Meloidae Research Articles

Norcantharidin enhances ABT-737-induced apoptosis in hepatocellular carcinoma cells by transcriptional repression of Mcl-1

Small-molecule cell-permeable Bcl-2/Bcl-xL antagonist ABT-737 has recently emerged as a novel cancer therapeutic agent because it potently induces apoptosis in certain cancer cells. However, since ABT-737 binds to Mcl-1 with low affinity, ABT-737-mediated apoptosis signaling is inhibited in hepatocellular carcinoma (HCC) cells and other solid cancer cells due to the elevated expression of Mcl-1. Accordingly, strategies that target Mcl-1 are explored for overcoming ABT-737-resistance. In this study, we reported that Norcantharidin (NCTD), a small-molecule anticancer drug derived from Chinese traditional medicine blister beetle (Mylabris), induced transcriptional repression of Mcl-1 and considerably enhanced ABT-737-triggered cell viability inhibition and apoptosis in multiple HCC cell lines. Moreover, we observed that the enhancement of ABT-737-mediated apoptosis by NCTD was associated with activation of mitochondrial apoptosis signaling pathway, which involved cytosolic release of cytochrome c, cleavage of caspase-9 and caspase-3. Additionally, knockdown of Bax/Bak, the key effectors permeabilizing mitochondrial outer membrane significantly attenuated the enhancement, indicating mitochondrial apoptosis pathway played an essential role in the execution of the apoptosis. Finally, knockdown of Mcl-1 substantially potentiated ABT-737-mediated apoptotic cell death, confirming the potency of Mcl-1 repression by NCTD in enhancing ABT-737-induced apoptosis. These results therefore suggest that combination treatment with NCTD can overcome ABT-737 resistance and enhance ABT-737 therapeutic efficacy in treating human HCC.

Farmers’ knowledge and perceptions of blister beetles, Hycleus spp. (Coleoptera: Meloidae), as pest herbivores of Desmodium legumes in western Kenya

A survey was undertaken to determine the pest status of herbivorous blister beetles, Hycleus spp., in western Kenya where they attack crops such as Desmodium spp., other leguminous plants and sweetpotato. Desmodium spp. are important intercrops in the ‘push–pull” strategy adopted for Striga and stemborer control in maize and sorghum. Production of desmodium seed is adversely affected by blister beetles, which feed on the flowers and negatively affect seed setting. To assess farmers’ knowledge and perceptions of Hycleus spp. as pests, a questionnaire survey was conducted in three sites in Bungoma district, western Kenya, in 2007. The survey was followed by field sampling of Desmodium spp. and sweetpotato to compare the results with the responses received from farmers. Hycleus spp. were mentioned by 75% of the respondents as major pests of Desmodium spp. During field sampling Hycleus spp. comprised 70% of the insect pests collected. According to farmers, blister beetles were more abundant on desmodium than on sweetpotato. However, field sampling revealed that differences in beetle abundance on the two crops were not consistent across different sites, suggesting that these crops may function as alternative hosts. The study provides baseline information for the development of a management strategy for blister beetles.

Bioactive component, cantharidin from Mylabris cichorii and its antitumor activity against Ehrlich ascites carcinoma

The anticancer activity of the extract of blister beetle, Mylabris cichorii has been documented earlier by us. In the present study, the active principle of M. cichorii was isolated and its anticancer efficacy was evaluated against murine Ehrlich ascites carcinoma (EAC). The isolated bioactive compound was characterized to be cantharidin which showed potent antitumor activity and inhibited the proliferation of Ehrlich ascites carcinoma, both in vivo and in vitro. Cantharidin-treated EAC-bearing mice showed about 82% increase in lifespan at the dose of 0.5mg/kg/day. In vitro cytotoxicity assay with the 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test revealed about 50% cell death at the concentration of 25.8μg/ml. The fluorescence and transmission electron microscopy revealed that EAC cells treated with cantharidin depicted typical apoptotic morphology with chromatin condensation, nuclear fragmentation into discrete masses, and plasma membrane blebbing which deduce towards the death of these cells. Histological examination of the kidney of cantharidin-treated mice showed glomerular and tubular congestion with abnormal Bowman's capsule, thus, indicating a renal toxicity in the host. Cantharidin-induced renal damage in the host was also manifested by the decreased lactate dehydrogenase isozymes and its possible release from the cells.

