New synthesized bis-imidazolium salts that are linked by xylyl derivatives moiety, 1-4 was reacted with Ag2O to facilitate the formation of dinuclear Ag(I)-N-heterocyclic carbene (NHC) complexes, 5-8, respectively. All the synthesized ligand salts and complexes were characterized by1H and13C NMR, FTIR spectroscopy and elemental analysis. Molecular structures of compounds 3, 5, and 7 were elucidated by single crystal X-ray diffraction analyses. Larvicidal studies against the Aedes aegypti and Culex quinquefasciatus were carried out on all synthesized compounds following the World Health Organization standard larval susceptibility test. All the imidazolium salts were found inactive while the activity of the dinuclear Ag(I)-NHC complexes on mosquito larvae are varies with the nature of the ligands. Complex 7 has high activity on Ae. aegypti and Cx. quinquefasciatus, emphasising its potential as a larvicidal compound.
Four different tests showed the effectiveness of Azolla pinnata plant extracts against Aedes aegypti and Aedes albopictus mosquitoes. In the adulticidal test, there was a significant increase in mortality as test concentration increases and A. pinnata extracts showed LC50 and LC95 values of 2572.45 and 6100.74 ppm, respectively, against Ae. aegypti and LC50 and LC95 values of 2329.34 and 5315.86 ppm, respectively, against Ae. albopictus. The ovicidal test showed 100% eggs mortality for both species tested for all the concentrations tested at 1500 ppm, 1000 ppm, 500 ppm, 250 ppm and 125 ppm. Both tested samples of Ae. aegypti and Ae. albopictus did not lay any eggs in the plastic cups filled with the A. pinnata extract but instead opted to lay eggs in the plastic cups filled with water during the oviposition deterrence test. Similarly, the non-choice test of Ae. aegypti mosquitoes laid eggs on the sucrose solution meant for the nutrient source of the mosquitoes instead of in the plastic cup that was designed to facilitate oviposition filled with the extract. This clearly indicates the presence of bioactive compounds which are responsible in adulticidal and ovicidal activity in Aedes mosquitoes and at the same time inducing repellence towards the mosquitoes. The LC-MS results showed mainly three important chemical compounds from A. pinnata extracts such as 1-(O-alpha-D-glucopyranosyl)-(1,3R,25R)-hexacosanetriol, Pyridate and Nicotinamide N-oxide. All these chemicals have been used for various applications such as both emulsion and non-emulsion type of cosmetics, against mosquito vector such as Culex pipens and Anopheles spp. Finally, the overall view of these chemical components from A. pinnata extracts has shown the potential for developing natural product against dengue vectors.
The presence of predators can have dramatic consequences on prey communities, not only by the direct effects of consumption but also through sublethal effects. We investigated the survival rate and subsequent life history of the mosquito Culex pervigilans Bergroth under the influence of its major predator, the backswimmer Anisops wakefieldi White. We established a field experiment with various treatments: 1) control without predators, 2) free-roaming A. wakefieldi (with one, three, or nine A. wakefieldi per container), 3) caged A. wakefieldi (empty cage without predators, with one, three, or nine A. wakefieldi in each cage, and 4) A. wakefieldi cues (with cues concentrations of one, three, or nine A. wakefieldi). Cx. pervigilans eggs were then taken from these four experimental treatments and reared in two different laboratory conditions: 1) in clean water without any traces of predators, or 2) in water with the same treatments as in field. The survival rate of Cx. pervigilans was significantly reduced by the presence of predators or their cues. Even after a brief exposure to waters containing predators or residual cues, the subsequent progeny and the ontogeny of the remaining survivors were still affected. The percentage of eggs that hatched, and the resulting mosquito population, was influenced by the presence of predators or their cues. Our results suggest that sublethal effects may be carried by surviving individuals primarily through the effects of stress, perhaps by epigenetic mechanisms. We may expect to observe similar plasticity in species or populations with high temporal or spatial variability in predation.
