Experimental hut evaluation of bednets treated with an organophosphate (chlorpyrifos-methyl) or a pyrethroid (lambdacyhalothrin) alone and in combination against insecticide-resistant Anopheles gambiae and Culex quinquefasciatus mosquitoes

Background

Pyrethroid resistant mosquitoes are becoming increasingly common in parts of Africa. It is important to identify alternative insecticides which, if necessary, could be used to replace or supplement the pyrethroids for use on treated nets. Certain compounds of an earlier generation of insecticides, the organophosphates may have potential as net treatments.

Methods

Comparative studies of chlorpyrifos-methyl (CM), an organophosphate with low mammalian toxicity, and lambdacyhalothrin (L), a pyrethroid, were conducted in experimental huts in Côte d'Ivoire, West Africa. Anopheles gambiae and Culex quinquefasciatus mosquitoes from the area are resistant to pyrethroids and organophosphates (kdr and insensitive acetylcholinesterase Ace.1 ^R ). Several treatments and application rates on intact or holed nets were evaluated, including single treatments, mixtures, and differential wall/ceiling treatments.

Results and Conclusion

All of the treatments were effective in reducing blood feeding from sleepers under the nets and in killing both species of mosquito, despite the presence of the kdr and Ace.1 ^R genes at high frequency. In most cases, the effects of the various treatments did not differ significantly. Five washes of the nets in soap solution did not reduce the impact of the insecticides on A. gambiae mortality, but did lead to an increase in blood feeding. The three combinations performed no differently from the single insecticide treatments, but the low dose mixture performed encouragingly well indicating that such combinations might be used for controlling insecticide resistant mosquitoes. Mortality of mosquitoes that carried both Ace.1 ^R and Ace.1 ^S genes did not differ significantly from mosquitoes that carried only Ace.1 ^S genes on any of the treated nets, indicating that the Ace.1 ^R allele does not confer effective resistance to chlorpyrifos-methyl under the realistic conditions of an experimental hut.     

High chlorpyrifos resistance in Culex pipiens mosquitoes: strong synergy between resistance genes

We investigated the genetic determinism of high chlorpyrifos resistance (HCR), a phenotype first described in 1999 in Culex pipiens mosquitoes surviving chlorpyrifos doses ⩾1 mg l⁻¹ and more recently found in field samples from Tunisia, Israel or Indian Ocean islands. Through chlorpyrifos selection, we selected several HCR strains that displayed over 10 000-fold resistance. All strains were homozygous for resistant alleles at two main loci: the ace-1 gene, with the resistant ace-1^R allele expressing the insensitive G119S acetylcholinesterase, and a resistant allele of an unknown gene (named T) linked to the sex and ace-2 genes. We constructed a strain carrying only the T-resistant allele and studied its resistance characteristics. By crossing this strain with strains harboring different alleles at the ace-1 locus, we showed that the resistant ace-1^R and the T alleles act in strong synergy, as they elicited a resistance 100 times higher than expected from a simple multiplicative effect. This effect was specific to chlorpyrifos and parathion and was not affected by synergists. We also examined how HCR was expressed in strains carrying other ace-1-resistant alleles, such as ace-1^V or the duplicated ace-1^D allele, currently spreading worldwide. We identified two major parameters that influenced the level of resistance: the number and the nature of the ace-1-resistant alleles and the number of T alleles. Our data fit a model that predicts that the T allele acts by decreasing chlorpyrifos concentration in the compartment targeted in insects.     

Mutations in acetylcholinesterase genes of Rhopalosiphum padi resistant to organophosphate and carbamate insecticides.

