Detection and isolation of Japanese encephalitis (JE) virus were attempted from female mosquitoes collected in Kampong Pasir Panjang, Sabak Bernam, Selangor, from May to November 1992. A total of 7,400 mosquitoes consisting of 12 species in 148 pools were processed and inoculated into Aedes albopictus clone C6/36 cell cultures. Of these, 26 pools showed the presence of viral antigens in the infected C6/36 cells by specific immunoperoxidase staining using an anti-JE virus polyclonal antibody. Presence of JE virus genome was confirmed in the infected culture fluid for 16 pools by using reverse transcriptase-polymerase chain reaction and JE virus-specific primers. Of these, 3 pools were from Culex tritaeniorhynchus, 4 from Culex vishnui, 3 from Culex bitaeniorhynchus, 2 from Culex sitiens, one from Aedes species, and 3 from Culex species. Isolation of JE virus from Cx. sitiens, Cx. bitaeniorhynchus, and Aedes sp. (Aedes butleri and Ae. albopictus) is reported for the first time in Malaysia.
Isolation of Japanese encephalitis virus (JEV) from mosquitoes in Sabak Bernam, Selangor, Malaysia, was attempted. An aliquot of homogenate from each pool of mosquitoes, 50 per tube, was inoculated into Aedes albopictus clone C6/36 cells for virus isolation. Each cell culture was tested for the presence of viral antigen by immunoperoxidase staining using an anti-JEV polyclonal antibody. Out of 4 Culex sitiens mosquito pools, 2 pools were positive for JEV by cell culture. Presence of JEV genome in the cell cultures for Cx. sitiens was confirmed by using reverse transcriptase-polymerase chain reaction and JEV-specific primers. This is the first report on the isolation of JEV from Cx. sitiens.
The control of filariasis vectors has been enhanced in several areas, but there are main challenges, including increasing resistance to insecticides and lack of cheap and eco-friendly products. The toxicity of iron (Fe0) and iron oxide (Fe2O3) nanoparticles has been scarcely investigated yet. We studied the larvicidal and pupicidal activity of Fe0 and Fe2O3 nanoparticles against Culex quinquefasciatus. Fe0 and Fe2O3 nanoparticles produced by green (using a Ficus natalensis aqueous extract) and chemical nanosynthesis, respectively, were analyzed by UV-Vis spectrophotometry, FT-IR spectroscopy, XRD analysis, SEM, and EDX assays. In larvicidal and pupicidal experiments on Cx. quinquefasciatus, LC50 of Fe0 nanoparticles ranged from 20.9 (I instar larvae) to 43.7 ppm (pupae) and from 4.5 (I) to 22.1 ppm (pupae) for Fe2O3 nanoparticles synthesized chemically. Furthermore, the predation efficiency of the guppy fish, Poecilia reticulata, after a single treatment with sub-lethal doses of Fe0 and Fe2O3 nanoparticles was magnified. Overall, this work provides new insights about the toxicity of Fe0 and Fe2O3 nanoparticles against mosquito vectors; we suggested that green and chemical fabricated nano-iron may be considered to develop novel and effective pesticides.
Laboratory-bred females of Culex quinquefasciatus, Aedes aegypti and Aedes albopictus from the insectarium, Unit of Medical Entomology, Institute for Medical Research were used in the experiment. The late third stage of the F0 larvae which survived the high selection pressure of malathion, permethrin and temephos were reared and colonies were established from adults that emerged. Cx. quinquefasciatus larvae were subjected to selection by malathion and permethrin for 40 generations, Ae. aegypti larvae to malathion, permethrin and temephos for 32 generations and Ae. albopictus larvae were selected against malathion and permethrin for 32 generations and 20 generations against temephos. The rate of resistance development was measured by LC50 value. Cx. quinquefasciatus larvae developed higher resistance to malathion and permethrin compared to Ae. aegypti and Ae. albopictus. On the whole, permethrin resistance developed at a faster rate than malathion and temephos.
