Phytophthora palmivora has caused disease in many crops including oil palm in the South America region. The pathogen has had a significant economic impact on oil palm cultivation in Colombia, and therefore poses a threat to oil palm cultivation in other regions of the World, especially in Southeast Asia, the largest producer of the crop. This study aimed to look at the ability of isolates from Malaysia, Colombia, and other regions to cross-infect Malaysian oil palm, durian, and cocoa and to develop specific biomarkers and assays for identification, detection, and diagnosis of P. palmivora as a key component for the oil palm biosecurity continuum in order to contain the disease especially at the ports of entry. We have developed specific molecular biomarkers to identify and detect Phytophthora palmivora using polymerase chain reaction (PCR) and real-time loop mediated isothermal amplification (rt-LAMP) in various sample types such as soil and plants. The limit of detection (DNA template, pure culture assay) for the PCR assay is 5.94 × 10-2 ng µl-1 and for rt-LAMP is 9.28 × 10-4 ng µl-1. Diagnosis using rt-LAMP can be achieved within 30 min of incubation. In addition, PCR primer pair AV3F/AV3R developed successfully distinguished the Colombian and Malaysian P. palmivora isolates.
Plants have been a good source of therapeutic agents for thousands of years;
an impressive number of modern drugs used for treating human diseases are derived from
natural sources. The Theobroma cacao tree, or cocoa, has recently garnered increasing
attention and become the subject of research due to its antioxidant properties, which are
related to potential anti-cancer effects. In the past few years, identifying and developing
active compounds or extracts from the cocoa bean that might exert anti-cancer effects
have become an important area of health- and biomedicine-related research. This review
provides an updated overview of T. cacao in terms of its potential anti-cancer compounds
and their extraction, in vitro bioassay, purification, and identification. This article also
discusses the advantages and disadvantages of the techniques described and reviews the
processes for future perspectives of analytical methods from the viewpoint of anti-cancer
compound discovery.
Gastrointestinal nematode infections can cause great losses in revenue due to decrease livestock production and animal death. The use of anthelmintic to control gastrointestinal nematode put a selection pressure on nematode populations which led to emergence of anthelmintic resistance. Because of that, this study was carried out to investigate the efficacy of aqueous and methanol extract of Cassia siamea against the motility of C. elegans Bristol N2 and C. elegans DA1316. Caenorhabditis elegans Bristol N2 is a susceptible strain and C. elegans DA1316 is an ivermectin resistant strain. In vitro bioassay of various concentrations of (0.2, 0.6, 0.8, 1.0 and 2.0 mg mL-1) aqueous and methanol extracts of C. siamea was conducted against the motility of L4 larvae of C. elegans Bristol N2 and C. elegans DA1316. The L4 larvae were treated with 0.02 μg mL-1 of ivermectin served as positive control while those in M9 solution served as negative control. The activity of the extracts was observed after 24 h and 48 h. A significant difference was recorded in the extract performance compared to control at (P < 0.001) after 48 h against the motility of the larvae of both strains. The methanol extracts inhibited the motility of C. elegans Bristol N2 by 86.7% as well as DA1316 up to 84.9% at 2.0 mg mL-1 after 48 h. The methanol extract was more efficient than aqueous extract (P < 0.05) against the motility of both strains of C. elegans. Cassia siamea may be used as a natural source of lead compounds for the development of alternative anthelmintic against parasitic nematodes as well ivermectin resistant strains of nematodes.
Residents in irrigated urban agricultural sites face numerous mosquito problems such as increased mosquito populations and reduced insecticides susceptibility due to the creation of mosquito breeding sites and agricultural use of insecticides and hence require effective protective products against them. In this study, the protection effectiveness of three pyrethroid formulated mosquito coils of Malaysian origin against Anopheles gambiae sensu lato from an irrigated urban agricultural site in Ghana were evaluated for their potential use. Sucrose fed An. gambiae s.l. were exposed to insecticide-containing coils in a 70 cm x 70 cm x 70 cm glass chamber to assess the insecticidal effect of the coils. The 0.005% metofluthrin coil caused the most rapid knockdown of 50% of the test mosquitoes. The mean lethal effect of the coils on An. gambiae s.l. were as follows; 0.005% metofluthrin (86%), 0.3% d-allethrin (74.33%), 0.15% d-trans allethrin (72%) and the 0.25% d-allethrin reference coil (69%). The 0.005% metofluthrin coil achieved the highest insecticidal effect on An. gambiae s.l. compared to the other coils and hence performed better than the others as an anti-mosquito product. All the three test coils were effective against An. gambaie s.l. from the irrigated agricultural site compared to the reference coil.
