Fitness costs associated with insecticide resistance

The chemical application is considered one of the most crucial methods for controlling insect pests, especially in intensive farming practices. Owing to the chemical application, insect pests are exposed to toxic chemical insecticides along with other stress factors in the environment. Insects require energy and resources for survival and adaptation to cope with these conditions. Also, insects use behavioral, physiological, and genetic mechanisms to combat stressors, like new environments, which may include chemicals insecticides. Sometimes, the continuous selection pressure of insecticides is metabolically costly, which leads to resistance development through constitutive upregulation of detoxification genes and/or target-site mutations. These actions are costly and can potentially affect the biological traits, including development and reproduction parameters and other key variables that ultimately affect the overall fitness of insects. This review synthesizes published in-depth information on fitness costs induced by insecticide resistance in insect pests in the past decade. It thereby highlights the insecticides resistant to insect populations that might help design integrated pest management (IPM) programs for controlling the spread of resistant populations.

Background Chemical control is still a major strategy to constrain vector density and mitigate pathogen transmission. However, insecticide overuse poses a high selective pressure, favouring the spread of resistance alleles in natural populations. In an insecticide-free environment, a fitness cost is expected in resistant insects when compared to susceptible counterparts. This study investigates whether insecticide resistance to an organophosphate (temephos) and a pyrethroid (deltamethrin) is associated with fitness traits in four Aedes aegypti wild populations sampled every three months over one year. Findings We measured development time from larvae to adult, female survival, wing length, fecundity, and adult resistance to starvation in field insecticide resistant Ae. aegypti populations four times over a year. These results were confronted with resistance levels to temephos and deltamethrin and with potentially related mechanisms, including a kdr mutation in the pyrethroid target site. No differences in fitness cost were found after contrasting mosquitoes from the same population collected throughout a year, irrespective of differences in insecticide resistance levels. Additionally, significant differences were not observed among field populations. However, compared to the reference strain Rockefeller, field females survived significantly less. Moreover, larval development was equal or slower in three out of four field populations. In no case differences were evidenced in starvation tolerance, wing length, and fecundity. Conclusions Overall, field resistant mosquitoes seemed to have a slight fitness disadvantage when compared with the Rockefeller susceptible strain which might represent a potential fitness cost of insecticide resistance. However, after comparing Ae. aegypti from the same population but sampled at different moments, or from different field populations, mosquito life-history traits varied independently of resistance ratios. The metabolic deviations necessary to overcome the adverse effects of insecticides may cause an energy trade-off that affects energy allocation and ultimately basic demands of insect biology. The extent of fitness cost due to insecticide resistance is critical information to delay the evolution of resistance in wild vector populations.

Insecticide resistance threatens control of agricultural and medically important insect pests. Resistance may come at a fitness cost to the insect pest, and whether these fitness costs exist may determine the persistence of resistance in the absence of insecticides. Not all resistant populations have shown fitness costs associated with resistance. Revealing possible patterns in the effects of insecticide resistance on fitness costs among insecticide classes, resistance levels, and insect orders would improve our understanding of evolutionary costs of resistance and may assist in optimizing existing resistance management strategies. We performed a comprehensive literature search to identify studies that examined fitness costs associated with insecticide resistance. Fitness data were collected on various life-history traits and subjected to multiple meta-analyses to determine overall effects of resistance. Generally, insecticide resistance often came with a cost to fitness; however, there were insecticide classes, orders of insects, and resistance magnitude levels that did not confer a fitness cost. The emerging patterns suggest that (i) resistant female insects exhibited a lower fecundity compared to susceptible females within the orders Diptera, Hemiptera, and Lepidoptera; (ii) resistant juvenile insects had prolonged development compared to susceptible juveniles within the orders Diptera, Hemiptera, and Lepidoptera; (iii) juvenile survival rate was much higher for susceptible than resistant individuals in the orders Lepidoptera, Diptera, and Hemiptera; (iv) female and male adult longevity were reduced for insecticide resistant individuals in the order Diptera, and (v) there was no clear and consistent trend between the magnitude of resistance and potential effects on fitness.