The development of new alternatives strategies to synthetic insecticides aimed at reducing pest populations by developing pesticides based on plant extracts without negative effects in non target organisms and environment. The present study was undertaken in order to assess the insecticidal activity of the crude methanolic extract of the Algerian Asteraceae Cotula cinerea, against the larval and the pupal stage of Culex pipiens (Diptera: Culicidae). It is also to determine the chemical composition of the used extract, and to understand the mechanism of toxic action of the tested extract. Based on the preliminary tests, five concentrations of the crude methanolic extract of C. cinerea (0.62, 1.25, 2.50, 3.75, and 5 mg/mL) were tested for their insecticidal activity according to the protocol recommended by the World Health Organization. The chemical profile of the extract was also obtained by high performance liquid chromatography (HPLC). Histopathological effects and inhibition of acetylcholinesterase activity in treated mosquitoes with LC90 were examined to elucidate the mechanism of the toxic effect of the tested extract (48 h post treatment). Eight compounds have been identified by HPLC. That includes four flavonoids (rutin, quercetin, myrcetin and cathechin), three phenolic acids (benzoic acid, vanillic acid, p-coumaric acid) and one alkaloid (berberine). C. cinerea methanolic extract showed good larvicidal and pupicidal activities with LC50 and LC90 values of 1.10 and 4.37 mg/mL respectively against pupae, 24h post treatment and 1.26, 2.35 mg/mL respectively against the fourth instar larvae. Data of enzymatic assay performed on LC50 and LC90 pupae and larvae revealed prominent neurotoxic effects. C. cinerea extract reduced the activity of acetylcholinesterase (AChE) enzyme in a concentration dependent manner. Obtained inhibition percentages, 48 h after treatment, were 35.11 ± 7.44 and 51.83 ± 4.04% for pupal stage and 30.98 ± 2.97 % and 48.77 ± 4.72% for the fourth instar larvae for LC50 and LC90 values respectively. Treated larvae and pupae showed also histopathological damages in the pupal cuticle and larval midgut. The results of this study showed that C. cinerea crude methanolic extract could be considered as an eco-friendly alternative for mosquito control.
A potentially novel actinobacterium isolated from forest soil, Streptomyces sp. KSF103 was evaluated for its insecticidal effect against several mosquito species namely Aedes aegypti, Aedes albopictus, Anopheles cracens and Culex quinquefasciatus. Mosquito larvae and adults were exposed to various concentrations of the ethyl acetate (EA) extract for 24 h. Considerable mortality was evident after the EA extract treatment for all four important vector mosquitoes. Larvicidal activity of the EA extract resulted in LC50 at 0.045 mg/mL and LC90 at 0.080 mg/mL for Ae. aegypti; LC50 at 0.060 mg/mL and LC90 at 0.247 mg/mL for Ae. albopictus; LC50 at 2.141 mg/mL and LC90 at 6.345 mg/mL for An. cracens; and LC50 at 0.272 mg/mL and LC90 at 0.980 mg/mL for Cx. quinquefasciatus. In adulticidal tests, the EA extract was the most toxic to Ae. albopictus adults (LD50 = 2.445 mg/mL; LD90 = 20.004 mg/mL), followed by An. cracens (LD50 = 5.121 mg/mL; LD90 = 147.854 mg/mL) and then Ae. aegypti (LD50 = 28.873 mg/mL; LD90 = 274.823 mg/mL). Additionally, the EA extract exhibited ovicidal activity against Ae. aegypti (LC50 = 0.715 mg/mL; LC90 = 6.956 mg/mL), Ae. albopictus (LC50 = 0.715 mg/mL; LC90 = 6.956 mg/mL), and An. cracens (LC50 = 0.715 mg/mL; LC90 = 6.956 mg/mL), evaluated up to 168 h post-treatment. It displayed no toxicity on the freshwater microalga Chlorella sp. Beijerinck UMACC 313, marine microalga Chlorella sp. Beijerinck UMACC 258 and the ant Odontoponera denticulata. In conclusion, the EA extract showed promising larvicidal, adulticidal and ovicidal activity against Ae. aegypti, Ae. albopictus, An. cracens, and Cx. quinquefasciatus (larvae only). The results suggest that the EA extract of Streptomyces sp. KSF103 has the potential to be used as an environmental-friendly approach in mosquito control. The current study would serve as an initial step toward complementing microbe-based bioinsecticides for synthetic insecticides against medically important mosquitoes.
Indoor bioefficacy of the thermal fogging application of Pesguard FG 161, a formulation containing both knockdown and killing agents (active ingredient [AI]: d-tetramethrin 4% [w/w] and cyphenothrin 12% [w/w]) was compared with Resigen5 (AI: s-bioallethrin 0.8% [w/w], permethrin 125/75] 18.7% [w/w], and piperonyl butoxide 16.8% [w/w]), another pyrethroid formulation, as larvicides and adulticides against Aedes aegypti, Aedes albopictus, Anopheles sinensis, and Culex quinquefasciatus using a portable Agrofog AF35 sprayer indoors in houses on Penang Island, Malaysia. Pesguard FG 161 at the concentrations tested was effective against all 4 mosquito species tested. The water-based Pesguard FG 161 performed far better as a larvicide than the diesel-based formulation. Resigen was also effective as a larvicide and adulticide against all 4 mosquito species tested. Larvae of Ae. aegypti were most susceptible to water-based Pesguard FG 161, followed by Cx. quinquefasciatus, An. sinensis, and Ae. albopictus. Even at the lowest concentrations tested, Pesguard FG 161 showed adequate adulticidal properties. At higher dosages, water-based Pesguard FG 161 proved effective as a larvicide against all 4 mosquito species.
