To evaluate the growth inhibition activity of the crude extract of Cyperus aromaticus (C. aromaticus) cultured cells against the 3rd instar larvae of Aedes aegypti (Linn.) and Aedes albopictus Skuse (Ae. albopictus) under laboratory conditions, and determine the sublethal effects (EI50) of the crude extract of C. aromaticus cultured cells on some biological and morphological parameters of both Aedes mosquito species during two generations as well.
Buprofezin is an insect growth regulator and widely used insecticide in Malaysia. The present study evaluated the toxic effects of buprofezin on the embryo and larvae of African catfish (Clarias gariepinus) as a model organism. The embryos and larvae were exposed to 7 different concentrations (0, 0.05, 0.5, 5, 25, 50 and 100 mg/L) of buprofezin. Each concentration was assessed in five replicates. Eggs were artificially fertilized and 200 eggs and larvae were subjected to a static bath treatment for all the concentrations. The mortality of embryos was significantly increased with increasing buprofezin concentrations from 5 to 100 mg/L (p< 0.05). However, the mortality was not significantly different (p<0.05) among the following concentrations: 0 (control), 0.05, 0.5 and 5 mg/L. Data obtained from the buprofezin acute toxicity tests were evaluated using probit analysis. The 24 h LC50 value (with 95% confidence limits) of buprofezin for embryos was estimated to be 6.725 (3.167-15.017) mg/L. The hatching of fish embryos was recorded as 68.8, 68.9, 66.9, 66.4, 26.9, 25.1 and 0.12% in response to 7 different concentrations of buprofezin, respectively. The mortality rate of larvae significantly (p<0.05) increased with increasing buprofezin concentrations exposed to 24-48 h. The 24 and 48 h LC50 values (with 95% confidence limits) of buprofezin for the larvae was estimated to be 5.702 (3.198-8.898) and 4.642 (3.264-6.287) mg/L respectively. There were no significant differences (p>0.05) in the LC50 values obtained at 24 and 48 h exposure times. Malformations were observed when the embryos and larvae exposed to more than 5 mg/L. The results emerged from the study suggest that even the low concentration (5 mg/L) of buprofezin in the aquatic environment may have adverse effect on the early embryonic and larval development of African catfish.
Two insular settlements (Kampung Pulau Ketam and Kampung Sungai Lima) were selected to study the population dynamics of Aedes aegypti and Aedes albopictus mosquitoes, vectors of dengue and chikungunya infections. Ovitrap surveillance was conducted between October 2007 and October 2008. There was an inverse negative association between ovitrap index and rainfall at the time of collection, probably because rainfall increased the number of available oviposition sites. Rainfall and ovitrap index were positively associates the 25th day after rainfall occurred. A minor, second peak was observed from the 38th to the 42nd day. The first peak was consistent with the minimum 18-day period between the hatching of eggs to the first oviposition. The second minor peak could be due to the second gonotrophic cycle of the female mosquitoes. Rainfall is an important environmental factor associated with Aedes breeding at the study sites.
