scholarly journals Comparative proteomics reveals complex insecticides-associated resistance mechanism of Culex pipiens pallens Coquillett

2020 ◽  
Author(s):  
Chongxing Zhang ◽  
Feng Miao ◽  
Qiqi Shi ◽  
Peng Cheng ◽  
Tao Li ◽  
...  
Keyword(s):  
Insecticide Resistance ◽  
Differential Expression ◽  
Mosquito Control ◽  
Mosquito Species ◽  
Resistance Mechanism ◽  
Susceptible Strain ◽  
Future Research ◽  
Protein Profiles ◽  
Metabolic Resistance ◽  
Pipiens Pallens

Abstract Background: Mosquito control based on chemical insecticides is considered as an important element in the current global strategies for the control of mosquito-borne diseases. Unfortunately, the development of insecticide resistance of important vector mosquito species jeopardizes the effectiveness of insecticide-based mosquito control. As opposed to target site resistance, other mechanisms are far from being fully understood.Results: Susceptible strain of Cx. pipiens pallen showed elevated resistance levels to after 25 generations insecticide-selected, through bioinformatics analysis allowed detecting 2,502 proteins, of which 1513 were differentially expression in insecticide-selected strains as compared to the susceptible strain. Finally, midgut differential expression protein profiles and 62 proteins were selected for verification of differential expression using parallel reaction monitoring strategy.Conclusions Significant molecular resources were developed for Cx. pipiens pallen potential candidates involved in metabolic resistance as well as those participating in lower penetration or sequestration of insecticide. Global protein profiles of change to three insecticide strains combined with midgut profiles revealed multiple insecticide resistance mechanisms operate simultaneously in resistant insects of Cx. pipiens pallens. Future research that is targeted towards RNA interference on the identified metabolic targets such as cuticular, cytochrome P450s and glutathione S-transferase proteins could lay the foundation for a better understanding of the genetic basis of insecticide resistance in Cx. pipiens pallen.

scholarly journals Comparative proteomics reveals mechanisms that underlie insecticide resistance in Culex pipiens pallens Coquillett

2021 ◽  
Vol 15 (3) ◽  
pp. e0009237
Author(s):  
Chongxing Zhang ◽  
Qiqi Shi ◽  
Tao Li ◽  
Peng Cheng ◽  
Xiuxia Guo ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Insecticide Resistance ◽  
Culex Pipiens ◽  
Mosquito Control ◽  
Expression Profiles ◽  
Susceptible Strain ◽  
Culex Pipiens Pallens ◽  
Metabolic Resistance ◽  
Tandem Mass Spectrometric ◽  
Pipiens Pallens

Mosquito control based on chemical insecticides is considered as an important element of the current global strategies for the control of mosquito-borne diseases. Unfortunately, the development of insecticide resistance of important vector mosquito species jeopardizes the effectiveness of insecticide-based mosquito control. In contrast to target site resistance, other mechanisms are far from being fully understood. Global protein profiles among cypermethrin-resistant, propoxur-resistant, dimethyl-dichloro-vinyl-phosphate-resistant and susceptible strain of Culex pipiens pallens were obtained and proteomic differences were evaluated by using isobaric tags for relative and absolute quantification labeling coupled with liquid chromatography/tandem mass spectrometric analysis. A susceptible strain of Culex pipiens pallens showed elevated resistance levels after 25 generations of insecticide selection, through iTRAQ data analysis detected 2,502 proteins, of which 1,513 were differentially expressed in insecticide-selected strains compared to the susceptible strain. Finally, midgut differential protein expression profiles were analyzed, and 62 proteins were selected for verification of differential expression using iTRAQ and parallel reaction monitoring strategy, respectively. iTRAQ profiles of adaptation selection to three insecticide strains combined with midgut profiles revealed that multiple insecticide resistance mechanisms operate simultaneously in resistant insects of Culex pipiens pallens. Significant molecular resources were developed for Culex pipiens pallens, potential candidates were involved in metabolic resistance and reducing penetration or sequestering insecticide. Future research that is targeted towards RNA interference of the identified metabolic targets, such as cuticular proteins, cytochrome P450s, glutathione S-transferases and ribosomal proteins proteins and biological pathways (drug metabolism—cytochrome P450, metabolism of xenobiotics by cytochrome P450, oxidative phosphorylation, ribosome) could lay the foundation for a better understanding of the genetic basis of insecticide resistance in Culex pipiens pallens.