Space spraying of chemical insecticides is still an important mean of controlling Aedes mosquitoes and dengue transmission. For this purpose, the bioefficacy of space-sprayed chemical insecticide should be evaluated from time to time. A simulation field trial was conducted outdoor in an open field and indoor in unoccupied flat units in Kuala Lumpur, to evaluate the adulticidal and larvicidal effects of Sumithion L-40, a ULV formulation of fenitrothion. A thermal fogger with a discharge rate of 240 ml/min was used to disperse Sumithion L-40 at 3 different dosages (350 ml/ha, 500 ml/ha, 750 ml/ha) against lab-bred larvae and adult female Aedes aegypti and Aedes albopictus. An average of more than 80% adult mortality was achieved for outdoor space spray, and 100% adult mortality for indoor space spray, in all tested dosages. Outdoor larvicidal effect was noted up to 14 days and 7 days at a dosage of 500 and 750 ml/ha for Ae. aegypti and Ae. albopictus, respectively. Indoor larvicidal effect was up to 21 days (500 ml/ha) and 14 days (750 ml/ha), respectively, after spraying with larval mortality > 50% against Ae. aegypti. This study concluded that the effective dosage of Sumithion L-40 thermally applied against adult Ae. aegypti and Ae. albopictus indoor and outdoor is 500 and 750 ml/ha. Based on these dosages, effective indoor spray volume is 0.4 - 0.6 ml/m³. Additional indoor and outdoor larvicidal effect will be observed at these application dosages, in addition to adult mortality.
There has been a worldwide surge in the number and severity of dengue in the past decades. In Singapore, relentless vector control efforts have been put in to control the disease since the 1960's. Space spraying, fogging, chemical treatment and source reduction are some commonly used methodologies for controlling its vectors, particularly Aedes aegypti. Here, as we explored the use of a commercially available delthamethrin-treated net as an alternative strategy and the efficacy of the treated net was found to be limited. Through bioassays and molecular studies, the failure of the treated net to render high mortality rate was found to be associated with the knockdown resistance (kdr) mutation. This is the first report of kdr- mutations in Singapore's Ae. aegypti. At least one point mutation, either homozygous or heterozygous, at amino acid residue V1016G of DIIS6 or F1269C of DIIIS6 was detected in 93% of field strains of Ae. aegypti. Various permutations of wild type and mutant amino acids of the four alleles were found to result in varying degree of survival rate among local field Ae. aegypti when exposed to the deltamethrin treated net. Together with the association of higher survival rate with the presence of both V1016G and F1269C, the data suggest the role of these mutations in the resistance to the deltamethrin. The high prevalence of these mutations were confirmed in a country wide survey where 70% and 72% of the 201 Ae. aegypti analysed possessed the mutations at residues 1016 and 1269 respectively. The highest mutated frequency combination was found to be heterozygous alleles (VG/FC) at both residues 1016 and 1269 (37.8%), followed by homozygous mutation at allele 1269 (24.4%) and homozygous mutation at allele 1016 (22.9%). The kdr- type of resistance among the vector is likely to undermine the effectiveness of pyrethroids treated materials against these mosquitoes.