Apple grain aphid, Rhopalosiphum padi (Linnaeus), is an important wheat pest. In China, it has been reported that R. padi has developed high resistance to carbamate and organophosphate insecticides. Previous work cloned from this aphid 2 different genes encoding acetylcholinesterase (AChE), which is the target enzyme for carbamate and organophosphate insecticides, and its insensitive alteration has been proven to be an important mechanism for insecticide resistance in other insects. In this study, both resistant and susceptible strains of R, padi were developed, and their AChEs were compared to determine whether resistance resulted from this mechanism and whether these 2 genes both play a role in resistance. Bioassays showed that the resistant strain used was highly or moderately resistant to pirimicarb, omethoate, and monocrotophos (resistance ratio, 263.8, 53.8, and 17.5, respectively), and showed little resistance to deltamethrin or thiodicarb (resistance ratio, 5.2 and 3.4, respectively). Correspondingly, biochemistry analysis found that AChE from resistant aphids was very insensitive to the first 3 insecticides (I50 increased 43.0-, 15.2-, and 8.8-fold, respectively), but not to thiodicarb (I50 increased 1.1-fold). Enzyme kinetics tests showed that resistant and susceptible strains had different AChEs. Sequence analysis of the 2 AChE genes cloned from resistant and susceptible aphids revealed that 2 mutations in Ace2 and 1 in Ace1 were consistently associated with resistance. Mutation F368(290)L in Ace2 localized at the same position as a previously proven resistance mutation site in other insects. The other 2 mutations, S329(228)P in Ace1 and V435(356)A in Ace2, were also found to affect the enzyme structure. These findings indicate that resistance in this aphid is mainly the result of insensistive AChE alteration, that the 3 mutations found might contribute to resistance, and that the AChEs encoded by both genes could serve as targets of insecticides.     

Contemporary evolution of resistance at the major insecticide target site gene Ace‐1 by mutation and copy number variation in the malaria mosquito Anopheles gambiae

Functionally constrained genes are ideal insecticide targets because disruption is often fatal, and resistance mutations are typically costly. Synaptic acetylcholinesterase (AChE) is an essential neurotransmission enzyme targeted by insecticides used increasingly in malaria control. In Anopheles and Culex mosquitoes, a glycine–serine substitution at codon 119 of the Ace‐1 gene confers both resistance and fitness costs, especially for 119S/S homozygotes. G119S in Anopheles gambiae from Accra (Ghana) is strongly associated with resistance, and, despite expectations of cost, resistant 119S alleles are increasing significantly in frequency. Sequencing of Accra females detected only a single Ace‐1 119S haplotype, whereas 119G diversity was high overall but very low at non‐synonymous sites, evidence of strong purifying selection driven by functional constraint. Flanking microsatellites showed reduced diversity, elevated linkage disequilibrium and high differentiation of 119S, relative to 119G homozygotes across up to two megabases of the genome. Yet these signals of selection were inconsistent and sometimes weak tens of kilobases from Ace‐1. This unexpected finding is attributable to apparently ubiquitous amplification of 119S alleles as part of a large copy number variant (CNV) far exceeding the size of the Ace‐1 gene, whereas 119G alleles were unduplicated. Ace‐1 CNV was detectable in archived samples collected when the 119S allele was rare in Ghana. Multicopy amplification of resistant alleles has not been observed previously and is likely to underpin the recent increase in 119S frequency. The large CNV compromised localization of the strong selective sweep around Ace‐1, emphasizing the need to integrate CNV analysis into genome scans for selection.     

A single nucleotide polymorphism in the acetylcholinesterase gene of the predatory mite Kampimodromus aberrans (Acari: Phytoseiidae) is associated with chlorpyrifos resistance

The predatory mite Kampimodromus aberrans (Oudemans) (Acari: Phytoseiidae) is one of the most important biocontrol agents of herbivorous mites in European perennial crops. The use of pesticides, such as organophosphate insecticides (OPs), is a major threat to the success of biocontrol strategies based on predatory mites in these cropping systems. However, resistance to OPs in K. aberrans has recently been reported. The present study investigated the target site resistance mechanisms that are potentially involved in OP insensitivity. In the herbivorous mite Tetranychus urticae Koch (Acari: Tetranychidae), resistance to OPs is due to a modified and insensitive acetylcholinesterase (AChE; EC: 3.1.1.7) that bears amino acid substitution F331W (AChE Torpedo numbering). To determine whether the predators and prey have evolved analogous molecular mechanisms to withstand the same selective pressure, the AChE cDNA from a putative orthologous gene was cloned and sequenced from susceptible and resistant strains of K. aberrans. No synonymous mutation coding for a G119S substitution was determined to be strongly associated with the resistant phenotype instead of the alternative F331W. Because the same mutation in T. urticae AChE was not associated with comparable levels of chlorpyrifos resistance, the role of the G119S substitution in defining insensitive AChE in K. aberrans remains unclear. G119S AChE genotyping can be useful in ecological studies that trace the fate of resistant strains after field release or in marker-assisted selection of improved populations of K. aberrans to achieve multiple resistance phenotypes through gene pyramiding. The latent complexity of the target site resistance in K. aberrans vs. that of T. urticae is also discussed in the context of data from the genome project of the predatory mite Metaseiulus occidentalis (Nesbitt) (Acari: Phytoseiidae).     