scholarly journals Insecticide Resistance Status of Aedes aegypti (Diptera: Culicidae) in California by Biochemical Assays

2020 ◽  
Vol 57 (4) ◽  
pp. 1176-1183
Author(s):  
Fan Yang ◽  
Samuel Schildhauer ◽  
Sarah A Billeter ◽  
Melissa Hardstone Yoshimizu ◽  
Robert Payne ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Insecticide Resistance ◽  
Southern California ◽  
Mosquito Control ◽  
Central Valley ◽  
Activity Levels ◽  
Metabolic Resistance ◽  
Organophosphate Resistance ◽  
Biochemical Assays ◽  
Effective Use

Abstract Insecticide resistance in Aedes aegypti mosquitoes poses a major threat to public health worldwide. There are two primary biological mechanisms that can lead to insecticide resistance, target site and metabolic resistance, both of which confer resistance to specific classes of insecticides. Due to the limited number of chemical compounds available for mosquito control, it is important to determine current enzymatic profiles among mosquito populations. This study assessed resistance profiles for three metabolic pathways, α-esterases, β-esterases, and mixed-function oxidases (MFOs), as well as insensitivity of the acetylcholinesterase (iAChE) enzyme in the presence of propoxur, among Ae. aegypti from the Central Valley and southern California. All field-collected Ae. aegypti demonstrated elevated MFOs and iAChE activity, indicating potential development of pyrethroid and organophosphate resistance, respectively. Although regional variations were found among α-esterase and β-esterase activity, levels were generally elevated, further suggesting additional mechanisms for developing organophosphate resistance. Furthermore, mosquito samples from southern California exhibited a higher expression level to all three metabolic enzymes and iAChE activity in comparison to mosquitoes from the central region. These results could help guide future mosquito control efforts, directing the effective use of insecticides while limiting the spread of resistance.

scholarly journals Diverse sensitivities of TRPA1 from different mosquito species to thermal and chemical stimuli

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Tianbang Li ◽  
Claire T. Saito ◽  
Tomoyuki Hikitsuchi ◽  
Yoshihiro Inoguchi ◽  
Honami Mitsuishi ◽  
...  
Keyword(s):  
Culex Pipiens ◽  
Transient Receptor Potential ◽  
Mosquito Control ◽  
Splice Variants ◽  
Mosquito Species ◽  
Temperature Threshold ◽  
Culex Pipiens Pallens ◽  
Chemical Stimuli ◽  
Pipiens Pallens

AbstractTemperature and odors profoundly affect the behavior of animals. Transient receptor potential channel, subfamily A, member 1 (TRPA1) functions as a polymodal nociceptor for sensing both vital environmental cues in insects. Mosquitoes are recognized as disease vectors, and many efforts have been devoted to investigations of their host-seeking behaviors and repellents. However, the physiological characteristics of mosquito TRPA1 have not been systematically studied. We identified multiple alternative splice variants of the TrpA1 gene from Anopheles gambiae, Anopheles stephensi, Aedes aegypti and Culex pipiens pallens mosquitoes. And we performed comparative analyses of the responses of mosquito TRPA1s to heat or chemical stimuli with calcium-imaging and whole-cell patch-clamp methods. Comparison of TRPA1 among four mosquito species from different thermal niches revealed that TRPA1 of Culex pipiens pallens inhabiting the temperate zone had a lower temperature threshold for heat-evoked activation, which was supported by the in vivo heat-avoidance test. Notably, the chemosensitivity of mosquito TRPA1 channels revealed differences not only between variants but also among species. Moreover, we discovered three novel mosquito TRPA1 agonists. Thermal niches selection and evolutionary trajectories significantly affect the functional properties of mosquito TRPA1, which represents a hallmark of the behaviors that may permit the design of improved mosquito control methods.

scholarly journals Absence of knockdown mutations in pyrethroid and DDT resistant populations of the main malaria vectors in Colombia