In this study, 13 weeks (October to December 2012) of ovitrap surveillance was conducted in two suburban residential areas in Kampar town, Perak. A total of 17,310 Aedes mosquitoes were found in Taman Kampar Jaya, whereas Taman Juloong recorded a higher number at 19,042. Less than 1% of these were identified as Aedes aegypti, with the remaining confirmed as Aedes albopictus. The female Ae. albopictus were subsequently subjected to WHO standard diagnostic test kits against two pyrethroids (0.05% deltamethrin and 0.75% permethrin) and two organophosphates (1% fenitrothion and 5% malathion). The Ae. albopictus from both research sites were the most susceptible to deltamethrin, recording KT50 and KT95 response values of 15.84 minutes and 16.18 minutes; and 48.18 minutes and 49.44 minutes respectively. This was followed by permethrin (20.57 minutes and 17.52 minutes; 29.54 minutes and 54.54 minutes) and malathion (48.46 minutes and 62.69 minutes; 87.72 minutes and 141.04 minutes). Fenitrothion was found to be least effective towards Ae. albopictus; recording KT50 and KT95 response values of 150.29 minutes and 293.41 minutes for Taman Kampar Jaya, and 203.32 minutes and 408.07 minutes respectively for Taman Juloong. All tested Ae. albopictus showed 100% mortality after 24 hours post exposure. As both residential areas were fogged periodically by the municipal council; alternating between organophosphates and pyrethroids, thus, constant monitoring is crucial in light of the emergence of resistance noted in Ae. albopictus towards fenitrothion.
Specification on residual action of a possible alternative insecticide derived from plant materials is important to determine minimum interval time between applications and the environmental persistence of the biopesticides. The objective of this study is to evaluate crude acethonilic extract of Ipomoea cairica leaves for its residual and persistence effects against Culex quinquefasciatus larvae. Wild strain of Cx. quinquefasciatus larvae were used for the purpose of the study. Two test designs, replenishment of water and without replenishment of water were carried out. For the first design, a total of 10 ml of test solution containing Ip. cairica extracts was replenished daily and replaced with 10 ml of distilled water. For the second design, treatment water was maintained at 1500 ml and only evaporated water was refilled. Larval mortality was recorded at 24 hours post-treatment after each introduction period and trials were terminated when mortality rate falls below 50%. Adult emergences from survived larvae were observed and number of survivals was recorded. For the non-replenishment design, mortality rate significantly reduced to below 50% after 28 days, meanwhile for replenishment of water declined significantly after 21 days (P < 0.05). There was no adult emergence observed up to seven days for non-replenishment and first two days for replenishment of water design. The short period of residual effectiveness of crude acethonilic extract of Ip. cairica leaves with high percentage of larval mortality on the first few days, endorses fewer concerns of having excess residues in the environment which may carry the risk of insecticide resistance and environmental pollution.
Bioprospecting of plant-based insecticides for vector control has become an area of interest within the last two decades. Due to drawbacks of chemical insecticides, phytochemicals of plant origin with mosquito control potential are being utilized as alternative sources in integrated vector control. In this regard, the present study aimed to investigate oviposition deterring and oviciding potentials of Ipomoea cairica (L.) (Family: Convolvulaceae) crude leaf extract against dengue vectors, Aedes aegypti and Aedes albopictus. Ipomoea cairica is an indigenous plant that has demonstrated marked toxicity towards larvae of Ae. aegypti and Ae. albopictus. Leaves of I. cairica were extracted using Soxhlet apparatus with acetone as solvent. Oviposition deterrent activity and ovicidal assay was carried out in oviposition site choice tests with three different concentrations (50, 100, 450 ppm). Acetone extract of I. cairica leaf strongly inhibited oviposition with 100% repellence to Ae. aegypti at lower concentration of 100 ppm, while for Ae. albopictus was at 450 ppm. The oviposition activity index (OAI) values which ranged from -0.69 to -1.00 revealed that I. cairica demonstrated deterrent effect. In ovicidal assay, similar trend was observed whereby zero hatchability was recorded for Ae. aegypti and Ae. albopictus eggs at 100 and 450 ppm, respectively. It is noteworthy that I. cairica leaf extract had significantly elicited dual properties as oviposition deterrent and oviciding agent in both Aedes species. Reduction in egg number through oviposition deterring activity, reduction in hatching percentage and survival rates, suggested an additional hallmark of this plant to be integrated in Aedes mosquito control. Ipomoea cairica deserved to be considered as one of the potential alternative sources for the new development of novel plant based insecticides in future.