Amino-acid substitutions in acetylcholinesterase 1 involved in insecticide resistance in mosquitoes

In natural populations of mosquitoes, high level of resistance to carbamates (CX) and organophosphates (OP) is provided by insensitive acetylcholinesterase (AChE1). Different alleles conferring resistance have been identified at the ace1 locus. They differ from the wild-type by only one amino-acid substitution. The comparison of the biochemical characteristics of mutated recombinant proteins and AChE1 in resistant mosquito extracts confirmed the role of each substitution in insensitivity. Selection of these different resistant alleles in field populations depends likely on the insecticides used locally. Theoretical modelling studies are initiated to develop novel strategies of mosquito control.     

Dot-blot test for identification of insecticide-resistant acetylcholinesterase in single insects

A test was developed to detect the presence of insecticide-resistant acetylcholinesterase (AChE) in single insects based on the quasipermanent binding of proteins onto blotting membranes. The method is simple, sensitive, requires inexpensive equipment, and produces a permanent record of results. AChE activity is revealed by the Karnovsky & Roots staining technique in the presence of propoxur, or after exposure of the membrane to paraoxon and rinsing with water. We chose insecticide concentrations that inhibited the sensitive AChE while allowing detectable residual activity of the resistant AChE to remain. By comparing the staining of insecticide-treated and control membranes, susceptible and resistant genotypes for the AChE gene could be distinguished in laboratory strains of mosquitoes (Culex spp. and Anopheles albimanus Wiedemann) and the house fly (Musca domestica L.). Resistant AChE from mosquitoes was less susceptible both to propoxur and paraoxon than the corresponding sensitive AChE, whereas resistant AChE from house fly was less susceptible mainly to paraoxon. The technique worked well for mosquito adults and house fly heads but not for mosquito larvae. Blotted AChE did not show detectable loss of activity during storage of the membranes for 3 wk at 25 degrees C. Storage is an important asset of the technique because transportation of live insect material to the laboratory may not be necessary.     

Mechanism behind Resistance against the Organophosphate Azamethiphos in Salmon Lice (Lepeophtheirus salmonis)

Acetylcholinesterase (AChE) is the primary target for organophosphates (OP). Several mutations have been reported in AChE to be associated with the reduced sensitivity against OP in various arthropods. However, to the best of our knowledge, no such reports are available for Lepeophtheirus salmonis. Hence, in the present study, we aimed to determine the association of AChE(s) gene(s) with resistance against OP. We screened the AChE genes (L. salmonis ace1a and ace1b) in two salmon lice populations: one sensitive (n=5) and the other resistant (n=5) for azamethiphos, a commonly used OP in salmon farming. The screening led to the identification of a missense mutation Phe362Tyr in L. salmonis ace1a, (corresponding to Phe331 in Torpedo californica AChE) in all the samples of the resistant population. We confirmed the potential role of the mutation, with reduced sensitivity against azamethiphos in L. salmonis, by screening for Phe362Tyr in 2 sensitive and 5 resistant strains. The significantly higher frequency of the mutant allele (362Tyr) in the resistant strains clearly indicated the possible association of Phe362Tyr mutation in L. salmonis ace1a with resistance towards azamethiphos. The 3D modelling, short term survival experiments and enzymatic assays further supported the imperative role of Phe362Tyr in reduced sensitivity of L. salmonis for azamethiphos. Based on all these observations, the present study, for the first time, presents the mechanism of resistance in L. salmonis against azamethiphos. In addition, we developed a rapid diagnostic tool for the high throughput screening of Phe362Tyr mutation using High Resolution Melt analysis.     

A Second Locus, Ixr B1 in Arabidopsis thaliana, that Confers Resistance to the Herbicide Isoxaben

An isoxaben resistant mutant of Arabidopsis thaliana is described whose locus, Ixr B1, is unlinked genetically to the previously described resistance locus Ixr A (DR Heim, JL Roberts, PD Pike, IM Larrinua [1989] Plant Physiol 90: 146-150). A cross of strains each homozygous for one of these two resistance loci gives rise to some isoxaben sensitive F₂ progeny. Growth curves versus isoxaben of this mutant, its F₁ progeny and the wild-type parent strain showed that this locus displays a weakly codominant Mendelian phenotype. Callus cultures were established from plants homozygous as well as heterozygous for this locus. Growth inhibition curves done with these cultures mimic the data obtained in vivo.     