Effects of temperature and photoperiod on termination of pseudopupal diapause in the bean blister beetle, Epicauta gorhami

Larvae of the bean blister beetle, Epicauta gorhami (Coleoptera: Meloidae), feed on grasshopper eggs in soil and undergo hypermetamorphosis. This beetle undergoes larval diapause in the fifth instar as a pseudopupa, a form characteristic of hypermetamorphosis in meloid beetles. This pseudopupal diapause is maintained for a long period if the larvae are kept under continuous diapause-inducing short-day conditions. In the present study, the effects of temperature and photoperiod on termination of pseudopupal diapause in E. gorhami were examined using pseudopupae obtained under 12L–12D at 25°C. Diapause was terminated by exposure to low or high temperature, but not by transfer to long-day conditions. The pseudopupal stage comprises diapause and post-diapause phases despite its unchanging appearance. The optimum low temperature for diapause termination was 10–15°C, and a higher or lower temperature was less effective. There was an individual variation in the low temperature requirement for diapause termination and post-diapause development may not occur until this requirement is satisfied. Although under natural conditions pseudopupae may encounter low temperatures that are effective for diapause termination at different ages, our results show that pseudopupae at various ages respond similarly to low temperature exposure. This response may ensure that resumption of development synchronizes in a population.

Origin, transfer and distribution of cantharidin-related compounds in the blister beetle Hycleus scabiosae

Cantharidin provides chemical protection for the coleopteran families Meloidae and Oedemeridae. In the present study, it was observed that cantharidin concentration in Hycleus scabiosae was slightly decreased from mated females (mean = 0.011 mg/mg of dry weight) to males (mean = 0.010 mg/mg) and considerably diminished in relation to virgin females (mean = 0.005 mg/mg). Significant concentrations of palasonin (21.69 ng/mg among virgins and 17.49 ng/mg in mated females) and palasoninimide (14.62 ng/mg in virgins and 9.17 ng/mg in mated females) were found in H. scabiosae. Palasonin, palasoninimide and cantharidinimide content of eggs were measured as 5.61, 7.69 and 7.80 ng/mg respectively. Surprisingly, males showed no trace of cantharidin-related compounds (CRCs); therefore CRCs in H. scabiosae could not be transferred from males to females and based on experiments employing its deuterated form, cantharidin is probably independently synthesized in females from the male nuptial transfer. An inseminated female incorporates about 38.5 ng of cantharidin (0.34% of the maternal content), 196.35 ng of palasonin (91.82% of maternal content) and 269.15 ng of palasoninimide (96.70% maternal content) into each egg mass during oviposition. It seems that eggs of this meloid species exploit a different array of protective chemicals by increasing the ratio of CRCs versus cantharidin. CRCs are less toxic than cantharidin; therefore, such compounds might have been deposited in eggs as a safer substitute for cantharidin to provide effective protection, but does not simultaneously harm the susceptible embryo.

Validation of serine/threonine protein phosphatase as the herbicide target site of endothall

Endothall, an older commercial herbicide, and cantharidin, a natural product from the blister beetle ( Epicauta spp.), are close chemical analogues. A comparison of the effect of endothall and cantharidin on plants revealed a similarity in their level of phytotoxicity on both Arabidopsis thaliana and Lemna paucicostata. Cantharidin is a potent inhibitor of animal serine/threonine protein phosphatases. Protein phosphatases and kinases maintain a sensitive balance between phosphorylated and dephosphorylated forms of proteins playing important roles in signal transduction pathways. In this study, we found endothall and cantharidin to both completely inhibit plant serine/threonine protein phosphatases, and their relative inhibitory activities were similar to their relative phytotoxicities. Both compounds acted as slow, irreversible inactivators of the serine/threonine protein phosphatase activities. Transcription of several genes determined to be affected by the inhibition of these protein phosphatases by cantharidin in A. thaliana by transcriptome analyses were affected similarly by endothall, but in a more pronounced way. Therefore, the molecular target site of endothall in plants is similar to that of cantharidin in animals, namely, serine/threonine protein phosphatases responsible for regulating an array of biochemical processes. This mode of action is unlike any other commercial herbicide.