Larvae of Aedes albopictus obtained from dengue endemic areas in Selangor, Malaysia were evaluated for their susceptibility to operational dosage of temephos (1 mg/L). Larval bioassays were carried out in accordance to modified WHO standard methods. Biochemical microassay of enzymes in Ae. albopictus was conducted to detect the emergence of insecticide resistance and to define the mechanisms involved in temephos resistance. The 50% mortality lethal time (LT50) for Ae. albopictus tested against temephos ranged between 58.65 to 112.50 minutes, with resistance ratio ranging from 0.75 - 1.45. This study addressed the fluctuation of time-related susceptibility status of Ae. albopictus towards insecticide. Significant difference on the weekly enzyme levels of non-specific esterases, mixed function oxidases and glutathione S-transferases was detected (p ≤ 0.05). No significant correlation was found between temephos resistance and enzyme activity (p > 0.05). Only glutathione S-transferases displayed high level of activity, indicating that Ae. albopictus may be resistant to other groups of insecticide. The insensitive acetylcholinesterase was detected in some field collected Ae. albopictus populations, indicating the possibility of emergence of carbamate or other organophosphate resistance in the field populations. Continuous resistance monitoring should be conducted regularly to confirm the efficacy of insecticides for dengue control.
A 14-months survey was carried out to identify the species composition of Anopheles mosquitoes from Kampung Bongor, Grik, Perak. Adding to that, a preliminary one month mosquito population screening was done at Kampung Tepin, Serian, Sarawak. Consequently, the insecticide susceptibility status of a pyrethroid was tested against two selected species of Anopheles collected from these two locations in Malaysia. A total of 4,497 Anopheles from 11 species were identified from collections in Kampung Bongor, whereas 2,654 An. letifer were collected from Kampung Tepin. The An. maculatus of Kampung Bongor and An. letifer of Kampung Tepin were then selected and tested using WHO standard diagnostic test kits and impregnated papers with 0.75% permethrin. The response values of KT50 and KT95 for An. maculatus were recorded at 28.09 minutes and 62.98 minutes respectively. Anopheles letifer recorded much slower response values of KT50 and KT95, which was at 35.09 minutes and 73.03 minutes respectively. Both An. maculatus and An. letifer showed 100% mortality after 24 hours holding period. The results indicate that both species were still susceptible to the tested pyrethroid. For effective vector control and resistance management, accurate and periodic insecticide resistance monitoring should be undertaken especially in rural areas with agricultural usage of insecticides.
Bioassay test against malathion had been carried out with larval and adult stages of Aedes aegypti. The mosquitoes were under selection pressure against malathion for forty-five consecutive generations. The rate of resistance development was measured by LC(50) and LT(50) values. The larvae and adult females, after subjection to malathion selection for 45 generations, developed high resistance level to malathion, with resistance ratio of 52.7 and 3.24 folds, respectively over control mosquitoes. Cross-resistance towards the same and different groups of insecticides was determined using the F44 and F45 malathion-selected adult females. Insecticides tested were DDT (4.0%), permethrin (0.75%), propoxur (0.1%), fenitrothion (1%), λ-cyhalothrin (0.05%) and cyfluthrin (0.15%). Results indicated that the mosquitoes were highly resistant to DDT and fenitrothion, moderately resistant to propoxur, tolerant to permethrin and λ-cyhalothrin, and very low resistant to cyfluthrin.
Routine surveillance on resistant status of field mosquito populations is important to implement suitable strategies in order to prevent pest outbreaks. WHO test kit bioassay is the most frequent bioassay used to investigate the susceptibility status of field-collected mosquitoes, as it is relatively convenient to be carried out in the field. In contrast, the topical application of active ingredient is less popular in investigating the susceptibility status of mosquitoes. In this study, we accessed the susceptibility status of Aedes albopictus Skuse collected from two dengue hotspots on Penang Island: Sungai Dua and Persiaran Mayang Pasir. Two active ingredients: permethrin and deltamethrin, were used. WHO test kit bioassay showed that both wild strains collected were susceptible to the two active ingredients; while topical application assay showed that they were resistant. This indicated that WHO test kit bioassay less sensitive to low level of resistance compared to topical application assay. Hence, topical application is expected to be more indicative when used in a resistance surveillance programme.