Bioefficacy of thermal fogging application of 2 Bacillus thuringiensis israelensis formulations, Vectobac ABG 6511 water-dispersible granules (3,000 international toxic unit [ITU]/mg) and Vectobac 12AS liquid (1,200 ITU/mg), was assessed for larvicidal activities against Aedes aegypti, Aedes albopictus, Anopheles dirus, and Culex quinquefasciatus. Portable Agrofog AF35 sprayers were used to apply the 2 formulations indoors in residential premises on Penang Island, Malaysia. Vectobac ABG 6511 showed good larvicidal effect against all 4 mosquito species at 3 of the higher doses tested (2.91 x 10(9), 1.45 x 10(9), and 0.71 x 10(9) ITU/ha), with more than 96% mortality at 48 h after spraying. As a comparative formulation, Vectobac 12AS also showed good larvicidal activity against all 4 mosquito species at 2 of the higher doses tested (2.87 x 10(9) and 1.46 x 10(9) ITU/ha), with more than 92.5% mortality at 48 h after spraying. Larvae of An. dirus were significantly more susceptible to both water-based Vectobac formulations when compared to the other 3 mosquito species. Both microbial formulations showed better efficacy at higher doses. However, even at the lowest dose tested, Vectobac ABG 6511 and Vectobac 12AS (both at 0.36 x 10(9) ITU/ha) showed larvicidal properties, with more than 66% mortality at 48 h after spraying. Overall, for this bacterial agent, the water-dispersible granule formulation has better prospects than the liquid formulation for the control of larvae of vector mosquitoes.
Among emerging zoonotic pathogens, mosquito-borne viruses (MBVs) circulate between vertebrate animals and mosquitoes and represent a serious threat to humans via spillover from enzootic cycles to the human community. Active surveillance of MBVs in their vectors is therefore essential to better understand and prevent spillover and emergence, especially at the human-animal interface. In this study, we assessed the presence of MBVs using molecular and phylogenetic methods in mosquitoes collected along an ecological gradient ranging from rural urbanized areas to highland forest areas in northern Thailand. We have detected the presence of insect specific flaviviruses in our samples, and the presence of the emerging zoonotic Tembusu virus (TMUV). Reported for the first time in 1955 in Malaysia, TMUV remained for a long time in the shadow of other flaviviruses such as dengue virus or the Japanese encephalitis virus. In this study, we identified two new TMUV strains belonging to cluster 3, which seems to be endemic in rural areas of Thailand and highlighted the genetic specificities of this Thai cluster. Our results show the active circulation of this emerging flavivirus in Thailand and the need for continuous investigation on this poorly known but threatening virus in Asia.
Information on the mosquito species that transmit canine filariosis is scanty. Hence, an experimental study was conducted to identify the potential vectors responsible for the transmission of D. immitis Leidy and B. pahangi Buckley & Edeson. A total of 367 mosquitoes belonging to six species containing both laboratory and field strains (i.e. Aedes togoi Theobald, Aedes aegypti Linnaeus, Aedes albopictus Skuse, Culex quinquefasciatus Say, Culex vishnui Theobald and Anopheles dirus Peyton & Harrison) were used in this study. All mosquitoes were artificially fed on either D. immitis or B. pahangi microfilariae (mfs) infected blood by using the Hemotek™ membrane feeding system. Out of 367 mosquitoes, 228 (64.9%) were fully engorged. After feeding on D. immitis (20%) and B. pahangi (33%) mfs positive blood, the mortality rates for Cx. quinquefasciatus were found to be slightly lower than that of other species of mosquitoes. On the other hand, majority of An. dirus were found to be incapable to withstand the infection of mfs as the mortality rates were relatively high (D. immitis = 71.4%; B. pahangi = 100.0%). Brugia pahangi was detected in Ae. togoi and Cx. quinquefasciatus with infection rates of 50% and 25%, respectively. Aedes togoi was the only species infected with D. immitis with an infection rate of 69%. Our results showed that Ae. togoi was an excellent experimental vector for both D. immitis and B. pahangi. This study also documented the observation of B. pahangi, for the first time in the head region of Cx. quinquefasciatus under a laboratory setting.