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Lorena I. Orjuela ◽  
Diego A. Álvarez-Diaz ◽  
Juliana A. Morales ◽  
Nelson Grisales ◽  
Martha L. Ahumada ◽  
...  
Keyword(s):  
Insecticide Resistance ◽  
Pyrethroid Resistance ◽  
Resistance Mechanism ◽  
Point Mutations ◽  
Resistance Mechanisms ◽  
Malaria Vectors ◽  
Anopheles Darlingi ◽  
Metabolic Resistance ◽  
Pyrethroid Insecticides ◽  
Voltage Gated Sodium Channels

Abstract Background Knockdown resistance (kdr) is a well-characterized target-site insecticide resistance mechanism that is associated with DDT and pyrethroid resistance. Even though insecticide resistance to pyrethroids and DDT have been reported in Anopheles albimanus, Anopheles benarrochi sensu lato (s.l.), Anopheles darlingi, Anopheles nuneztovari s.l., and Anopheles pseudopunctipennis s.l. malaria vectors in Latin America, there is a knowledge gap on the role that kdr resistance mechanisms play in this resistance. The aim of this study was to establish the role that kdr mechanisms play in pyrethroid and DDT resistance in the main malaria vectors in Colombia, in addition to previously reported metabolic resistance mechanisms, such as mixed function oxidases (MFO) and nonspecific esterases (NSE) enzyme families. Methods Surviving (n = 62) and dead (n = 67) An. nuneztovari s.l., An. darlingi and An. albimanus mosquitoes exposed to diagnostic concentrations of DDT and pyrethroid insecticides were used to amplify and sequence a ~ 225 bp fragment of the voltage-gated sodium channels (VGSC) gene. This fragment spanning codons 1010, 1013 and 1014 at the S6 segment of domain II to identify point mutations, which have been associated with insecticide resistance in different species of Anopheles malaria vectors. Results No kdr mutations were detected in the coding sequence of this fragment in 129 samples, 62 surviving mosquitoes and 67 dead mosquitoes, of An. darlingi, An. nuneztovari s.l. and An. albimanus. Conclusion Mutations in the VGSC gene, most frequently reported in other species of the genus Anopheles resistant to pyrethroid and DDT, are not associated with the low-intensity resistance detected to these insecticides in some populations of the main malaria vectors in Colombia. These results suggest that metabolic resistance mechanisms previously reported in these populations might be responsible for the resistance observed.

scholarly journals Susceptibility to insecticides and resistance mechanisms in three populations of Aedes aegypti from Peru

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Jesus Pinto ◽  
Miriam Palomino ◽  
Leonardo Mendoza-Uribe ◽  
Carmen Sinti ◽  
Kelly A. Liebman ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Insecticide Resistance ◽  
Disease Transmission ◽  
Molecular Mechanisms ◽  
Mosquito Control ◽  
Resistance Mechanisms ◽  
Activity Levels ◽  
Metabolic Resistance ◽  
Glutathione S Transferases ◽  
Target Site Resistance

Abstract Background Epidemics of dengue, chikungunya and Zika are a growing threat to areas where Aedes aegypti are present. The efficacy of chemical control of Ae. aegypti is threatened by the increasing frequency of insecticide resistance. The objective of this study was to determine the susceptibility status as well as the biochemical and molecular mechanisms underlying insecticide resistance in three populations of Ae. aegypti in high risk areas of dengue, chikungunya, and Zika in Peru. Methods Bioassays were conducted on adult Ae. aegypti to evaluate their susceptibility to insecticides used currently or historically for mosquito control in Peru, including six pyrethroids, three organophosphates and one organochlorine, in populations of Ae. aegypti from the districts of Chosica (Department of Lima), Punchana (Department of Loreto) and Piura (Department of Piura). Resistance mechanisms were determined by biochemical assays to assess activity levels of key detoxification enzyme groups (nonspecific esterases, multi-function oxidases, glutathione S-transferases and insensitive acetylcholinesterase). Real-time PCR assays were used to detect two kdr mutations (V1016I and F1534C) on the voltage-gated sodium channel gene. Results Resistance to DDT was detected in all three populations, and resistance to pyrethroids was detected in all populations except the population from Chosica, which still exhibited susceptibility to deltamethrin. Resistance to organophosphates was also detected, with the exception of populations from Punchana and Piura, which still demonstrated susceptibility to malathion. In general, no increase or alteration of activity of any enzyme group was detected. Both 1016I and 1534C alleles were detected in Punchana and Piura, while only the 1534C allele was detected in Chosica. Conclusions The results suggest that resistance to multiple classes of insecticides exist in areas important to Ae. aegypti-borne disease transmission in Peru. The F1534C mutation was present in all 3 populations and the V1016I mutation was present in 2 populations. To our knowledge, this is the first report of the presence of 1016I and 1534C in Ae. aegypti in Peru. The absence of highly elevated enzymatic activity suggests that target site resistance is a key mechanism underlying insecticide resistance in these populations, although further research is needed to fully understand the role of metabolic resistance mechanisms in these populations.