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.
House flies were collected from April 2007-April 2008 from two poultry farms (Balik Pulau and Juru) in the state of Penang. The resistance level of the first generation offspring was evaluated against DDT, malathion, propoxur, and permethrin using the topical application method. The resistance ratio (RR) of the Balik Pulau strain house flies for propoxur, malathion and DDT ranged from 10.28 to 99.00, 7.83 to 47.01 and 6.05 to 31.10, respectively. Resistance to propoxur and malathion in house fly was attributed to cross resistance to organophosphate insecticides used in the farm. Increased metabolic detoxification might be the mechanism involved in DDT resistance due to excessive application of cypermethrin formulation. The RR of the Juru strain for propoxur, malathion and DDT was in a decreasing pattern throughout the study period, ranging from 5.58 to 83.38, 15.19 to 27.82, and 10.04 to 22.69, respectively. Permethrin appeared to be the most potent insecticide in controlling house fly in both the Balik Pulau (RR=0.50 to 1.96) and Juru poultry farms (RR=0.64 to 2.40). The fluctuations of insecticides resistance in house fly was also found to correlate with climatic factors due to its rapid breeding. Relative humidity exhibited positive correlation indices with the changes in the resistance level for DDT (r=0.481, p<0.05), malathion (r=0.698, p<0.01), and permethrin (r=0.580, p<0.05) in Balik Pulau. Similarly, relative humidity in Juru also showed strong correlation with the RR for DDT (r=0.900, p<0.01), malathion (r=0.762, p<0.05), permethrin (r=0.760, p<0.05), and propoxur (r=0.897, p<0.01).
Vegetative proteins from Malaysian strains of Bacillus thuringiensis israelensis strains (Bt 11, Bt 12, Bt 15, Bt 16, Bt 17, Bt 21 and Bt 22) and Bacillus sphaericus H-25 strains (Bs 1 and Bs 2) were screened for haemolytic, cytotoxic and larvicidal activity. SDS-PAGE profiles of the Bacillus thuringiensis strains studied consistently showed major bands of 33-37 kDa and 47 kDa. Bt 16 also showed two bands of 66 kDa and 45 kDa similar to the previously reported binary vegetative protein, Vip1Ac (66 kDa) and Vip 2Ac (45 kDa). Both the Bacillus sphaericus strains showed a 35 kDa band that was similiar to a previously reported vegetative protein, the Mtx2 protein. Bs 2 also contains a 37 kDa band, similar to another vegetative protein, the Mtx 3 protein. With the exception of Bt 17 and Bt 21, vegetative proteins from all Bacillus thuringiensis and Bacillus sphaericus strains were highly haemolytic to human erythrocytes, causing more than 75% haemolysis at the highest concentration of 200 microg/ml. High haemolytic activity was associated with high cytotoxic activity with most of the haemolytic strains being indiscriminately cytotoxic to both CEM-SS (human T lymphoblastoid) and HeLa (human uterus cervical cancer) cell lines. Interestingly, the less haemolytic vegetative proteins from Bt 17 and Bt 21 demonstrated cytotoxic activity comparable to that of the highly haemolytic vegetative proteins. Bt 21 displayed toxicity towards both cell lines while Bt 17 was more toxic towards CEM-SS cells. Bioassay against Aedes aegypti and Culex quinquefasciatus larvae revealed that vegetative proteins from the Bacillus thuringiensis strains had activity against both species of larvae but vegetative proteins from Bacillus sphaericus were weakly larvicidal towards Cx. quinquefasciatus only.