The Southern house mosquito, Culex quinquefasciatus (Cx. quinquefasciatus) is an important vector that transmit multiple diseases including West Nile encephalitis, Japanese encephalitis, St. Louis encephalitis and lymphatic filariasis. Long noncoding RNAs (lncRNAs) involve in many biological processes such as development, infection, and virus-host interaction. However, there is no systematic identification and characterization of lncRNAs in Cx. quinquefasciatus. Here, we report the first lncRNA identification in Cx. quinquefasciatus. By using 31 public RNA-seq datasets, a total of 4763 novel lncRNA transcripts were identified, of which 3591, 569, and 603 were intergenic, intronic, and antisense respectively. Examination of genomic features revealed that Cx. quinquefasciatus shared similar characteristics with other species such as short in length, low GC content, low sequence conservation, and low coding potential. Furthermore, compared to protein-coding genes, Cx. quinquefasciatus lncRNAs had lower expression values, and tended to be expressed in temporally specific fashion. In addition, weighted correlation network and functional annotation analyses showed that lncRNAs may have roles in blood meal acquisition of adult female Cx. quinquefasciatus mosquitoes. This study presents the first systematic identification and analysis of Cx. quinquefasciatus lncRNAs and their association with blood feeding. Results generated from this study will facilitate future investigation on the function of Cx. quinquefasciatus lncRNAs.
Observation on predation activities of guppies (Poecilia reticulata) on the larvae of three species of mosquito, namely Aedes albopictus, Aedes aegypti, and Culex quinquefasciatus was carried out under laboratory conditions. Male and female guppies were used as predators for predation experiments on the 4th instars of mosquito larvae. The daily feeding rates comparing male and female guppies on mosquito larvae were different; the female guppies consumed more mosquito larvae than male guppies did. The daily feeding rates of female guppies were 121.3 for Ae. aegypti, 105.6 for Ae. albopictus, and 72.3 for Cx. quinquefasciatus. The daily feeding rates of male guppies were 98.6 for Ae. aegypti, 73.6 for Ae. albopictus, and 47.6 for Cx. quinquefasciatus. In terms of prey preference, there was greater preference towards mosquito larvae of Ae. aegypti, followed by Ae. albopictus, and the least preferred was Cx. quinquefasciatus. Male and female guppies consumed more mosquito larvae during lights on (day time) compared with lights off (night time). The water volume, prey species, number of fish predators available, prey densities, and prey's sex also influenced the predation activities.
Japanese encephalitis (JE) is a global public health issue that has spread widely to more than 20 countries in Asia and has extended its geographic range to the south Pacific region including Australia. JE has become the most important cause of viral encephalitis in the world. Japanese encephalitis viruses (JEV) are divided into five genotypes, based on the nucleotide sequence of the envelope (E) gene. The Muar strain, isolated from patient in Malaya in 1952, is the sole example of genotype V JEV. Here, the XZ0934 strain of JEV was isolated from Culex tritaeniorhynchus, collected in China. The complete nucleotide and amino acid sequence of XZ0934 strain have been determined. The nucleotide divergence ranged from 20.3% to 21.4% and amino acid divergence ranged from 8.4% to 10.0% when compared with the 62 known JEV isolates that belong to genotype I-IV. It reveals low similarity between XZ0934 and genotype I-IV JEVs. Phylogenetic analysis using both complete genome and structural gene nucleotide sequences demonstrates that XZ0934 belongs to genotype V. This, in turn, suggests that genotype V JEV is emerging in JEV endemic areas. Thus, increased surveillance and diagnosis of viral encephalitis caused by genotype V JEV is an issue of great concern to nations in which JEV is endemic.
Using the reverse transcription-polymerase chain reaction (RT-PCR) and direct sequencing, capsid protein and non-structural protein 1 (nsP1) regions of Sagiyama virus and eight Getah virus strains were analysed. The viruses were isolated from Malaysia and various areas of Japan over a period of 30 years. Based on the available published sequence data, oligonucleotide primers were designed for RT-PCR and the sequences were determined. Our findings showed that though there were differences in the nucleotide sequences in the nsP1 region, there was 100% amino acid homology. On the other hand, in the capsid region, the nucleotide differences caused a major difference in the amino acid sequence. Therefore, the difference in the capsid region is one of the useful markers in the genetic classification between Sagiyama virus and strains of Getah virus, and might be responsible for the serological difference in complement fixation test. The genomic differences among the Getah virus strains are due to time factor rather than geographical distribution.