Possible cantharidin poisoning of a great bustard (Otis tarda)

A possible cantharidin intoxication of a great bustard (Otis tarda) was described. This wild bird died by a traumatism, but also presented diarrhoea, congestion of the gastrointestinal tract and kidneys and had ingested several blister beetles of the species Berberomeloe majalis. The analysis of the stomach content by GC–MS revealed the presence of cantharidin at a concentration of 1.37 μg/g of wet weight, a similar level than in other birds poisoned in captivity.

Analysis of cantharidin in false blister beetles (Coleoptera: Oedemeridae) by headspace solid-phase microextraction and gas chromatography–mass spectrometry

A headspace solid-phase microextraction (HS-SPME) coupled to gas chromatography–mass spectrometry (GC–MS) method was developed to determine a type of terpenoid named as cantharidin in the false blister beetles, family Oedemeridae. The experimental parameters for HS-SPME method were optimized. Six commercial fibers for HS-SPME method development were tested and the divinylbenzene/carboxene/polydimethylsiloxane fiber was selected to provide the best detection of analyzed compound. The calibration curve showed linearity in the range of 0.1–50 μg mL −1, correlation coefficient ( R 2 = 0.992), limit of detection (0.01 ng mL −1) and quantitation (0.04 ng mL −1) were obtained for the proposed method. The relative standard deviations of intra-day and inter-day assays were 7.8 and 3.4%, respectively. The recovery values, obtained after spiking the beetle samples by three concentration levels of standard solution, were higher than 87%. The results indicated the successful application of the proposed method on the analysis of cantharidin from the false blister beetles.

Transcriptional responses to cantharidin, a protein phosphatase inhibitor, in Arabidopsis thaliana reveal the involvement of multiple signal transduction pathways

Cantharidin is a natural compound isolated from the blister beetle (Epicauta spp.). It is a potent inhibitor of protein serine/threonine phosphatases (PPPs), especially PP2A and PP4. Protein phosphatases and kinases maintain a sensitive balance between dephosphorylated and phosphorylated forms of appropriate proteins, thereby playing important roles in signal transduction pathways and regulation of gene expression, cellular proliferation, cell differentiation, apoptosis and other processes. The foliage of 12-day-old Arabidopsis thaliana seedlings was treated with 200 µM (IC(30) ) of the PPP inhibitor cantharidin, and the entire transcriptome profile was determined by microarray analysis at 2, 10 and 24 h after treatment. The transcription of approximately 10% (2577) of the 24 000 genes of Arabidopsis changed significantly (P≤ 0.05 and signal log ratios: ≥1 or ≤-1) after treatment. Inhibition of PPPs significantly reduced transcription of genes associated with auxin and light signaling and induced expression of genes involved in the hypersensitive response and in flagellin and abscisic acid signaling. The great variety of up- and downregulated genes in this microarray experiment implied that cantharidin interfered with the activities of PPPs that interact directly or indirectly with receptors or are located near the beginning of signal transduction pathways. In many cases, PPPs interact with protein complexes of various receptors such as ethylene or light sensors localized in different cell compartments. They function as negative regulators modifying receptor functions, thus altering signaling that influences transcriptional responses.

Redefinition of the vittata species group of Epicauta Dejean (1834) (Coleoptera: Meloidae) and taxonomic revision of the species from southern South America

The Epicauta vittata group are commonly known as striped blister beetles and was defined by previous authors to include 32 species, 18 from North America, Central America and northern South America, and 14 from southern South America. In the present revision we revised 22 species from South America, excluding the following southern South American species: E. borgmeieri Denier, 1935; E. floydwerneri Martínez, 1955; E. franciscana Denier, 1935; E. fulginosa (Oliver, 1795); E. purpureiceps (Berg, 1889); E. rutilifrons Borchmann, 1930; and E. zebra (Dohrn, 1876) because they do not have the diagnostic characters of the group. The species of the E. vittata group from southern South America are: E. bosqi Denier, 1935; E. clericalis (Berg, 1881); E. grammica (Fischer, 1827); E. leopardina (Haag-Rutemberg, 1880); E. luteolineata Pic, 1933; E. missionum (Berg, 1881); E. monachica (Berg, 1883); E. rutilifrons Borchmann, 1930; plus two more species E. excavata (Klug, 1825); and E. semivittata (Fairmaire, 1875) until now not included in other groups. We provide a complete diagnosis of the E. vittata group from southern South America, redescribing and illustrating all included species. Detailed descriptions and illustrations of female and male genitalia are presented for the first time for these species. Finally, we provide an identification key for the ten species presently included in the E. vittata group, and update the geographic distribution of each species.