The emergence of multidrug-resistant bacterial strains has prompted the reintroduction of maggot therapy in the treatment of chronic, infected wounds. Many previous studies have demonstrated the potent antibacterial activity of larval excretions/secretions of the blowfly Lucilia sericata (Meigen) (Diptera:Calliphoridae) against bacteria. However, the antibacterial activity of its sibling species, Lucilia cuprina (Wiedemann) (Diptera:Calliphoridae) against a wide range of pathogenic bacteria has never been determined. The aim of this study was to develop a new procedure to produce whole body extract of larvae of L. cuprina via methanol extraction as well as to demonstrate the in vitro antibacterial activity of this extract against seven selected wound pathogens (Staphylococcus aureus, methicillin-resistant S. aureus, S. epidermidis, Streptococcus pyogenes, Klebsiella pneumoniae, Pseudomonas aeruginosa and Escherichia coli). The turbidimetric assay demonstrated that L. cuprina larval extract was significantly potent against all bacteria tested (P
Plant derived insecticides have considerable potential for mosquito control because these products are safer than conventional insecticides. This study aimed to investigate sublethal activities of Ipomoea carica or railway creeper crude acethonilic extract against life history trait of dengue vectors, Aedes albopictus and Aedes aegypti. The late third instar larvae of Ae. albopictus and Ae. aegypti were exposed to a sublethal dose at LC50 and larvae that survived were further cultured. Overall, Ipomea cairica crude extracts affected the whole life history of both Aedes species. The study demonstrated significantly lower egg production (fecundity) and eggs hatchability (fertility) in Ae. albopictus. The sublethal dose of crude extracts reduced significantly the width of larval head capsule and the wing length of both sexes in both Aedes species. The significance of sublethal effects of I. cairica against Aedes mosquitoes was an additional hallmark to demonstrate further activity of this plant despite its direct toxicity to the larvae. The reduced reproductive capacity as well as morphological and physiological anomalies are some of the effects that make I. cairica a potential candidate to be used as a new plant-based insecticide to control dengue vectors.
Paraquat poisoning is commonly associated with suicide or homicide in Malaysia. In a case involving advanced body decomposition, pathological analysis regarding the cause of death may become difficult or almost impossible. Insects serve as common alternative matrix for poison detection in forensic analysis. Paraquat detection via secondary bioaccumulation in fly larvae tissue has never been reported. In this study, tissues from blowfly larvae collected from a rabbit carcass with paraquat poisoning were analyzed for secondary bioaccumulation. Larvae samples were collected and analyzed using liquid-liquid extraction. The detection was performed via reduction of quaternary ammonium presence in paraquat and analyzed using gas chromatography-mass spectrometry (GC-MS) with selected ion monitoring mode (SIM mode). GC-MS showed the elution of reduced paraquat was at retention time 12.8 min. Blowfly larvae tissue has proven useful as a secondary detector in paraquat-related deaths.
Animals use signals to coordinate a wide range of behaviors, from feeding offspring to predator avoidance. This poses an evolutionary problem, because individuals could potentially signal dishonestly to coerce others into behaving in ways that benefit the signaler. Theory suggests that honest signaling is favored when individuals share a common interest and signals carry reliable information. Here, we exploit the opportunities offered by bacterial signaling to test these predictions with an experimental evolution approach. We show that: (1) reduced relatedness leads to the relative breakdown of signaling, (2) signaling breaks down by the invasion of mutants that show both reduced signaling and reduced response to signal, (3) the genetic route to signaling breakdown is variable, and (4) the addition of artificial signal, to interfere with signal information, also leads to reduced signaling. Our results provide clear support for signaling theory, but we did not find evidence for previously predicted coercion at intermediate relatedness, suggesting that mechanistic details can alter the qualitative nature of specific predictions. Furthermore, populations evolved under low relatedness caused less mortality to insect hosts, showing how signal evolution in bacterial pathogens can drive the evolution of virulence in the opposite direction to that often predicted by theory.
The ever-increasing threat from infectious diseases and the development of insecticide resistance in mosquito populations drive the global search for new natural insecticides. The aims of this study were to evaluate the mosquitocidal activity of the extracts of seaweed Bryopsis pennata against dengue vectors Aedes aegypti and Aedes albopictus, and determine the seaweed's toxic effect on brine shrimp nauplii (as a non-target organism). In addition, the chemical compositions of the active larvicidal extract and fraction were analyzed by using liquid chromatography-mass spectrometry (LC-MS). Chloroform extract exhibited strong ovicidal activity (with LC50 values of 229.3 and 250.5 µg/mL) and larvicidal activity against Ae. aegypti and Ae. albopictus. The larvicidal potential of chloroform extract was further ascertained when its A7 fraction exhibited strong toxic effect against Ae. aegypti (LC50 = 4.7 µg/mL) and Ae. albopictus (LC50 = 5.3 µg/mL). LC-MS analysis of the chloroform extract gave a tentative identification of 13 compounds; Bis-(3-oxoundecyl) tetrasulfide was identified as the major compound in A7 fraction. Methanol extract showed strong repellent effect against female oviposition, along with weak adulticidal activity against mosquito and weak toxicity against brine shrimp nauplii. The mosquitocidal results of B. pennata suggest further investigation for the development of effective insecticide.