Insecticide resistance in Anopheles mosquitoes is a major obstacle in maintaining the momentum in reducing the malaria burden; mitigating strategies require improved understanding of the underlying mechanisms. Mutations in the target site of insecticides (the voltage gated sodium channel for the most widely used pyrethroid class) and over-expression of detoxification enzymes are commonly reported, but their relative contribution to phenotypic resistance remain poorly understood. Here we present a genome editing pipeline to introduce single nucleotide polymorphisms in An. gambiae which we have used to study the effect of the classical kdr mutation L1014F (L995F based on An. gambiae numbering), one of the most widely distributed resistance alleles. Introduction of 1014F in an otherwise fully susceptible genetic background increased levels of resistance to all tested pyrethroids and DDT ranging from 9.9-fold for permethrin to >24-fold for DDT. The introduction of the 1014F allele was sufficient to reduce mortality of mosquitoes after exposure to deltamethrin treated bednets, even as the only resistance mechanism present. When 1014F was combined with over-expression of glutathione transferase Gste2, resistance to permethrin increased further demonstrating the critical combined effect between target site resistance and detoxification enzymes in vivo. We also show that mosquitoes carrying the 1014F allele in homozygosity showed fitness disadvantages including increased mortality at the larval stage and a reduction in fecundity and adult longevity, which can have consequences for the strength of selection that will apply to this allele in the field.

Insecticide resistance in Anopheles mosquitoes is a major obstacle in maintaining the momentum in reducing the malaria burden; mitigating strategies require improved understanding of the underlying mechanisms. Mutations in the target site of insecticides (the voltage gated sodium channel for the most widely used pyrethroid class) and over-expression of detoxification enzymes are commonly reported, but their relative contribution to phenotypic resistance remain poorly understood. Here we present a genome editing pipeline to introduce single nucleotide polymorphisms in An. gambiae which we have used to study the effect of the classical kdr mutation L1014F (L995F based on An. gambiae numbering), one of the most widely distributed resistance alleles. Introduction of 1014F in an otherwise fully susceptible genetic background increased levels of resistance to all tested pyrethroids and DDT ranging from 9.9-fold for permethrin to >24-fold for DDT. The introduction of the 1014F allele was sufficient to reduce mortality of mosquitoes after exposure to deltamethrin treated bednets, even as the only resistance mechanism present. When 1014F was combined with over-expression of glutathione transferase Gste2, resistance to permethrin increased further demonstrating the critical combined effect between target site resistance and detoxification enzymes in vivo. We also show that mosquitoes carrying the 1014F allele in homozygosity showed fitness disadvantages including increased mortality at the larval stage and a reduction in fecundity and adult longevity, which can have consequences for the strength of selection that will apply to this allele in the field.

Insecticide resistance in insects severely threatens both human health and agriculture, making insecticides less compelling and valuable, leading to frequent pest management failures, rising input costs, lowering crop yields, and disastrous public health. Insecticide resistance results from multiple factors, mainly indiscriminate insecticide usage and mounted selection pressure on insect populations. Insects respond to insecticide stress at the cellular level by modest yet significant genetic propagations. Transcriptional, co-transcriptional, and post-transcriptional regulatory signals of cells in organisms regulate the intricate processes in gene expressions churning the genetic information in transcriptional units into proteins and non-coding transcripts. Upregulation of detoxification enzymes, notably cytochrome P450s (CYPs), glutathione S-transferases (GSTs), esterases [carboxyl choline esterase (CCE), carboxyl esterase (CarE)] and ATP Binding Cassettes (ABC) at the transcriptional level, modification of target sites, decreased penetration, or higher excretion of insecticides are the noted insect physiological responses. The transcriptional regulatory pathways such as AhR/ARNT, Nuclear receptors, CncC/Keap1, MAPK/CREB, and GPCR/cAMP/PKA were found to regulate the detoxification genes at the transcriptional level. Post-transcriptional changes of non-coding RNAs (ncRNAs) such as microRNAs (miRNA), long non-coding RNAs (lncRNA), and epitranscriptomics, including RNA methylation, are reported in resistant insects. Additionally, genetic modifications such as mutations in the target sites and copy number variations (CNV) are also influencing insecticide resistance. Therefore, these cellular intricacies may decrease insecticide sensitivity, altering the concentrations or activities of proteins involved in insecticide interactions or detoxification. The cellular episodes at the transcriptional and post-transcriptional levels pertinent to insecticide resistance responses in insects are extensively covered in this review. An overview of molecular mechanisms underlying these biological rhythms allows for developing alternative pest control methods to focus on insect vulnerabilities, employing reverse genetics approaches like RNA interference (RNAi) technology to silence particular resistance-related genes for sustained insect management.