Salivary Gland Proteome Analysis Reveals Modulation of Anopheline Unique Proteins in Insensitive Acetylcholinesterase Resistant Anopheles gambiae Mosquitoes

Insensitive acetylcholinesterase resistance due to a mutation in the acetylcholinesterase (ace) encoding ace-1 gene confers cross-resistance to organophosphate and carbamate insecticides in Anopheles gambiae populations from Central and West Africa. This mutation is associated with a strong genetic cost revealed through alterations of some life history traits but little is known about the physiological and behavioural changes in insects bearing the ace-1^R allele. Comparative analysis of the salivary gland contents between An. gambiae susceptible and ace-1^R resistant strains was carried out to charaterize factors that could be involved in modifications of blood meal process, trophic behaviour or pathogen interaction in the insecticide-resistant mosquitoes. Differential analysis of the salivary gland protein profiles revealed differences in abundance for several proteins, two of them showing major differences between the two strains. These two proteins identified as saglin and TRIO are salivary gland-1 related proteins, a family unique to anopheline mosquitoes, one of them playing a crucial role in salivary gland invasion by Plasmodium falciparum sporozoites. Differential expression of two other proteins previously identified in the Anopheles sialome was also observed. The differentially regulated proteins are involved in pathogen invasion, blood feeding process, and protection against oxidation, relevant steps in the outcome of malaria infection. Further functional studies and insect behaviour experiments would confirm the impact of the modification of the sialome composition on blood feeding and pathogen transmission abilities of the resistant mosquitoes. The data supports the hypothesis of alterations linked to insecticide resistance in the biology of the primary vector of human malaria in Africa.     

Novel AChE Inhibitors for Sustainable Insecticide Resistance Management

Resistance to insecticides has become a critical issue in pest management and it is particularly chronic in the control of human disease vectors. The gravity of this situation is being exacerbated since there has not been a new insecticide class produced for over twenty years. Reasoned strategies have been developed to limit resistance spread but have proven difficult to implement in the field. Here we propose a new conceptual strategy based on inhibitors that preferentially target mosquitoes already resistant to a currently used insecticide. Application of such inhibitors in rotation with the insecticide against which resistance has been selected initially is expected to restore vector control efficacy and reduce the odds of neo-resistance. We validated this strategy by screening for inhibitors of the G119S mutated acetylcholinesterase-1 (AChE1), which mediates insensitivity to the widely used organophosphates (OP) and carbamates (CX) insecticides. PyrimidineTrione Furan-substituted (PTF) compounds came out as best hits, acting biochemically as reversible and competitive inhibitors of mosquito AChE1 and preferentially inhibiting the mutated form, insensitive to OP and CX. PTF application in bioassays preferentially killed OP-resistant Culex pipiens and Anopheles gambiae larvae as a consequence of AChE1 inhibition. Modeling the evolution of frequencies of wild type and OP-insensitive AChE1 alleles in PTF-treated populations using the selectivity parameters estimated from bioassays predicts a rapid rise in the wild type allele frequency. This study identifies the first compound class that preferentially targets OP-resistant mosquitoes, thus restoring OP-susceptibility, which validates a new prospect of sustainable insecticide resistance management.     

The acetylcholinesterase gene and organophosphorus resistance in the Australian sheep blowfly, Lucilia cuprina