Since ancient times, honey has been used for medical purposes and the treatment of various disorders. As a high-quality food product, the honey industry is prone to fraud and adulteration. Moreover, limited experimental studies have investigated the impact of adulterated honey consumption using zebrafish as the animal model. The aims of this study were: (1) to calculate the lethal concentration (LC50) of acid-adulterated Apis mellifera honey on embryos, (2) to investigate the effect of pure and acid-adulterated A. mellifera honey on hatching rate (%) and heart rate of zebrafish (embryos and larvae), (3) to elucidate toxicology of selected adulterated honey based on lethal dose (LD50) using adult zebrafish and (4) to screen the metabolites profile of adulterated honey from blood serum of adult zebrafish. The result indicated the LC50 of 31.10 ± 1.63 (mg/ml) for pure A. mellifera honey, while acetic acid demonstrates the lowest LC50 (4.98 ± 0.06 mg/ml) among acid adulterants with the highest mortality rate at 96 hpf. The treatment of zebrafish embryos with adulterated A. mellifera honey significantly (p ≤ 0.05) increased the hatching rate (%) and decreased the heartbeat rate. Acute, prolong-acute, and sub-acute toxicology tests on adult zebrafish were conducted at a concentration of 7% w/w of acid adulterants. Furthermore, the blood serum metabolite profile of adulterated-honey-treated zebrafish was screened by LC-MS/MS analysis and three endogenous metabolites have been revealed: (1) Xanthotoxol or 8-Hydroxypsoralen, (2) 16-Oxoandrostenediol, and (3) 3,5-Dicaffeoyl-4-succinoylquinic acid. These results prove that employed honey adulterants cause mortality that contributes to higher toxicity. Moreover, this study introduces the zebrafish toxicity test as a new promising standard technique for the potential toxicity assessment of acid-adulterated honey in this study and hazardous food adulterants for future studies.
Metisa plana is a widespread insect pest infesting oil palm plantations in Malaysia. Farnesyl acetate (FA), a juvenile hormone analogue, has been reported to exert in vitro and in vivo insecticidal activity against other insect pests. However, the insecticidal mechanism of FA on M. plana remains unclear. Therefore, this study aims to elucidate responsive genes in M. plana in response to FA treatment. The RNA-sequencing reads of FA-treated M. plana were de novo-assembled with existing raw reads from non-treated third instar larvae, and 55,807 transcripts were functionally annotated to multiple protein databases. Several insecticide detoxification-related genes were differentially regulated among the 321 differentially expressed transcripts. Cytochrome P450 monooxygenase, carboxylesterase, and ATP-binding cassette protein were upregulated, while peptidoglycan recognition protein was downregulated. Innate immune response genes, such as glutathione S-transferases, acetylcholinesterase, and heat shock protein, were also identified in the transcriptome. The findings signify that changes occurred in the insect's receptor and signaling, metabolic detoxification of insecticides, and immune responses upon FA treatment on M. plana. This valuable information on FA toxicity may be used to formulate more effective biorational insecticides for better M. plana pest management strategies in oil palm plantations.
Acinetobacter baumannii, a top-priority WHO pathogen, causes life-threatening infections in immunocompromised patients, leading to prolonged hospitalisation and high mortality. Here, we used the Galleria mellonella model to investigate community strain C98 (Ab-C98) virulence via transcriptomic analysis. Ab-C98 showed greater killing and faster colonisation in larvae than the clinical reference strain (ATCC BAA1605). Genes in three iron clusters, acinetobactin, baumannoferrin and the Feo system, were significantly up-regulated. Targeted knockout of siderophore genes (basC, bfnD, and the gene encoding isochorismatase) significantly increased the survival of infected larvae by at least 35.16%, identifying these genes as potential targets for developing anti-virulence agents against A. baumannii.