Widespread insecticide resistance in African malaria vectors raises concerns over the potential to compromise malaria vector control interventions. Understanding the evolution of resistance mechanisms, and whether the selective disadvantages are large enough to be useful in resistance management or designing suitable control strategies is crucial. This study assessed whether insecticide resistance to pyrethroids has an effect on the gonotrophic cycle and reproductive potential of malaria vector Anopheles gambiae. Comparative tests were performed with pyrethroid-resistant and susceptible colonies of Anopheles gambiae colonized from the same geographical area, and the reference Kisumu strain was used as a control. Adult females aged 3 days old were given a blood meal and kept separately for individual egg-laying. The number of days taken to lay eggs post-blood-feeding was recorded to determine the length of the gonotrophic cycle. To measure adult longevity and reproduction potential, newly emerged males and females of equal numbers were aspirated into a cage and females allowed to blood feed daily. The number of eggs laid and the surviving mosquitoes were recorded daily to determine fecundity, net reproduction rate, intrinsic growth rate and adult longevity. Overall, the resistant females had a significantly longer (1.8 days) gonotrophic cycle than susceptible females (F2, 13 = 9. 836, P < 0.01). The proportion of resistant females that laid eggs was lower 31.30% (94/300) compared to 54% (162/300) in the susceptible colony and 65.7% (197/300) in the Kisumu strain. The mean number of eggs laid per female was significantly lower in the resistant colony (88.02 ± 20) compared to the susceptible colony (104.9 ± .28.8) and the Kisumu strain (97.6 ± 34.8). The adult longevity was significantly higher for resistant (39.7 ± 1.6 days) compared to susceptible (29.9 ± 1.7 days) and the Kisumu strain was (29.6 ± 1.1 days) (F2,8 = 45.05, P < 0.0001). Resistant colony exhibited a lower fecundity (4.3 eggs/females/day) and net reproductive rate (2.6 offsprings/female/generation) compared to the susceptible colony (8.6 eggs/female/day; 4.7 offsprings/female/generation respectively) and Kisumu strain (9.7 eggs/female/day; 4.1 offsprings/female/generation respectively). The study suggests high fitness cost on reproductive parameters of pyrethroid-resistant mosquitoes particularly on the duration of gonotrophic cycle, fecundity and net reproductive rate. These fitness costs are likely associated with maintaining both target site and metabolic mechanisms of resistance to pyrethroids. Despite these costs, resistant mosquitoes had longer longevity. These results give insights to understanding the fitness cost of insecticide resistance and thus are critical when predicting the epidemiological impact of insecticide resistance.