scholarly journals Evidence of Insecticide Resistance to Pyrethroids and Bendiocarb in Anopheles funestus from Tsararano, Marovoay District, Madagascar

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Tsiriniaina Rakotondranaivo ◽  
Solohery Fanomezana Randriamanarivo ◽  
Mihajarilala Rakotoniaina Tanjona ◽  
Inès Vigan-Womas ◽  
Milijaona Randrianarivelojosia ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Insecticide Resistance ◽  
Resistance Mechanism ◽  
Anopheles Funestus ◽  
Indoor Residual Spraying ◽  
Enzymatic Activities ◽  
Eastern Coast ◽  
Bed Nets ◽  
Metabolic Resistance ◽  
Central Highland

Introduction. In Madagascar, malaria control relies on the countrywide use of long lasting insecticide treated bed nets (LLINs) and on indoor residual spraying (IRS) in the central highland area as well as a small area on the eastern coast. We tested insecticide resistance mechanisms of Anopheles funestus from Tsararano, a malaria endemic village in the coastal health district of Marovoay. Methods. Insecticide susceptibility bioassays were done in July 2017 on first-generation Anopheles funestus (F1) to assess (i) the susceptibility to permethrin (0.05%), deltamethrin (0.05%), DDT (4%), malathion (5%), fenitrothion (1%), and bendiocarb (0.1%); (ii) the effect of preexposure to the piperonyl butoxide (PBO) synergist; and (iii) the enzymatic activities of cytochrome P450, esterases, and glutathione S-transferases (GST). Results. Our results demonstrated that An. funestus was phenotypically resistant to pyrethroids and bendiocarb, with a mortality rate (MR) of 33.6% (95%CI: 24.5-43.7%) and 86% (95%CI: 77.6-92.1%), respectively. In contrast, An. funestus were 100% susceptible to DDT and organophosphates (malathion and fenitrothion). Preexposure of An. funestus to PBO synergist significantly restored the susceptibility to bendiocarb (MR=100%) and increased the MR in the pyrethroid group, from 96% (95%CI: 90.0-98.9%) to 100% for deltamethrin and permethrin, respectively (χ2 = 43, df = 3, P< 0.0001). Enzymatic activities of cytochrome P450 and α-esterases were significantly elevated among An. funestus compared with the IPM reference strain (Mann-Whitney U= 30, P<0.0001; U = 145.5, P <0.0001, respectively). No significant differences of β-esterases activities compared to the IPM reference strain were observed (Mann-Whitney U = 392.5, P = 0.08). Conclusion. In Tsararano, despite the absence of an IRS programme, there is evidence of high levels of insecticide resistance to pyrethroids and bendiocarb in An. funestus. Biochemical data indicated that a metabolic resistance mechanism through the cytochrome P450 genes is operating in the An. funestus population.

scholarly journals Physiological and Environmental Factors Affecting the Composition of the Ejaculate in Mosquitoes and Other Insects

Insects ◽  
2019 ◽  
Vol 10 (3) ◽  
pp. 74 ◽  
Author(s):  
Megan Meuti ◽  
Sarah Short
Keyword(s):  
Reproductive Biology ◽  
Mosquito Control ◽  
Seminal Fluid ◽  
Control Strategies ◽  
Abiotic Factors ◽  
Mosquito Species ◽  
Future Research ◽  
Factors Affecting ◽  
Host Seeking ◽  
The Individual