Despite being a major human pathogen, limited studies have reported RNA modifications in Acinetobacter baumannii. These post-transcriptional modifications play crucial regulatory roles in bacteria and have also been shown to modulate bacterial virulence. Using nanopore sequencing, we characterized RNA modifications in a virulent A. baumannii strain (Ab-C98) under free-living (mid-exponential phase in vitro culture) and during an early stage of infection (3 h post-infection) in Galleria mellonella larvae. Analysis revealed that m5C methylations are essential for ribosome synthesis, while m6A and Ψ are involved in metabolic pathways and translation processes. Iron-chelating genes exbD (m5C and m6A) and feoB (m6A and Ψ) and RNA polymerase subunit rpoC (m6A and Ψ) were selectively modified during infection. This first transcriptome-wide study highlights the potential regulatory roles of m5C, m6A and Ψ modifications in A. baumannii during infection.
This study aims to improve the larvicidal activity of crude Chisocheton erythrocarpus extract (CCEE) against Aedes aegypti larvae via nano-emulsion formulation method. The fruits of C. erythrocarpus possesses limonoids with good larvicidal activity against Aedes mosquitoes. Using ternary phase diagrams, four selected formulations were prepared using non-ionic surfactants, methylester and water. The optimized nano-emulsion (S5) was discovered to possess submicron particle size (36.32 nm), low polydispersity index (0.348) and higher zeta potential value (-18.2 mV). After 48-hours, the CCEE and S5 demonstrated respective LC50 values of 139.44 ppm and 75.83 ppm against Ae. aegypti larvae. Higher larvicidal activity of S5 signified improved cuticular penetration of the active ingredient. Moreover, morphological deformities of treated larvae indicated possible interference with the larvae's breathing mechanism, contributing to their death. Thus, the nano-emulsion formulation of C. erythrocarpus extract has the potential to be used as a vector control agent to prevent the spread of dengue infections.
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.
Babesia bigemina is a tick-borne protozoan that affects cattle in almost all regions of the world. Despite its importance, there is no report of its prevalence in cattle using molecular detection methods in Peninsular Malaysia. This study describes the prevalence, distribution, and risk factors associated with B. bigemina infection using molecular diagnostic methods. Also, the species of ticks infesting cattle and the attitude of cattle farmers towards tick control in Peninsular Malaysia were studied. Blood samples were collected from 1045 cattle from 43 herds throughout the country, and were subjected to molecular studies to detect B. bigemina. Tick samples for entomological studies were also collected and identified. Epidemiological information of each cattle and farm were obtained using a well-structured questionnaire containing open-ended and closed-ended questions. Data were statistically analyzed using Univariate and Multivariate models. The 211-base pair of AMA-1 gene of B. bigemina was amplified and confirmed in 30.5 % (319/1045; 95 % CI = 27.8-33.4) of the sampled population, with the haemoprotozoan detected in all the sampled herds. Breed, age, physiological status, management type, rate of de-ticking, and closeness to human settlement were the risk factors significantly (p < 0.05) associated with the prevalence of B. bigemina in cattle. Rhipicephalus (Boophilus) microplus and Haemaphysalis bispinosa were the species of ticks collected from cattle, with the former been more prevalent. A large number of cattle farmers (12/43; 28 %) do not control ticks in their herds. The findings of this study will create baseline data on the epidemiology of the haemoprotozoan and control patterns of its tick vectors that will guide the government in enacting policies that will improve food security and the economy of the nation.
In this study, larvicidal activity of silver nanoparticles (AgNPs) synthesised using apple extract against fourth instar larvae of Aedes aegypti was determined. As a result, the AgNPs showed moderate larvicidal effects against Ae. aegypti larvae (LC50 = 15.76 ppm and LC90 = 27.7 ppm). In addition, comparison of larvicidal activity performance of AgNPs at high concentration prepared using two different methods showed that Ae. aegypti larvae was fully eliminated within the duration of 2.5 h. From X-ray diffraction, the AgNP crystallites were found to exhibit face centred cubic structure. The average size of these AgNPs as estimated by particle size distribution was in the range of 50-120 nm. The absorption maxima of the synthesised Ag showed characteristic Ag surface plasmon resonance peak. This green synthesis provides an economic, eco-friendly and clean synthesis route to Ag.
