Bioassays (at generation G2) with a newly collected field population (designated CH3) of Plutella xylostella L. from farmers' fields in the Cameron Highlands, Malaysia, indicated resistance ratios of 813-, 79-, 171-, 498- and 1285-fold for indoxacarb, fipronil, spinosad, deltamethrin and Bacillus thuringiensis toxin Cry1Ac respectively compared with a laboratory susceptible population (Lab-UK). At G2 the field-derived population was divided into two subpopulations: one was selected (G2 to G7) with indoxacarb (indoxa-SEL), while the second was left unselected (UNSEL). A significant reduction in the resistance ratio for each compound was observed in UNSEL at G8. For indoxa-SEL, bioassays at G8 found that selection with indoxacarb gave a resistance ratio of 2594 compared with Lab-UK and of 90 compared with UNSEL. The toxicity of fipronil, spinosad and deltamethrin was not significantly different in indoxa-SEL at G8 compared with G2 but was significantly greater than UNSEL at G8. The toxicity of Cry1Ac was significantly reduced in indoxa-SEL at G8 compared with G2 but was also significantly greater than UNSEL at G8. This suggests that indoxacarb selection maintained resistance to these compounds in the indoxa-SEL population. Synergist studies indicated that resistance to indoxacarb in indoxa-SEL was esterase associated. Logit regression analysis of F1 reciprocal crosses between indoxa-SEL and Lab-UK indicated that resistance to indoxacarb was inherited as an autosomal, incompletely recessive (D(LC) = 0.35) trait. Tests of monogenic inheritance suggested that resistance to indoxacarb was controlled by a single locus.
Aedes albopictus was bioassayed to determine resistance development to malathion (OP). Two methods were applied, including WHO larval bioassay to determine the susceptibility to lethal concentration (LC), and adult bioassay to determine lethal time (LT). Larvae from colonies that had undergone selection pressure with malathion to yield 50% mortality were further subjected to selection for subsequent 10 generations. Selection of Ae. albopictus with malathion could relatively induce a consistent resistance ratio of 1.0 throughout 10 generations. It was noted that Ae. albopictus larvae showed less susceptibility to malathion compared to adults. The susceptibility test of adult mosquitoes to diagnostic dosage of 5.0% malathion-impregnated paper showed a variety of susceptibility to malathion when compared to the susceptible strain. Bioassay results indicated that the LT50 values of malathion-selected Ae. albopictus ranged between 11.5 - 58.8 minutes for ten consecutive generations. Biochemical enzyme studies indicated that there was a significant difference (p < 0.05) in esterase level in malathion-selected mosquitoes compared to non-selected control. Electrophoretic patterns of non-specific esterases at different life stages in malathion-selected Ae. albopictus suggested that non-specific esterases do not play a role in resistance of malathion-selected Ae. albopictus.
Larvae and adults of Culex quinquefasciatus were used for the test undertaken for malathion resistant strain (F61 - F65) and permethrin resistant strain (F54 - F58). The results showed that the LC50 for both malathion (F61 - F65) and permethrin (F54 - F58) resistant Cx. quinquefasciatus increased steadily throughout the subsequent five generations, indicating a marked development of resistance. The adult female malathion resistant strain have developed a high resistance level to malathion diagnostic dosage with a resistance ratio of 9.3 to 17.9 folds of resistance compared with the susceptible Cx. quinquefasciatus. Permethrin resistance ratio remained as 1.0 folds of resistance at every generation. It was obvious that malathion resistance developed at a higher rate in adult females compared to permethrin. Enzyme-based metabolic mechanisms of insecticide resistance were investigated based on the biochemical assay principle. From the results obtained obviously shows that there is a significant difference (p < 0.05) in esterase level in both malathion and permethrin selected strains. Female malathion selected strain has the higher level of esterase activity compared to the female permethrin selected strain at (0.8 to 1.04) alpha-Na micromol/min/mg protein versus (0.15 to 0.24) alpha-Na micromol/min/mg protein respectively. This indicated increased level of non-specific esterase is playing an important role in resistance mechanism in female malathion selected strain. Permethrin selected strain exhibited non-specific esterase activity at a very low level throughout the different life stages compared to malathion selected strain. This study suggests that life stages play a predominant role in conferring malathion and permethrin resistance in Cx. quinquefasciatus.