Revision of the Afrotropical Mylabrine GenusLydocerasMarseul, 1870 (Coleoptera: Meloidae)

The blister beetle genus Lydoceras Marseul, 1870, tribe Mylabrini, is revised. Its four species are redescribed and figured. Faunistic and ecological records of each species also are summarized. Results of a morphology-based cladistic analysis show Lydoceras lictor (Gerstacker, 1885) and L. fasciatus (Fabricius, 1775) as most closely related. These two species and L. flavosellatus (Fairmaire, 1887) are placed in the nominate subgenus. The group occurs in eastern Africa and western Arabian Peninsula. The highly distinctive L. stanleyanus (Duvivier, 1890), endemic to central Africa, is placed in the resurrected subgenus Denierus Pic.

Cantharidin induces G2/M phase arrest and apoptosis in human colorectal cancer colo 205 cells through inhibition of CDK1 activity and caspase-dependent signaling pathways

Cantharidin (CTD) is a traditional Chinese medicine and an effective component isolated from blister beetle, and it has been demonstrated to have anticancer, antibiotic, antivirus activities and immune-regulated functions. It has been reported that CTD induces cell cycle arrest and apoptosis in many cancer cell types. However, there are no reports showing that CTD would induce cell cycle arrest and apoptosis in human colorectal cancer colo 205 cells. In this study, we studied colo 205 cells which were treated with CTD and demonstrated its molecular mechanisms in apoptosis. CTD induced growth inhibition, G2/M phase arrest and apoptosis in colo 205 cells. The IC50 is 20.53 µM in CTD-treated colo 205 cells. DAPI/TUNEL double staining and Annexin V assays were used to confirm the apoptotic cell death in colo 205 cells after CTD exposure. CTD caused G2/M arrest, down-regulated CDK1 activity, decreased Cyclin A, Cyclin B, CDK1 and increased CHK1 and p21 protein levels. Colorimetric assays also indicated that CTD triggered activities of casapse-8, -9 and -3 in colo 205 cells. Moreover, CTD increased ROS production and decreased the level of mitochondrial membrane potential (ΔΨm) in colo 205 cells. Consequently, CTD-induced growth inhibition was significantly attenuated by N-acetylcysteine (NAC, a scavenger). CTD stimulated the protein levels of Fas/CD95, the caspase-3 active form, cytochrome c and Bax, but suppressed the protein levels of pro-caspase-8, pro-caspase-9 and Bcl-2, determined by Western blot analysis. Based on our observations, we suggest that CTD is able to induce G2/M phase arrest and apoptosis in colo 205 cells through inhibition of CDK1 activity and caspase-dependent signaling pathways.

Influences of the biophysical environment on blister rust and mountain pine beetle, and their interactions, in whitebark pine forests

Aim To understand how the biophysical environment influences patterns of infection by non-native blister rust (caused by Cronartium ribicola) and mortality caused by native mountain pine beetles (Dendroctonus ponderosae) in whitebark pine (Pinus albicaulis) communities, to determine how these disturbances interact, and to gain insight into how climate change may influence these patterns in the future. Location High-elevation forests in south-west Montana, central Idaho, eastern and western Oregon, USA. Methods Stand inventory and dendroecological methods were used to assess stand structure and composition and to reconstruct forest history at sixty 0.1-ha plots. Patterns of blister rust infection and mountain pine beetle-caused mortality in whitebark pine trees were examined using nonparametric Kruskal–Wallis ANOVA, Mann–Whitney U-tests, and Kolmogorov–Smirnov two-sample tests. Stepwise regression was used to build models of blister rust infection and mountain pine beetle-related mortality rates based on a suite of biophysical site variables. Results Occurrence of blister rust infections was significantly different among the mountain ranges, with a general gradient of decreasing blister rust occurrence from east to west. Evidence of mountain pine beetle-caused mortality was identified on 83% of all dead whitebark pine trees and was relatively homogenous across the study area. Blister rust infected trees of all ages and sizes uniformly, while mountain pine beetles infested older, larger trees at all sites. Stepwise regressions explained 64% and 58% of the variance in blister rust infection and beetle-caused mortality, respectively, indicating that these processes are strongly influenced by the biophysical environment. More open stand structures produced by beetle outbreaks may increase the exposure of surviving whitebark pine trees to blister rust infection. Main conclusions Variability in the patterns of blister rust infection and mountain pine beetle-caused mortality elucidated the fundamental dynamics of these disturbance agents and suggests that the effects of climate change will be complex in whitebark pine communities and vary across the species’ range. Interactions between blister rust and beetle outbreaks may accelerate declines or facilitate the rise of rust resistance in whitebark pine depending on forest conditions at the time of the outbreak.