In addition to transferring sperm, male mosquitoes deliver several proteins, hormones and other factors to females in their seminal fluid that inhibit remating, alter host-seeking behaviors and stimulate oviposition. Recently, bioinformatics, transcriptomics and proteomics have been used to characterize the genes transcribed in male reproductive tissues and the individual proteins that are delivered to females. Thanks to these foundational studies, we now understand the complexity of the ejaculate in several mosquito species. Building on this work, researchers have begun to identify the functions of various proteins and hormones in the male ejaculate, and how they mediate their effects on female mosquitoes. Here, we present an overview of these studies, followed by a discussion of an under-studied aspect of male reproductive physiology: the effects of biotic and abiotic factors on the composition of the ejaculate. We argue that future research in this area would improve our understanding of male reproductive biology from a physiological and ecological perspective, and that researchers may be able to leverage this information to study key components of the ejaculate. Furthermore, this work has the potential to improve mosquito control by allowing us to account for relevant factors when implementing vector control strategies involving male reproductive biology.

scholarly journals Coagulation-Flocculation: A potential Application for Mosquito Larval Source Management (LSM) with No Insecticide Resistance

2020 ◽  
Author(s):  
XinRan Li ◽  
Minsheng Huang ◽  
PeiEn Leng ◽  
XinYu Lu ◽  
Bing Xiao
Keyword(s):  
Insecticide Resistance ◽  
Culex Pipiens ◽  
Control Method ◽  
Mosquito Control ◽  
Tap Water ◽  
Oviposition Preference ◽  
Mosquito Larvae ◽  
Culex Pipiens Pallens ◽  
Lethal Time ◽  
Pipiens Pallens

Abstract Background: Mosquito control is an essential step to eliminate mosquito-borne diseases. Larval mosquitoes have a more limited home range and lower resistance to adverse environment than adults, thus can be ideal targets for vector control in some cases. Coagulation-flocculation technology, which could be used for water treatment in breeding sites of several vector mosquito species, can significantly change both the distribution of organic particles and surface sediment characteristics in water environment. The aim of this study was to explore the effect, principle and possibility of using coagulation-flocculation technology in immature mosquitoes killing.Method: In laboratory, chlorine-free tap water was treated with Poly Aluminum Chloride (PACl, sewage treatment using). The oviposition preference of gravid Culex pipiens pallens, the hatching of mosquito eggs and the survival amount of mosquito larvae were observed, and the pupa amounts were recorded each day.Results: Coagulation-flocculation treatment could improve the oviposition preference of Culex pipiens pallens to some extent (compared with ordinary chlorine-free tap water), but not significantly (p=0.345). After treatment, mosquitoes laid eggs in chlorine-free tap water were 31.88% more than those laid in untreated water. Coagulation-flocculation affected the larvae’s survival by physical means: Ⅰ) alum floc layer increases the difficulty of larvae foraging, leads larvae starving to death; (Ⅱ) the little floc particles adhere to the surface of larvae, which stops larvae from floating upward to breathe. As a result, the alum floc layer had a good killing effect on the mosquito larvae, presented the half lethal time (LT50) of 2d, the 90% lethal time (LT90) of 8.7±7.3 ~ 14±4.5 d, and the pupation rate of 0 ~ (6.5±0.5)%, respectively.Conclusions: PACl coagulation-flocculation produced lots of alum flocs, which may attract more gravid mosquitoes for laying eggs, and was shown to be highly active against 1st~2nd instar larvae. The principle of this technology illustrates this method won’t develop insecticide resistance. In this study, coagulation-flocculation technology is considered to be a new potential approach to a sustainable, low-impact and low-cost mosquito control method.

scholarly journals Insecticide resistance status of malaria vectors Anopheles gambiae (s.l.) of southwest Burkina Faso and residual efficacy of indoor residual spraying with microencapsulated pirimiphos-methyl insecticide

