albus and Clostridium thermocellum. The gene comprised 828 nucleotides encoding a polypeptide of 275 amino acids. The coding sequence was cloned into the pET30a expression vector and overexpressed in E. coli BL21 (DE3). Gene product Fae1A was found to exhibit activity against a number of substrates including naphthyl fatty acid esters, p‐nitrophenyl fatty acid esters and hydroxylcinnamic acid esters. Conclusions: Fae1A exhibited a lower K_m and higher catalytic efficiency (k_cat/K_m) on ferulic acid esters than on α‐NA or p‐nitrophenyl acetate, suggesting that it has a higher affinity for ethyl and methyl ferulate than for the acetyl esters. It releases ferulic acid and p‐coumaric acid from barley straw. Activity of Fae1A was inhibited by the serine‐specific protease inhibitor, phenylmethylsulfonyl fluoride, indicating that a serine residue plays a role in its activity. Significance and Impact of the Study: To our knowledge, this is the first report of characterization of carbohydrate esterase gene from the genus of Anaeromyces.     

敏感和抗性棉铃虫对溴氰菊酯毒力反应的机理研究

通过生物测定和生物化学方法比较了棉铃虫玎Helicoverpa armigera敏感和抗性种群对溴氰菊酯毒力反应及其3种解毒酶的差异。结果表明,田间抗性种群和室内药剂汰选的抗性种群对溴氰菊酯均有较高的抗性,其抗性倍数分别达到195.8和37 375倍。水解酯酶和多功能氧化酶是导致棉铃虫对溴氰菊酯产生高抗性的重要酶系。特异性抑制剂活体内外抑制作用测试发现,敏感种群和抗性种群均含有较高量的乙酰胆碱酯酶,但两个种群对抑制剂的亲和力反应不同,表明乙酰胆碱酯酶在敏感种群和抗性种群中发生了不同的变化,这种变化可能与棉铃虫对溴氰菊酯的抗性有关。由此推断,棉铃虫对拟除虫菊酯这类中枢神经系统神经毒剂产生抗性,乙酰胆碱酯酶发生变化可能也是一个重要因子。     

甜菜夜蛾对氰戊菊酯和顺式氯氰菊酯的抗性机理

通过对活体增效作用进行测定和生化分析,探讨了甜菜夜蛾对氰戊菊酯和顺式氯氰菊酯的抗性机理.结果表明:增效醚(PBO)、增效磷(SV1)、磷酸三苯酯(TPP)和顺丁烯二酸二乙酯(DEM)对甜菜夜蛾抗氰戊菊酯品系(Fen-R)和敏感品系(S)的增效倍数之比分别为10.2、7.8、12.5和1.1,对抗顺式氯氰菊酯品系(Cyp-R)和敏感品系(S)的增效倍数之比分别为21.6、15.5、8.6和1.2.PBO、SV1和TPP对氰戊菊酯和顺式氯氰菊酯均有显著增效作用,表明多功能氧化酶和羧酸酯酶均参与了甜菜夜蛾对氰戊菊酯和顺式氯氰菊酯的抗性.Fen-R品系和Cyp-R品系4龄幼虫羧酸酯酶的活性分别是S品系的1.9和2.2倍,而谷胱甘肽-S-转移酶活性与S品系差异不显著,表明羧酸酯酶活性的提高是甜菜夜蛾对氰戊菊酯和顺式氯氰菊酯产生抗性的重要原因,谷胱甘肽-S-转移酶与两种药剂的抗性无关.Fen-R品系和Cyp-R品系的Na-K-ATPase活性与S品系均无显著差异,但在相同浓度下氰戊菊酯和顺式氯氰菊酯对S品系Na-K-ATPase的抑制作用显著高于抗性品系,表明抗性品系Na-K-ATPase对杀虫剂的敏感性已明显降低.     

Identification of a point mutation in an esterase gene in different populations of the southern cattle tick, Boophilus microplus

Two esterase cDNA sequences were obtained from susceptible and organophosphorus resistant strains of Boophilus microplus. Both sequences have a high degree of homology to carboxylesterase B. One gene has identical sequences in both strains and the other showed two point mutations. One mutation produces an amino acid substitution when the amino acid sequence is deduced, this mutation was detected in six different populations susceptible and resistant to insecticides, but a pyrethroid resistant strain was the only one that showed only the mutant allele. Identification of this mutation and the strong signal detected in southern blot with this strain, suggest that esterases are contributing to detoxification of pyrethroid compounds, as a resistant mechanism in Mexican strains of the southern cattle tick.     

Bidirectional selection for body mass and correlated response of pyrethroid resistance and fitness in Sitophilus zeamais

Responses to artificial selection on body mass in the maize weevil Sitophilus zeamais (Coleoptera: Curculionidae) were investigated to determine whether changes in body mass are associated with insecticide susceptibility, rate of population growth, and metabolic rate. Two strains of the maize weevil differing in susceptibility to pyrethroid insecticides were subjected to bidirectional selection on body mass. The susceptible strain responded to selection resulting in individuals with lower or higher body mass, but the resistant strain responded significantly only to selection for lower body mass. The resistant strain selected for low body mass increased its level of deltamethrin resistance in 44 × . In contrast, selection for low body mass in the susceptible parental strain led to increased deltamethrin susceptibility (50 × ) and selection for high body mass increased deltamethrin resistance (4 × ). Thus, the correlated response of insecticide resistance to selection for body mass differed between strains, a likely consequence of their distinct genetic background. Regardless, body mass was positively correlated with fitness (reproductive output) (r = 0.79; P < 0.001), while such correlation with respiration rate was significant only at P = 0.07 (r = 0.44). Therefore, the association between body mass and deltamethrin resistance is population‐dependent in the maize weevil, and the confluence of deltamethrin resistance and high body mass in a given strain will likely favour its energy metabolism and lead to the mitigation of fitness costs usually associated with insecticide resistance. The genetic background and selection history of insecticide resistant populations should not be neglected since they may favour the confluence of insecticide resistance with mitigation mechanisms of its associated fitness costs limiting the tactics available to their management.