First-instar larva of Palaestra rufipennis (Westwood, 1841) and other Australian blister beetles (Coleoptera, Meloidae, Nemognathinae).

This is the first study of Australian blister beetle larvae. The first‐instar larva of one species of the endemic genus Palaestra Laporte de Castelnau, 1840, is described as well as those of other phoretic species without adult association and consequently unplaced to genus. Two main larval types (Groups 1 and 2) are recognised, as are distinct morphotypes within each group. Although both groups are assignable to the Nemognathinae, they are characterised by traits not paralleled by subfamily representatives from other regions of the world. Group 1, which includes Palaestra, is the more distinctive, whereas Group 2 shares most characteristics with members of the cosmopolitan tribe Nemognathini. A tabular comparison of larval groups and morphotypes is included.

Behavioural study of Mylabris phalerata (Meloidae:Coleoptera) in field and laboratory conditions

Certain behavioural aspects of Mylabris phalerata (Pallas) the most widely distributed species of blister beetles (Meloidae) in India have been presented. These beetles, like blister beetles in general, are attracted to yellow petals but M. phalerata abandon the yellow Canna as soon as Ipomoea carnea is in bloom, although this flower is not yellow at all. Mating and egg laying have been studied. These beetles mate on Calotropis procera, Acacia arabica and Cassia sopherea but not on their food plant Ipomoea carnea.

Effects of temperature, photoperiod and soil humidity on induction of pseudopupal diapause in the bean blister beetle Epicauta gorhami

Larvae of the bean blister beetle Epicauta gorhami Marseul (Coleoptera: Meloidae) feed on grasshopper eggs in soil and undergo hypermetamorphosis. This beetle undergoes larval diapause in the fifth instar as a pseudopupa, a form characteristic of hypermetamorphosis in meloid beetles. The effects of temperature, photoperiod and soil humidity on larval development of E. gorhami are examined in a population in Miyazaki, Japan, using egg pods of Locusta migratoria L. as food. At lower temperatures (20 and 22.5 °C), all larvae become pseudopupae, regardless of the photoperiod. By contrast, at higher temperatures (27.5 and 30 °C), almost all larvae pupate at the end of the fourth instar, again regardless of the photoperiod. A long‐day photoperiodic response occurs only at an intermediate temperature (25 °C): under an LD 12 : 12 h photocycle, all larvae enter diapause, although the diapause incidence tends to decrease as the day length becomes longer. Pseudopupae are immobile and remain in diapause for ≥120 days when they are kept under the same conditions, except that diapause terminates within a relatively short time at 30 °C. Although lower soil humidity retards post‐feeding development, soil humidity has no effect on the diapause incidence. On the basis of the short developmental period and diapause avoidance under summer conditions, it is suggested that this beetle partially produces two generations a year in southwestern Japan.

Open conformation of adipokinetic hormone receptor from the malaria mosquito facilitates hormone binding

Insect flight requires rapid mobilization of energy reserves during flight, which is mediated and regulated by hormonal control via adipokinetic hormones. The structure of the G-protein receptors to which these hormones bind, are crucial in understanding many of the physiological processes in which they play a central role. To date no 3D structure of an insect G-protein coupled receptor (GPCR) is available. Here, the first models of the 3D structures of a GPCR from the malaria mosquito are presented. Homology modeling of the receptor identified from the genome of Anopheles gambiae was used to construct two models of the receptor. The 7 transmembrane helical bundles of these two models are based on the crystal structures of beta2-adrenergic receptor and rhodopsin. The flexible loop regions were modeled using high temperature simulated annealing and constrained molecular dynamic simulations. The two receptor models differ in a number of critical features, the most important of which is that the rhodopsin-based model has a ‘closed’ structure while the beta2-based structure is ‘open’. The ‘open’ conformation provides easy access of the hormone to the binding pocket. Docking calculations with the insect adipokinetic hormones, AKH-1 (pGlu-Leu-Thr-Phe-Thr-Pro-Ala-Trp-NH 2) from the malaria mosquito and Del-CC (pGlu-Lys-Asn-Phe-Ser-Pro-Asn-Trp-Gly-Asn-NH 2) from the blister beetle showed that while the binding motif of the two is similar, AKH-1 has more than 30 times higher affinity than Del-CC, which strongly suggests that the binding is specific, and that the correct binding site was identified. Using these models it is possible to design antagonists, which block the binding site and are thus species-specific insecticides.