Acetylcholinesterase (AChE), encoded by the Ace gene, is the primary target of organophosphorous (OP) and carbamate insecticides. Ace mutations have been identified in OP resistants strains of Drosophila melanogaster. However, in the Australian sheep blowfly, Lucilia cuprina, resistance in field and laboratory generated strains is determined by point mutations in the Rop-1 gene, which encodes a carboxylesterase, E3. To investigate the apparent bias for the Rop-1/E3 mechanism in the evolution of OP resistance in L. cuprina, we have cloned the Ace gene from this species and characterized its product. Southern hybridization indicates the existence of a single Ace gene in L. cuprina. The amino acid sequence of L. cuprina AChE shares 85.3% identity with D. melanogaster and 92.4% with Musca domestica AChE. Five point mutations in Ace associated with reduced sensitivity to OP insecticides have been previously detected in resistant strains of D. melanogaster. These residues are identical in susceptible strains of D. melanogaster and L. cuprina, although different codons are used. Each of the amino acid substitutions that confer OP resistance in D. melanogaster could also occur in L. cuprina by a single non-synonymous substitution. These data suggest that the resistance mechanism used in L. cuprina is determined by factors other than codon bias. The same point mutations, singly and in combination, were introduced into the Ace gene of L. cuprina by site-directed mutagenesis and the resulting AChE enzymes expressed using a baculovirus system to characterise their kinetic properties and interactions with OP insecticides. The K(m) of wild type AChE for acetylthiocholine (ASCh) is 23.13 microM and the point mutations change the affinity to the substrate. The turnover number of Lucilia AChE for ASCh was estimated to be 1.27x10(3) min(-1), similar to Drosophila or housefly AChE. The single amino acid replacements reduce the affinities of the AChE for OPs and give up to 8.7-fold OP insensitivity, while combined mutations give up to 35-fold insensitivity. However, other published studies indicate these same mutations yield higher levels of OP insensitivity in D. melanogaster and A. aegypti. The inhibition data indicate that the wild type form of AChE of L. cuprina is 12.4-fold less sensitive to OP inhibition than the susceptible form of E3, suggesting that the carboxylesterases may have a role in the protection of AChE via a sequestration mechanism. This provides a possible explanation for the bias towards the evolution of resistance via the Rop-1/E3 mechanism in L. cuprina.     

A novel point mutation of acetylcholinesterase in a trichlorfon-resistant strain of the oriental fruit fly Bactrocera dorsalis (Diptera: Tephritidae)

Acetylcholinesterase (AChE) is the target enzyme of organophosphorus and carbamate insecticides. We applied trichlorfon to select resistant strains of Bactrocera dorsalis Hendel in the laboratory. Two trichlorfon-resistant strains, the Tri-R₁ strain with 18.23-fold resistance and the Tri-R₂ strain with 69.5-fold resistance, were obtained. Three known mutations, I159V, G433S and Q588R were identified in AChE of two resistant strains, and a novel mutation, G365A, was identified in the more resistant Tri-R₂ strain. The modeled 3-D-structure of AChE showed that G365A and G433S are closely adjacent in the gorge above the catalytic site S235. Mutations of G365A and G433S resulted in a steric hindrance by stronger Van der Waals force between two sites. Such a minor structural change might block insecticides from squeezing through the gorge to reach the active site, but not the natural substrate. Compared with the susceptible strain, the AChE activity of the Tri-R₁ strain and the Tri-R₂ strain was 0.87- and 0.67-fold, the K _m value of the Tri-R₁ strain and the Tri-R₂ strain was 0.11- and 0.10-fold, the V _max value of two resistant strains was 0.26- and 0.15-fold, whereas, the I ₅₀ to trichlorfon significantly increased by 9.07- and 13.19-fold. These results suggested that the novel point mutation G365A of AChE might be involved in increasing resistance to trichlorfon in the resistant strain of oriental fruit fly.     

Comparison of two acetylcholinesterase gene cDNAs of the lesser mealworm, Alphitobius diaperinus, in insecticide susceptible and resistant strains

Two cDNAs encoding different acetylcholinesterase (AChE) genes (AdAce1 and AdAce2) were sequenced and analyzed from the lesser mealworm, Alphitobius diaperinus. Both AdAce1 and AdAce2 were highly similar (95 and 93% amino acid identity, respectively) with the Ace genes of Tribolium castaneum. Both AdAce1 and AdAce2 have the conserved residues characteristic of AChE (catalytic triad, intra-disulfide bonds, and so on). Partial cDNA sequences of the Alphitobius Ace genes were compared between two tetrachlorvinphos resistant (Kennebec and Waycross) and one susceptible strain of beetles. Several single nucleotide polymorphisms (SNPs) were detected, but only one non-synonymous mutation was found (A271S in AdAce2). No SNPs were exclusively found in the resistant strains, the A271S mutation does not correspond to any mutations previously reported to alter sensitivity of AChE to organophosphates or carbamates, and the A271S was found only as a heterozygote in one individual from one of the resistant A. diaperinus strains. This suggests that tetrachlorvinphos resistance in the Kennebec and Waycross strains of A. diaperinus is not due to mutations in either AChE gene. The sequences of AdAce1 and AdAce2 provide new information about the evolution of these important genes in insects.     