The development of insecticide resistance in pest insects is an increasing problem for agriculture, forestry and public health. Aphids are ubiquitous herbivorous insects, with approximately 4700 known species, of which less than 5% exploit the agricultural environment successfully. Of these, the peach‐potato aphid Myzus persicae Sulzer is recognized as one of the most important pests worldwide because it has acquired resistance to many insecticides. Although resistance to insecticides provides important benefits for pests in agricultural fields that are treated with insecticides, it may be associated with fitness (or other) costs in environments that are insecticide free. In the present study, the fitness and energy costs that might be experienced by M. persicae in an insecticide‐free environment when carrying at least one insecticide resistance mutation (IRM), or by having an increased production of esterases, are evaluated. The study investigates whether genotypes that have an IRM also have enhanced esterase production, whether there is any metabolic cost associated with insecticide resistance, and whether there are any fitness costs associated with insecticide resistance and metabolic expenditure. The intrinsic rate of increase, standard metabolic rate (i.e. a measure of maintenance costs) and constitutive esterase activity are determined for 30 different multilocus genotypes carrying (or not carrying) at least one of the two most frequent insecticide resistance mutations (MACE and kdr/super‐kdr) that occur in Chile. The results show that genotypes carrying at least one IRM have higher levels of total esterase activity than genotypes without an IRM, that there is no evidence of an energy cost associated with total esterase activity or IRM, and no evidence for a reproductive fitness cost associated with total esterase activity, IRM or metabolic rate. The results agree with previous studies showing linkage disequilibrium between insecticide resistance mechanisms, although they contrast with those of studies that report fitness costs associated with insecticide resistance in Myzus persicae.

The intensive use of chemicals through decades has been selecting resistant popula‐ tions of several insect species to distinct classes of insecticides, like neurotoxics, in‐ sect growth regulators, and toxins derived from bacteria. Insecticide resistance is nowadays a huge challenge for control programs of pests of rural practices and principally to the management of arthropod vector-borne diseases. Several behavio‐ ral, physiological, and molecular mechanisms can be selected for avoiding toxic ef‐ fects of insecticides in the insect organism. These changes are genetic traits that arise randomly and spread throughout the population along time, under an envi‐ ronment with insecticide selective pressure. However, new rapidly achieved char‐ acteristics can present a fitness cost to their harbors, with negative effects in development and reproductive aspects. In this way, in the absence of insecticides, susceptible individuals may present reproductive advantages and then the popula‐ tion resistance levels would tend to decrease. If the selection pressure persists, how‐ ever, compensatory genes known as modifiers can be selected, ameliorating the negative effects caused by the resistance genes themselves or their pleiotropic ef‐ fects. In this chapter, we present a review of research articles that describe some fitness costs associated with insecticide resistance, trying to correlate with the known se‐ lected mechanisms whenever possible, under an evolutionary perspective. Exam‐ ples from natural population, as well as lineages artificially selected for resistance in the laboratory, were considered. Although new tools of vector control are currently being tested under field conditions, the use of insecticides will remain with an im‐ portant role in the near future at least. In this sense, the knowledge of evolutionary processes of insecticide resistance is crucial to try to revert the resistant status of natural populations and to avoid resistance to new compounds, maintaining this strategy as an effective alternative of insect control.

The small hive beetle (Aethina tumida Murray) is a serious threat to beekeeping and crops that rely on honeybees for pollination. The small hive beetle not only causes significant damage to honeybees by feeding on pollen and honey, attacking bee brood and causing stored honey to ferment, but also might serve as a vector of diseases. In addition, the small hive beetle has developed resistance to the pyrethroid and organophosphate insecticides registered for control of honeybee pests in the United States. The development of resistance in small hive beetle populations is a great concern to the beekeeping industry; thus, there is an urgent need for strategies to manage that resistance. Therefore, we used synergist probes to determine the mechanisms of resistance in a small hive beetle population to these insecticides. Our studies on the toxicity of insecticides alone or with the synergists piperonyl butoxide (PBO) and S,S,S,-tributyl phosphorotrithionate (DEF) suggested that mixed-function oxidases and esterases were the major resistance factors to these insecticides in a studied population of the small hive beetle. In contrast, there was no synergism with diethyl maleate (DEM), triphenyl phosphate (TPP) and formamidine. Therefore, glutathione-S-transferase, carboxylesterase and target site were not involved in insecticide resistance in the small hive beetle. Rotation of classes of insecticides (with different modes of action) and metabolic synergists were suggested for the development of successful resistance management programs. To the best of our knowledge, this is the first study of the mechanisms of resistance in small hive beetle populations in Florida and suggests an urgent need for alternative control strategies for these serious pests of honeybee colonies.