Larvae obtained from Taman Samudera (Gombak, Selangor), Kampung Banjar (Gombak, Selangor), Taman Lembah Maju (Cheras, Kuala Lumpur) and Kampung Baru (City centre, Kuala Lumpur) were bioassayed with diagnostic dosage (0.012 mg/L) and operational dosage (1 mg/L) of temephos. All strains of Aedes aegypti and Aedes albopictus showed percentage mortality in the range of 16.00 to 59.05 and 6.4 to 59.50 respectively, after 24 hours. LT50 values for the 6 strains of Ae. aegypti and Ae. albopictus were between 41.25 to 54.42 minutes and 52.67 to 141.76 minutes respectively, and the resistance ratio for both Aedes species were in the range of 0.68 to 1.82 when tested with operational dosage, 1 mg/L temephos. These results indicate that Aedes mosquitoes have developed some degree of resistance. However, complete mortality for all strains were achieved after 24 hours when tested against 1 mg/L temephos.
Larvae of Aedes aegypti and Aedes albopictus obtained from 6 consecutive ovitrap surveillance (OS) in Taman Samudera and Kg. Banjar were evaluated for their susceptibility to temephos. Larval bioassays were carried out in accordance with WHO standard methods, with diagnostic dosage (0.012 mg/L) and operational dosage (1 mg/L) of temephos respectively. Aedes aegypti and Ae. albopictus obtained from six OS in Taman Samudera showed resistance to diagnostic dosage of temephos with percentage mortality between 5.3 to 72.0 and 9.3 to 56.0, respectively, while Ae. aegypti and Ae. albopictus obtained from Kg. Banjar showed resistance to temephos with percentage mortality between 16.0 to 72.0 and 0 to 50.6, respectively. Only two strains of Ae. aegypti from Kg. Banjar were susceptible to temephos with 93.3% (OS 2) and 100% (OS 3) mortality. The 50% mortality at lethal time (LT50) for all strains of Ae. aegypti and Ae. albopictus tested against operational dosage of temephos showed range between 36.07 to 75.69 minutes and 58.65 to 112.50 minutes, respectively, and complete mortality was achieved after 24 hours. Our results indicated that there is weekly variations of the resistance status for Ae. aegypti and Ae. albopictus. Aedes susceptibility to temephos is changing from time to time in these two study sites. It is essential to continue monitoring the resistance of this vector to insecticides in order to ensure the efficiency of program aimed at vector control and protection of human health.
The bioefficacy of a commercial formulation of temephos, Creek against Aedes aegypti larvae was studied in the laboratory. Earthen jars were filled with 10 L tap water each. One g of temephos (Creek) sand granule formulation was added into each earthen jar as recommended by the manufacturer. The final test concentration of Creek was 1 mg a.i./L. One earthen jar was filled with 10 L tap water and served as a test control (untreated). Thirty late 3(rd) or early 4(th) instar of lab-bred Ae. aegypti larvae were added into each earthen jar. Mortality of the larvae was recorded after 24 hours and percent mortality was calculated. Test was repeated every week. The results showed that complete larval mortality was achieved after 24 hours. The residual effect lasted 15 weeks (105 days), indicating that Creek is effective at the dosage recommended by the manufacturer which is 1 mg a.i./L.
The adulticidal activity of methanol extracts from three Malaysian plants namely Acorus calamus Linn., Litsea elliptica Blume and Piper aduncum Linn. against adult of Aedes aegypti (L.) were studied. Standard WHO bioassay tests were used to evaluate the effectiveness of these plant extracts. The hexane fraction from methanol extract of Acorus calamus rhizome was the most effective, exhibiting LC50 and LC90 values of 0.04 mgcm(-2) and 0.09 mgcm(-2) respectively. For L. elliptica, the methanol fraction also displayed good adulticidal property with the LC50 and LC90 values of 0.11 mgcm(-2) and 6.08 mgcm(-2) respectively. It is found that hexane fraction of the P. aduncum crude extract was the least effective among the three plants showing LC50 and LC90 values of 0.20 mgcm(-2) and 5.32 mgcm(-2), respectively. However, although A. calamus showed lowest LC values, the LT50 results indicated that the methanol fraction of L. elliptica was most potent extract among the extracts tested.