Mutations in acetylcholinesterase associated with insecticide resistance in the cotton aphid, Aphis gossypii Glover

Two acetylcholinesterase genes, Ace1 and Ace2, have been fully cloned and sequenced from both organophosphate-resistant and susceptible clones of cotton aphid. Comparison of both nucleic acid and deduced amino acid sequences revealed considerable nucleotide polymorphisms. Further study found that two mutations occurred consistently in all resistant aphids. The mutation F139L in Ace2 corresponding to F115S in Drosophila acetylcholinesterase might reduce the enzyme sensitivity and result in insecticide resistance. The other mutation A302S in Ace1 abutting the conserved catalytic triad might affect the activity and insecticide sensitivity of the enzyme. Phylogenetic analysis showed that insect acetylcholinesterases fall into two subgroups, of which Ace1 is the paralogous gene whereas Ace2 is the orthologous gene of Drosophila AChE. Both subgroups contain resistance-associated AChE genes. To avoid confusion in the future work, a nomenclature of insect AChE is also suggested in the paper.     

Evidence of Biological Control of Agrobacterium tumefaciens Strains Sensitive and Resistant to Agrocin 84 by Different Agrobacterium radiobacter Strains on Stone Fruit Trees

The effectiveness of Agrobacterium radiobacter K84, 0341, and a K84 non-agrocin-producing mutant (K84 Agr^-) in biological control of crown gall on rootstocks of stone fruit trees was determined in three experiments. In experiment 1, K84 and 0341 controlled crown gall on plum plants in soil inoculated with two strains of Agrobacterium tumefaciens resistant to agrocin 84. In experiment 2, K84 controlled crown gall on peach plants in soils inoculated with strains of A. tumefaciens sensitive or resistant to agrocin 84 or with a mixture of both. However, the effectiveness of K84 was higher against the sensitive strain than against the resistant strain. There was a residual effect of K84 from one year to another in soil inoculated with the sensitive strains. In experiment 3, K84 and K84 Agr^- controlled crown gall on plum and peach plants in soils inoculated with strains of A. tumefaciens sensitive or resistant to agrocin 84. The control afforded by K84 was higher than that provided by K84 Agr^- against the sensitive strain but was similar against the resistant strain.     

Miltefosine and Antimonial Drug Susceptibility of Leishmania Viannia Species and Populations in Regions of High Transmission in Colombia

Background

Pentavalent antimonials have been the first line treatment for dermal leishmaniasis in Colombia for over 30 years. Miltefosine is administered as second line treatment since 2005. The susceptibility of circulating populations of Leishmania to these drugs is unknown despite clinical evidence supporting the emergence of resistance.

Methodology/Principal Findings

In vitro susceptibility was determined for intracellular amastigotes of 245 clinical strains of the most prevalent Leishmania Viannia species in Colombia to miltefosine (HePC) and/or meglumine antimoniate (Sb^V); 163, (80%) were evaluated for both drugs. Additionally, susceptibility to Sb^V was examined in two cohorts of 85 L. V. panamensis strains isolated between 1980–1989 and 2000–2009 in the municipality of Tumaco. Susceptibility to each drug differed among strains of the same species and between species. Whereas 68% of L. V. braziliensis strains presented in vitro resistance to HePC, 69% were sensitive to Sb^V. Resistance to HePC and Sb^V occurred respectively, in 20% y 21% of L. panamensis strains. Only 3% of L. V. guyanensis were resistant to HePC, and none to Sb^V. Drug susceptibility differed between geographic regions and time periods. Subpopulations having disparate susceptibility to Sb^V were discerned among L. V. panamensis strains isolated during 1980–1990 in Tumaco where resistant strains belonged to zymodeme 2.3, and sensitive strains to zymodeme 2.2.

Conclusions/Significance

Large scale evaluation of clinical strains of Leishmania Viannia species demonstrated species, population, geographic, and epidemiologic differences in susceptibility to meglumine antimoniate and miltefosine, and provided baseline information for monitoring susceptibility to these drugs. Sensitive and resistant clinical strains within each species, and zymodeme as a proxy marker of antimony susceptibility for L. V. panamensis, will be useful in deciphering factors involved in susceptibility and the distribution of sensitive and resistant populations.