The possible insecticide resistance mechanisms of four Malaysian field-collected strains of the German cockroach, Blattella germanica (Linnaeus) (Dictyoptera: Blattellidae), were characterized with biochemical assays and native polyacrylamide gel electrophoresis (PAGE). Elevated esterase activity (at low to moderate frequency) and altered acetylcholinesterase (low frequency) were detected in all field strains, while elevated glutathione S-transferase levels were present in only two strains. Seven esterase bands were separated by native PAGE; a greater intensity occurred in three bands in the resistant strains compared to the susceptible strain. Inhibition studies using specific inhibitors on polyacrylamide gels suggested that the slowest of these three esterases is a cholinesterase, while the other two are carboxylesterases with a preference for beta- over alpha-naphthyl acetate.
Wild caught female Culex quinquefasciatus (Say) from Kuala Lumpur were blood fed and reared in the insectarium. The late third stage of the F1 larvae which survived the high selection pressure of malathion and permethrin were reared and colonies were established from adults that emerged. Larvae from these colonies were then subjected in the subsequent 9 generations to higher selection pressure. The rate of resistance development were measured by LC50 value of larval bioassay, LT50 value of adult bioassay and the frequency of the elevated esterase levels. In another set of experiments using the same batch of Culex mosquitos, the larvae were not exposed to any insecticides and the decrease in resistance rate was monitored in each subsequent 9 generations by using similar methods. The heterozygous standard laboratory strain was selected for susceptibility using the single raft sib-selection method. The result showed that the field collected F1 generation was 96.0 and 6.3 fold more resistant to malathion and permethrin, respectively. After selection for about 9 generations the resistance ratio to malathion and permethrin was 6.2 and 767.3 fold more compared to the LC50 values of F1 generations, respectively. Esterase in F1 larvae was 6.0 fold more than the standard laboratory strain.
We have developed the methodologies for typing and family studies to establish the modes of inheritance of water buffalo red cell acid phosphatase (Acp), protease inhibitor (Pi), and group-specific component (Gc) on isoelectric focusing and albumin (Alb), red cell alpha-esterase-3 (Est-3), and catalase (Cat) on polyacrylamide gel electrophoresis. Family studies showed that Pi, Gc, Alb, and Cat are coded by autosomal genes with two codominant alleles, while Est-3 is autosomal with two codominant alleles and a recessive null allele and Acp exhibits three codominant alleles.
Despite the public health importance of Culex pipiens pipiens, their resistance to pirimiphos-methyl insecticides has not been explored enough. Late third and early fourth larvae of Culex pipiens pipiens were collected from three localities between 2003 and 2005 in Northern and Southern Tunisia. All bioassays were carried out using pirimiphosmethyl and propoxur insecticides. Populations of Culex pipiens pipiens were susceptible, moderate and resistant to pirimiphos-methyl insecticide. Resistance to this compound ranged from 2.62 in sample # 2 to 19.9 in sample # 1. The moderate resistance (5.25) was recorded in sample # 3. Synergist's tests showed that the resistance to pirimiphos-methyl was not affected by detoxification enzymes. However, biochemical assays showed the involvement of both metabolic (esterases) and target site (insensitive acetylcholinesterase) resistance mechanisms. The highest frequencies of the resistant phenotypes ([RS] and [RR]) (<0.74) were detected in the most resistant samples (#1). Four esterases enzymes including C1 encoded by the Est-1 locus and three esterases encoded by the Ester super locus: A2-B2, A4-B4 (or A5-B5, which has the same electrophoretic mobility) and B12 were detected. The highest (0.61) and the lowest (0.22) frequencies of these esterases were recorded in samples # 1 (Sidi Hcine) and # 2 (El Fahs) which recorded the highest and the lowest level of resistance, respectively. Monitoring of insecticide resistance should be evaluated regularly for management of vector control.
The aim of this study was to evaluate the resistance status of Culex pipiens pipiens to pirimiphos-methyl insecticide. Three field populations of mosquitoes were collected from Tunisia and analyzed in laboratory. The samples studied showed low level of resistance not exceeding 5-folds. The low resistance recorded is particularly interesting, because it leaves a range of tools useable by vector control services. Both metabolic and target-site resistance mechanisms were identified. Different esterases of high activity including A2-B2, A4-B4 (and/or A5-B5) and B12 were observed in studied field samples using starch electrophoresis although opposite results were found using synergists tests on samples # 1 and 3. The polymorphism of AChE1 (Acetylcholinesterase) was analyzed and three phenotypes were detected: susceptible (ACHE1S, phenotype [SS]), resistant (ACHE1R, phenotype [RR]), and heterozygous (phenotype [RS]) of ACHE1. The resistance of Culex pipiens pipiens to pirimiphos-methyl remains low although the occurrences of multiple resistance mechanisms are able to confer high resistance levels to organophosphate insecticides. Therefore, continuous monitoring of resistance is fundamental for rational use of insecticides and mosquito control programs.