The effects of eight diets (atta flour, wheat flour, self-rising flour, rice flour, custard powder, corn flour, tapioca starch, and potato starch) on the development of the red flour beetle, Tribolium castaneum (Herbst), reared at 29-31 degrees C and 66-70% RH were assessed. Five pairs of male and female T. castaneum were reared on the respective diets for 28 d before the experimental setup was dismantled and adult counts were recorded. In another experiment, the insects were allowed to mate and oviposit in each flour or starch type over a period of 7 d before being removed. The counting of pupae and adult emergence began on the day of emergence and was continued on a daily basis until day 140. Proximate analysis was performed for chemical composition of each diet, and the numbers of new adults that developed were found to be positively correlated (r2 = 0.97; P < 0.05) with the protein content and negatively correlated (r2 = 0.93; P < 0.05) with the carbohydrate content. For T. castaneum, the suitable diets were ranked as follows: atta flour > wheat flour > self-rising flour > rice flour > custard powder > corn flour > tapioca starch > potato starch. T. castaneum larval development to the pupal and adult stages developed significantly faster in atta flour (P < 0.05) than in the other diets, and the greatest number of progeny was produced from beetles reared on atta flour. Fewer adults emerged from wheat flour, self-rising flour, and rice flour, and no new emergences were recorded for the remaining diets. Developmental rate was much slower in beetles reared on diets in which a low number in progeny was produced. These data illustrate that different diets can influence the sustainability of these insects and affect their development and growth.
Forensic entomology applies knowledge about insects associated with decedent in crime scene investigation. It is possible to calculate a minimum postmortem interval (PMI) by determining the age and species of the oldest blow fly larvae feeding on decedent. This study was conducted in Malaysia to identify maggot specimens collected during crime scene investigations. The usefulness of the molecular and morphological approach in species identifications was evaluated in 10 morphologically identified blow fly larvae sampled from 10 different crime scenes in Malaysia. The molecular identification method involved the sequencing of a total length of 2.2 kilo base pairs encompassing the 'barcode' fragments of the mitochondrial cytochrome oxidase I (COI), cytochrome oxidase II (COII) and t-RNA leucine genes. Phylogenetic analyses confirmed the presence of Chrysomya megacephala, Chrysomya rufifacies and Chrysomya nigripes. In addition, one unidentified blow fly species was found based on phylogenetic tree analysis.
Feeding of bacterially encapsulated heat shock proteins (Hsps) to invertebrates is a novel way to limit Vibrio infection. As an example, ingestion of Escherichia coli overproducing prokaryotic Hsps significantly improves survival of gnotobiotically cultured Artemia larvae upon challenge with pathogenic Vibrio campbellii. The relationship between Hsp accumulation and enhanced resistance to infection may involve DnaK, the prokaryotic equivalent to Hsp70, a major molecular chaperone in eukaryotic cells. In support of this proposal, heat-stressed bacterial strains LVS 2 (Bacillus sp.), LVS 3 (Aeromonas hydrophila), LVS 8 (Vibrio sp.), GR 8 (Cytophaga sp.), and GR 10 (Roseobacter sp.) were shown in this work to be more effective than nonheated bacteria in protecting gnotobiotic Artemia larvae against V. campbellii challenge. Immunoprobing of Western blots and quantification by enzyme-linked immunosorbent assay revealed that the amount of DnaK in bacteria and their ability to enhance larval resistance to infection by V. campbellii are correlated. Although the function of DnaK is uncertain, it may improve tolerance to V. campbellii via immune stimulation, a possibility of significance from a fundamental perspective and also because it could be applied in aquaculture, a major method of food production.