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Dieudonné Diloma Soma ◽  
Barnabas Zogo ◽  
Domonbabele François de Sales Hien ◽  
Aristide Sawdetuo Hien ◽  
Didier Alexandre Kaboré ◽  
...  
Keyword(s):  
Insecticide Resistance ◽  
Burkina Faso ◽  
Anopheles Gambiae ◽  
Local Population ◽  
Residual Activity ◽  
Indoor Residual Spraying ◽  
Susceptible Strain ◽  
Malaria Vectors ◽  
Pirimiphos Methyl ◽  
Residual Efficacy

Abstract Background The rapid spread of insecticide resistance in malaria vectors and the rebound in malaria cases observed recently in some endemic areas underscore the urgent need to evaluate and deploy new effective control interventions. A randomized control trial (RCT) was conducted with the aim to investigate the benefit of deploying complementary strategies, including indoor residual spraying (IRS) with pirimiphos-methyl in addition to long-lasting insecticidal nets (LLINs) in Diébougou, southwest Burkina Faso. Methods We measured the susceptibility of the Anopheles gambiae (s.l.) population from Diébougou to conventional insecticides. We further monitored the efficacy and residual activity of pirimiphos-methyl on both cement and mud walls using a laboratory susceptible strain (Kisumu) and the local An. gambiae (s.l.) population. Results An. gambiae (s.l.) from Diébougou was resistant to DDT, pyrethroids (deltamethrin, permethrin and alphacypermethrin) and bendiocarb but showed susceptibility to organophosphates (pirimiphos-methyl and chlorpyrimiphos-methyl). A mixed-effect generalized linear model predicted that pirimiphos-methyl applied on cement or mud walls was effective for 210 days against the laboratory susceptible strain and 247 days against the local population. The residual efficacy of pirimiphos-methyl against the local population on walls made of mud was similar to that of cement (OR = 0.792, [0.55–1.12], Tukey’s test p-value = 0.19). Conclusions If data on malaria transmission and malaria cases (as measured trough the RCT) are consistent with data on residual activity of pirimiphos-methyl regardless of the type of wall, one round of IRS with pirimiphos-methyl would have the potential to control malaria in a context of multi-resistant An. gambiae (s.l.) for at least 7 months.

scholarly journals At the tip of an iceberg: citizen science and active surveillance collaborating to broaden the known distribution of Aedes japonicus in Spain

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Roger Eritja ◽  
Sarah Delacour-Estrella ◽  
Ignacio Ruiz-Arrondo ◽  
Mikel A. González ◽  
Carlos Barceló ◽  
...  
Keyword(s):  
Citizen Science ◽  
Active Surveillance ◽  
Mosquito Control ◽  
Mosquito Species ◽  
Surveillance Systems ◽  
Northern Spain ◽  
Surveillance Strategy ◽  
Aedes Japonicus ◽  
Invasive Mosquito Species ◽  
Invasive Mosquito

Abstract Background Active surveillance aimed at the early detection of invasive mosquito species is usually focused on seaports and airports as points of entry, and along road networks as dispersion paths. In a number of cases, however, the first detections of colonizing populations are made by citizens, either because the species has already moved beyond the implemented active surveillance sites or because there is no surveillance in place. This was the case of the first detection in 2018 of the Asian bush mosquito, Aedes japonicus, in Asturias (northern Spain) by the citizen science platform Mosquito Alert. Methods The collaboration between Mosquito Alert, the Ministry of Health, local authorities and academic researchers resulted in a multi-source surveillance combining active field sampling with broader temporal and spatial citizen-sourced data, resulting in a more flexible and efficient surveillance strategy. Results Between 2018 and 2020, the joint efforts of administrative bodies, academic teams and citizen-sourced data led to the discovery of this species in northern regions of Spain such as Cantabria and the Basque Country. This raised the estimated area of occurrence of Ae. japonicus from < 900 km2 in 2018 to > 7000 km2 in 2020. Conclusions This population cluster is geographically isolated from any other population in Europe, which raises questions about its origin, path of introduction and dispersal means, while also highlighting the need to enhance surveillance systems by closely combining crowd-sourced surveillance with public health and mosquito control agencies’ efforts, from local to continental scales. This multi-actor approach for surveillance (either passive and active) shows high potential efficiency in the surveillance of other invasive mosquito species, and specifically the major vector Aedes aegypti which is already present in some parts of Europe. Graphical abstract

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