Taxonomía y distribución de <i>Meloe (Eurymeloe) saharensis</i> Chobaut, 1898 (Coleoptera, Meloidae), con nuevas sinonimias y primeros registros para Europa y la Macaronesia

A small group of species of the genus Meloe related to M. saharensis Chobaut, 1898 stands out amongst the rarest species of the family Meloidae in the Mediterranean Basin. The taxonomic study of nine specimens (including type material of M. saharensis, M. otini Peyerimhoff, 1949 and M. vignai Bologna, 1990), allows us to conclude that previously reported specific differences between M. saharensis, M. otini and M. marianii Kaszab, 1983, particularly the appearance of the metanotum, fall within the variability of a single species. The study of a broader set of characters, including male genitalia, does not contribute to the separation of taxa except for M. vignai . Consequently, we propose the formal synonymy of M. marianii ( syn. nov. ) and M. otini ( syn. nov. ) with M. saharensis , and therefore this species complex is reduced to two taxa: M. saharensis and M. vignai . Specimens examined allow us to assign a broad geographic range to M. saharensis , which extends from Israel and Saudi Arabia to the Canary Islands (Tenerife), with presence in southeastern Spain and most of the northern African countries (Egypt, Tunisia, Algeria and Morocco). Despite its antiquity, dating back more than 80 years, the record of M. saharensis in Murcia represents the first known data about its presence in Europe.

Time of diversification in the Cape fauna endemisms, inferred by phylogenetic studies of the genusIselma(Coleoptera: Meloidae: Eleticinae)

The endemic diversification of the Cape zone fauna and the phylogenetic relationships among the 30 species of the blister beetle genus Iselma are investigated. We analysed morphological, molecular (mtDNA 16S) and combined datasets of characters using a number of approaches (maximum parsimony, maximum likelihood, Bayesian inference analyses). We propose hypotheses of diversification times among taxa from molecular clock analyses. Morphological and molecular analyses produced similar results. According to maximum likelihood molecular studies, radiation within the genus Iselma occurred during the Pliocene and Pleistocene, roughly contemporaneously with the shift in African climate, vegetation and faunal assemblages. Two main lineages, one Namibian and one South African, separated c. 4.9 Ma. Within the South African lineage we identify two groups of endemic species, one in Little Karoo and one that extend towards southern Namaqualand. The remaining South African groups of species are spread through Namaqualand and the southwestern Cape area. These include several endemisms, with different times of diversification during the Pleistocene, probably related in part to glacial cycles. These endemisms are distributed in small areas of the following ecosystems: coastal Strandveld, Succulent Karoo and particularly Fynbos.

Leishmania major: In vitro and in vivo anti-leishmanial effect of cantharidin

Cantharidin is a natural poisonous compound secreted by male blister beetles. The effect of different doses of cantharidin on Leishmania major (MRHO/IR/75/ER) were investigated both in vitro (promastigote and amastigote viability) and in experimentally-infected BALB/c mice (skin lesions) using ointment or soluble cantharidin. In this study, cantharidin with concentrations of 0.5, 1, 2, 5, 10, 20 and 50 μg/ml inhibited the growth of L. major promastigotes after 24 h and the resultant inhibition levels were 39.22%, 41.95%, 49.88%, 54.78%, 58.01%, 68.30% and 80.04%, respectively. After 72 h, the mean number of amastigotes per macrophage in the culture using 2 μg/ml of cantharidin, (the 50% inhibitory concentration dose (IC 50)), was 1.2 while in the control group it was 2.7. In order to perform the inflammatory blister technique, 500 μg of cantharidin were solved in 25 μl of DMSO to show the formation of the blister which leads to treatment of cutaneous leishmaniasis. Using the blister technique, the small lesions (<5 mm) healed after one session. Two weeks of topical treatment with 0.1% cantharidin ointment was an effective method for treating cutaneous leishmaniasis in infected BALB/c mice.