Morphological and host-plant relationship studies were conducted to differentiate two sympatric populations of brown planthopper (BPH), Nilaparvata lugens, one from rice (Oryza sativa) and the other from Leersia hexandra, a weed grass. In morphometric studies based on esterase activities, an UPGMA dendrogram using 17 quantitative morphological characters, including stridulatory organs (courtship signal-producing organs) between two sympatric populations of N. lugens, one from rice and the other from L. hexandra, a weed grass revealed that both populations were separated from each other. An out-group, N. bakeri, was found to be completely different from the two sympatric populations of N. lugens. Rice plants were best suited for the establishment of the rice-infesting population, and L. hexandra was a favourable host for the Leersia-infesting population. The individuals derived from one host did not thrive on the other host, as shown by a significant reduction in survival and nymphal development, ovipositional preferences, ovipositional response, and egg hatchability. Therefore, morphological and host-plant relationship studies indicate that rice-associated population with high esterase activities and L. heaxandra-associated population with low esterase activities are two closely related sibling species.
A combination of a modified Feret' (Silvae Genet. 1971, 20, 46-50) extraction buffer and two types of electrophoresis with acrylamide and starch gels were used to characterize allozymes in mature vegetative tissue of a commercially high value species of rattans (Calamus subinermis). From the analysis of allelic segregation from single maternal rattans and their offspring, genetic control of the 16 observed banding zones, which were consistently scorable, was assumed. Seventeen gene loci were identified. The percentage of polymorphic loci within Calamus subinermis was much higher (70.5%) than expected levels of genetic diversity for tropical woody and non-woody species. It is thought that the protocol described may be applied to the analysis of the genetic diversity of all the endangered Calamus species.
The resistance status of Selangor Aedes aegypti (Linnaeus) larvae against four major groups of insecticides (i.e., organochlorines, carbamates, organophosphates and pyrethroids) was investigated. Aedes aegypti were susceptible against temephos (organophosphate), although resistance (RR50 = 0.21-2.64) may be developing. The insecticides susceptibility status of Ae. aegypti larvae were found heterogeneous among the different study sites. Results showed that Ae. aegypti larvae from Klang, Sabak Bernam and Sepang were susceptible against all insecticides tested. However, other study sites exhibited low to high resistance against all pyrethroids (RR50 = 1.19-32.16). Overall, the application of synergists ethacrynic acid, S.S.S.- tributylphosphorotrithioate and piperonyl butoxide increased the toxicity of insecticides investigated. However, the application failed to increase the mortality to susceptible level (>97%) for certain populations, therefore there are chances of alteration of target site resistance involved. Biochemical assays revealed that α-esterase, (Gombak, Kuala Langat, Kuala Selangor and Sabak Bernam strains) β-esterase (Klang and Sabak Bernam strains), acetylcholinesterase (Kuala Selangor and Sabak Bernam strains), glutathione-S-transferase (Kuala Selangor and Sabak Bernam strains) and mono-oxygenases (Gombak, Hulu Langat, Hulu Selangor and Kuala Langat strains) were elevated. Spearman rank-order correlation indicated a significant correlation between resistance ratios of: DDT and deltamethrin (r = 0.683, P = 0.042), cyfluthrin and deltamethrin (r = 0.867, P =0.002), cyflyuthrin and lambdacyhalothrin (r = 0.800, P =0.010), cyfluthrin and permethrin (r = 0.770, P =0.015) deltamethrin and permethrin (r = 0.803, P =0.088), propoxur and malathion (r = 0.867, P = 0.002), malathion and temephos (r = 0.800, P = 0.010), etofenprox and MFO enzyme (r = 0.667, P =0.050). The current study provides baseline information for vector control programs conducted by local authorities. The susceptibility status of Ae. aegypti should be monitored sporadically to ensure the effectiveness of current vector control strategy in Selangor.