The aphid Aphis gossypii Glover is an important pest of Australian cotton and has developed resistance to many chemicals used for its control. Its resistance management is partially based on chemical rotation that relies on insecticide resistance being associated with fitness costs. Therefore, understanding fitness costs associated with insecticide resistance is critical to its sustainable resistance management. We studied the fitness cost of pirimicarb resistance in A. gossypii caused by a single mutation in the acetylcholinesterase gene ACE1 by mixing different ratios of susceptible and resistant aphids. This was achieved by establishing A. gossypii populations of a known starting allele frequency in aphid proof cages and measuring allele frequency change over time via qPCR. Unlike traditional cohort fitness studies, we used competitive fitness as a measure of relative fitness of resistant versus susceptible aphids in the same environment. We demonstrate that competitive fitness measured in this study is an accurate predictor of overall relative fitness. We found that pirimicarb resistance had a significant fitness cost in the presence of susceptible aphids in the absence of insecticide pressure and that the fitness cost was related to the initial resistance allele frequency. By using the competitive fitness measure and knowing the initial allele frequency, it is possible to predict the likely time from resistant to an essentially susceptible population. As resistance was stable in the absence of susceptible competition, we recommend the use of resistance management tactics that do not completely eliminate the susceptible genotype such as complimentary integrated pest management.

Cancer cells often acquire resistance to therapy due to genetic mutations. However, without drug selection, these mutations would likely have a fitness cost to cancer cells and we hypothesize that the severity of this fitness cost is correlated with the effectiveness of drug treatment, with more effective treatments leading to more drastic genetic lesions. The size of a genetic lesion often correlates with its fitness cost - point mutation having little cost, aneuploidy having a large cost. However, aneuploidy is fast because the search space is only 46 gains or losses, while point mutations are slow with billions of base pairs to search before finding the right mutations. We designed experimental and computational systems to understand the selection pressures that would lead to resistance by these different genetic mechanisms. Mice were implanted with xenograft tumors from the HT1080 fibrosarcoma cell line that were heterozygous for DR5 (TNFRSF10B) and extremely sensitive to treatment with a DR5 specific monoclonal antibody, which induced extrinsic apoptosis. Treatment with three doses of anti-DR5 cured 3/11 mice, while 8/11 tumors relapsed. Isolation and culture of the relapse tumors showed they were completely resistant to DR5-mediated killing and had lost the functional copy of DR5. Assaying copy number variation using next generation sequencing (CNVSeq) showed that the relapse lines had lost one copy of chromosome 8p, where the functional DR5 gene was located. Treatment with two doses of anti-DR5 lead to relapses in all mice with loss of 8p. Finally, treatment with a single dose resulted in 3/4 tumors with loss of 8p. However 1/4 tumors became more resistant to anti-DR5 but did not lose the DR5 gene, presumably becoming resistant by smaller genetic changes or by non-genetic means that altered DR5 signaling. Thus, in most cases, treatment of xenograft tumors led to resistance by the loss of a chromosome arm, while resistance was rarely induced by other mechanisms. In contrast, cultured HT1080 cells treated with three doses of anti-DR5 became resistant but never lost 8p. In culture, treatment with anti-DR5 only kills a maximum of 80% of the cells and was therefore weaker than in mice where anti-DR5 could be curative. Thus, we observed an association between the strength of drug selection and the genetic mechanism by which cells became resistant. Experiments to further decrease selective pressure in xenografts and increase it in culture are ongoing. Next, we developed a computational model of tumor evolution to further explore the interplay between strength of selection and type of adaptation. In this simulation, cells were under two selective pressures, drug selection and competition for nutrients. Drug sensitive tumor cells could become resistant by two mechanisms analogous to point mutations or loss of 8p. Point mutations in this model caused resistance incrementally thereby taking several generations to acquire resistance, but they had no fitness cost in terms of competing for nutrients. Loss of 8p in this model caused complete resistance but had a fitness cost. Simulations with strong selective pressure led to the emergence of tumors that had completely lost 8p, even when the fitness cost was high. This result occurred because cells that did not lose 8p were eliminated by the drug. In contrast, simulations with selective pressure similar to culture (80% kill) led to tumors that became resistant without loss of 8p. In this case, sensitive cells incrementally acquired resistance mutations and then out competed cells that lost 8p for nutrients. Thus, simulation showed that resistance mutations with a large fitness cost will only arise under strong drug selection. In conclusion, our data suggest that the closer a therapy is to curing a tumor, the more extreme and costly resistance mutations will be. Citation Format: Lee A. Albacker, Rainer Kohler, Ralph Weissleder, Peter K. Sorger. The strength of drug selection determines the maximum fitness cost of resistance mutations in culture and xenografts. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr PR07.