Japanese encephalitis (JE) virus activity is an important cause of viral encephalitis in Southeast Asia. In Malaysia, JEV activity has been first detected in Culex gelidus in 1976. Since then, no study has fully addressed the seasonal dynamics of this mosquito. As irrigated rice production expands, the incidence of JEV vectors, particularly Cx. gelidus is expected to increase. We surveyed Penang Island to determine the breeding patterns of Cx. gelidus and their potential insect predators, in relation to habitat/niche and rice growing period. Six rice fields proper (RFP) and related drainage canals (DC) were visited through three cultivation cycles (CCs) over 17 months. Weekly visits were performed to each of the 36 sites and mosquito larvae and aquatic insects were sampled from RFP and DCs using dippers. Culex gelidus was abundant in RFP and almost absent in DCs. Its densities usually were high during the first and 3rd CC and when the RFs were in Fp, Pp and Gp. In DCs, the mosquito was abundant during Mp, e.g., 2nd CC. Predators, especially those belonging to the families Corixidae, Coenagrionidae and Dytiscidae, were more present in RFP. Predator numbers usually were high during the first CC; in some cases predator abundance peaked during other CCs, e.g., corixids and dysticids. In RFP, neither corixids nor coenagrionids showed any positive correlation with densities of Cx. gelidus. However, dytiscids' population peaked when the mosquito densities were on the rise. These observations suggest that Cx. gelidus is active during the period of rice cultivation. Operational vector control through bio-control or with insecticides near the end of the rice cultivation season in RFP may prove beneficial in reducing the density of Cx. gelidus, but also the amount of bio-agent or insecticide applied on riceland.
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 standard laboratory strain was found to be heterozygous for susceptibility. Hence, an attempt was made to obtain a homozygous susceptible strain in Culex quinquefasciatus (Say) using single raft sib-selection method. Lab-bred females of Cx. quinquefasciatus from insectariums, Unit of Medical Entomology were used in the experiment. After blood feeding Cx. quinquefasciatus mosquitoes laid eggs in raft form, ten rafts selected randomly for the test. Each egg raft was introduced into a plastic tray from number one to number ten. Twenty-five third stage larvae from each tray were exposed to 17.5 microl from 500mg/l malathion in a paper cup label number 1 to number ten. In the bioassay, which had 100% mortality, the respective larva in that particular tray was bred to adult stage for the following generation. Less than 7days old female mosquitoes that emerged from F(0) were used in the test. The F(0) and the subsequent adult and larval stage generations were subjected to adult and larval bioassay. After selection for about 10 generations, a homozygous susceptible strain in Cx. quinquefasciatus was obtained.
To determine resistance level and characterize malathion and permethrin resistance in Culex quinquefasciatus, two methods were used namely: WHO procedures of larval bioassay to determine the susceptibility of lethal concentration (LC) and adult bioassay to determine the lethal time (LT) which are resistant to malathion and permethrin. These mosquito strains were bred in the Insectarium, Division of Medical Entomology, IMR. Thousands of late fourth instar larvae which survived the selection pressure to yield 50% mortality of malathion and permethrin were reared and colonies were established from adults that emerged. Larvae from these colonies were then subjected to the subsequent 10 generations in the test undertaken for malathion resistant strain (F61 - F70) and permethrin resistant strain (F54 - F63). Selection pressure at 50% - 70% mortality level was applied to the larvae of each successive generation. The rate of resistance development and resistance ratio (RR) were calculated by LC5 0 for larval bioassay and LT50 value for adult bioassay. The lab bred Cx. quinquefasciatus was used as a susceptible strain for comparison purpose. The adult bioassay test was carried out by using diagnostic dosages of malathion 5.0%, permethrin 0.75% and with propoxur 0.1%. All bioassay results were subjected to probit analysis. The results showed that LC5 0 for both malathion (F61 - F70) and permethrin (F54 - F63) resistant Cx. quinquefasciatus increased steadily to the subsequent 10 generations indicating a marked development of resistance. The adult female malathion resistant strain have developed high resistance level to malathion diagnostic dosage with resistance ratio 9.3 to 9.6 folds of resistance. Permethrin resistance ratio remained as 1.0 folds of resistance at every generation. It was obvious that malathion resistance developing at a higher rate in adult females compared to permethrin. Female adults exposed to 2 hours of exposure period for propoxur 0.1% showed presence of cross-resistance among the both strains of mosquitoes towards propoxur and it was indicated by 70%-100% mortality at 24 hours post-recovery period.
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.
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.