Nepenthes is a genus comprising carnivorous tropical pitcher plants that have evolved trapping organs at the tip of their leaves for nutrient acquisition from insect trapping. Recent studies have applied proteomics approaches to identify proteins in the pitcher fluids for better understanding the carnivory mechanism, but protein identification is hindered by limited species-specific transcriptomes for Nepenthes. In this study, the proteomics informed by transcriptomics (PIT) approach was utilized to identify and compare proteins in the pitcher fluids of Nepenthes ampullaria, Nepenthes rafflesiana, and their hybrid Nepenthes × hookeriana through PacBio isoform sequencing (Iso-Seq) and liquid chromatography-mass spectrometry (LC-MS) proteomic profiling. We generated full-length transcriptomes from all three species of 80,791 consensus isoforms with an average length of 1,692 bp as a reference for protein identification. The comparative analysis found that transcripts and proteins identified in the hybrid N. × hookeriana were more resembling N. rafflesiana, both of which are insectivorous compared with omnivorous N. ampullaria that can derive nutrients from leaf litters. Previously reported hydrolytic proteins were detected, including proteases, glucanases, chitinases, phosphatases, nucleases, peroxidases, lipid transfer protein, thaumatin-like protein, pathogenesis-related protein, and disease resistance proteins. Many new proteins with diverse predicted functions were also identified, such as amylase, invertase, catalase, kinases, ligases, synthases, esterases, transferases, transporters, and transcription factors. Despite the discovery of a few unique enzymes in N. ampullaria, we found no strong evidence of adaptive evolution to produce endogenous enzymes for the breakdown of leaf litter. A more complete picture of digestive fluid protein composition in this study provides important insights on the molecular physiology of pitchers and carnivory mechanism of Nepenthes species with distinct dietary habits.
Candida glabrata has been reported as the second or third most common yeast species isolated from patients with vaginitis and invasive candidiasis. This study was aimed to determine the genetic diversity, antifungal susceptibility and enzymatic profiles of C. glabrata isolated from vaginal and blood samples in the Medical Microbiology Diagnostic Laboratory, University Malaya Medical Centre. A random amplified polymorphic DNA (RAPD) analysis method, using M13 and (GTG)5 primers, was used for strain differentiation of C. glabrata isolates. Antifungal susceptibility testing of C. glabrata isolates was determined using E-test against amphotericin B, caspofungin, fluconazole and voriconazole and microbroth dilution method against clotrimazole. The enzymic profiles of C. glabrata were determined using APIZYM semi-quantitation kit and egg-yolk agar method. A total of 14 RAPD patterns were identified amongst C. glabrata isolates investigated this study. Susceptibility to amphotericin B, caspofungin, fluconazole and voriconazole was noted. Approximately one third of the isolates demonstrated resistance to clotrimazole (MIC>=1 µg/ml). A single isolate of C. glabrata was resistant to caspofungin (MIC:1.5 µg/ml). Enzymatic activities of acid and alkaline phosphatase, aminopeptidases, esterase and lipase and phospholipase were detected in the C. glabrata isolates. The genetic diversity and antifungal susceptibility profiles of C. glabrata isolates were presented in this study. Continued surveillance and monitoring of the incidence and antifungal resistance in C. glabrata isolates is necessary.
Purple urine bag syndrome (PUBS) is a rare presentation of urinary tract infections (UTIs). It is commonly seen in constipated patients. There is a deep purple discoloration of contents of urine bag due to presence of indigo and indirubin pigments which are metabolites of tryptophan. We would like to describe an interesting case of purple urine bag syndrome of 88-year-old woman who presented with catheter-related urinary tract infection. She had low-grade fever and suprapubic discomfort for three days duration. She had increased white cell count and C-reactive peptide (CRP). Urinalysis showed protein 2+, nitrite and leucocyte esterase positive. Urine culture grew Escherichia coli and Klebsiella pneumoniae. She was treated with oral cefuroxime and recovered. This case report may be the first case of PUBS reported in this region.
Recurring reports of a highly allergenic 42-46-kDa protein in Hevea brasiliensis latex appeared to have been resolved with the discovery of a 43-kDa allergenic latex protein that was a homologue to patatin. However, the low to moderate prevalence of sensitization to the protein, designated Hev b 7, among latex-allergic patients could not adequately explain the frequent observations of the 42-46-kDa allergen. This led to the hypothesis that another, more allergenic protein of a similar molecular mass existed in Hevea latex. We report the isolation and purification of a 42.98-kDa latex glycoprotein showing homology to the early nodule-specific protein (ENSP) of the legumes Medicago sativa, Medicago truncatula, and Glycine max. The protein is allergenic, being recognized by immunoglobulin E (IgE) in sera from latex-allergic patients. The IgE epitope resides on the carbohydrate moiety of the protein, and the presence of a similar carbohydrate component on potato tuber patatin enables the latter to inhibit IgE binding to the ENSP homologue. The cDNA encoding the ENSP homologue was isolated by reverse transcription-PCR and cloned. The protein predicted from the cDNA sequence has 391 amino acids, the first 26 of which constitute a putative signal peptide. The deduced molecular mass of the mature protein is 40.40 kDa, while its isoelectric point is estimated at 5.0. The discrepancy between the predicted and observed molecular mass might be due to glycosylation, for which three N-sites on the protein are predicted. The purified protein showed lipase and esterase activities and may be involved in plant defense.