Mathematics of an epidemiology-genetics model for assessing the role of insecticides resistance on malaria transmission dynamics

Fitness cost is usually associated with insecticide resistance and may be mitigated by increased energy accumulation and mobilization. Preliminarily evidence from tests with Sitophilus zeamais Motschulsky, the maize weevil (Coleoptera: Curculionidae) suggested possible involvement of proteinases and amylases in such a phenomenon. Therefore, trypsin-like serine-proteinases, cysteine-proteinases and α-amylases were purified and characterized from an insecticide-susceptible and two insecticide-resistant strains (one with associated fitness cost [resistant cost strain], and the other without it [resistant no-cost strain]). Trypsin-like serine-proteinases were purified by aprotinin-agarose affinity chromatography, while cysteine-proteinases were purified using thiol-sepharose affinity chromatography, and the main α-amylase of each strain was purified by glycogen precipitation and ion-exchange chromatography. The activity and inhibition profile differed among strains for each group of purified enzyme. The higher levels of activity observed for trypsin-like proteinases and amylase in the resistant no-cost strain, as well as their susceptibility to inhibition provide support for the hypothesis that enhanced trypsin-like protease and α-amylase activity may be playing a major role in mitigating fitness costs associated with insecticide resistance. In contrast, enhanced cysteine-proteinase activity is likely to play only a secondary role, if any, in mitigating the costs usually associated with insecticide resistance. Keywords : Fitness cost mitigation, Insecticide resistance, Digestive enzymes, Amylases, Proteinases.

BackgroundThe New World screw-worm (NWS), Cochliomyia hominivorax, is one of the most important myiasis-causing flies, causing severe losses to the livestock industry. In its current geographical distribution, this species has been controlled by the application of insecticides, mainly organophosphate (OP) compounds, but a number of lineages have been identified that are resistant to such chemicals. Despite its economic importance, only limited genetic information is available for the NWS. Here, as a part of an effort to characterize the C. hominivorax genome and identify putative genes involved in insecticide resistance, we sampled its transcriptome by deep sequencing of polyadenylated transcripts using the 454 sequencing technology.ResultsDeep sequencing on the 454 platform of three normalized libraries (larval, adult male and adult female) generated a total of 548,940 reads. Eighteen candidate genes coding for three metabolic detoxification enzyme families, cytochrome P450 monooxygenases, glutathione S-transferases and carboxyl/cholinesterases were selected and gene expression levels were measured using quantitative real-time polymerase chain reaction (qRT-PCR). Of the investigated candidates, only one gene was expressed differently between control and resistant larvae with, at least, a 10-fold down-regulation in the resistant larvae. The presence of mutations in the acetylcholinesterase (target site) and carboxylesterase E3 genes was investigated and all of the resistant flies presented E3 mutations previously associated with insecticide resistance.ConclusionsHere, we provided the largest database of NWS expressed sequence tags that is an important resource, not only for further studies on the molecular basis of the OP resistance in NWS fly, but also for functional and comparative studies among Calliphoridae flies. Among our candidates, only one gene was found differentially expressed in resistant individuals, and its role on insecticide resistance should be further investigated. Furthermore, the absence of mutations in the OP target site and the high frequency of mutant carboxylesterase E3 indicate that metabolic resistance mechanisms have evolved predominantly in this species.