Clostridium bifermentans serovar malaysia (C.b.m.) is highly toxic to mosquito larvae. In this study, the following aquatic nontarget invertebrates were treated with high C.b.m. concentrations (up to 1,600-fold the toxic concentration for Anopheles stephensi) to study their susceptibility towards the bacterial toxin: Planorbis planorbis (Pulmonata); Asellus aquaticus (Isopoda); Daphnia pulex (Cladocera); Cloeon dipterum (Ephemeroptera); Plea leachi (Heteroptera); and Eristalis sp., Chaoborus crystallinus, Chironomus thummi, and Psychoda alternata (Diptera). In addition, bioassays were performed with mosquito larvae (Aedes aegypti, Anopheles stephensi, and Culex pipiens). Psychoda alternata larvae were very susceptible, with LC50/LC90 values comparable to those of mosquito larvae (about 10(3)-10(5) spores/ml). The tests with Chaoborus crystallinus larvae showed significant mortality rates at high concentrations, but generally not before 4 or 5 days after treatment. The remaining nontarget organisms did not show any susceptibility. The investigation confirms the specificity of C.b.m. to nematocerous Diptera.
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.
Mosquito collections were carried out from May to June 1992 and from September to December 1992 in an area where a case of Japanese encephalitis was confirmed. A total of 40,072 mosquitoes belonging to 35 species and 8 genera were collected. The dominant species in that locality were Culex vishnui, Culex tritaeniorhynchus, Culex pseudovishnui, Culex gelidus, Aedes butleri, and Mansonia uniformis.
Two new repellent formulations, KBR 3023 10% and 20% from Bayer AG, Germany, were evaluated together with DEET 10% and 20% as standard repellent formulations. Evaluation was based on two separate field studies: a daytime study (0900-1700 hr) in a forested orchard on Penang Island and a nighttime study (2100-0100 hr) in a squatter residential area on the adjacent mainland of peninsular Malaysia. Both studies were carried out by exposing humans with bare arms and legs to mosquitoes landing/biting for an eight hour period. Right arms and legs of the human baits were treated with different repellent formulations (KBR 3023 10%, 20% and DEET 10%, 20%) and the left limbs were left untreated to act as controls. The daytime study indicated that all four formulations were equally effective (P < 0.05) as repellents against the predominant Aedes albopictus with greater than 88.5% reduction in landing/biting in the first four hours and not less than 65.0% in the next four hours of the assessment period. In the night study, all four formulations were also found to be equally effective (P < 0.05) in repelling Culex quinquefasciatus, the predominant species. All four formulations provided complete protection against Cx. quinquefasciatus in the first two hours of exposure. The percentage reduction values were maintained above 90.0% for the next six hours of the assessment period. In conclusion, both the KBR 3023 and DEET formulations were found to be equally effective (P < 0.05) in providing a long-lasting reduction in human-mosquito contact in both the day and night field studies.
Eighteen strains of Getah virus isolated from mosquitoes, swine and horses in Japan (1956 to 1981), and one strain isolated in Malaysia (1955), were analysed by RNase T1-resistant oligonucleotide fingerprinting. All fingerprints showed a poly(A) tract. The fingerprint pattern of the Malaysian strain was quite different from those of the Japanese strains. Although most of the recent Japanese isolates shared many large oligonucleotide spots in common, the patterns were not identical even among the strains obtained in one locality in the same year. These results suggest that the Getah virus genome undergoes mutation rather frequently. However, there is a tendency for the isolates of the same year to show greater similarity. The fingerprint patterns of certain host-dependent temperature-sensitive (ts) mutants differed from that of the parental strain. Also, there were some differences in large oligonucleotide spots between strain JaNAr12380M isolated in suckling mouse brain (SMB) and strain JaNAr12380A isolated in C6/36 cells, despite the fact that both strains were derived from the same wild mosquito homogenate. In addition, many host-dependent ts mutants were present in strain JaNAr12380A, whereas no such mutants were observed in strain JaNAr12380M. It is concluded that there is considerable variation in the strains of Getah virus infecting mosquitoes in the wild, and also that the variants or mutants present in mosquitoes might be subject to selection during viral multiplication in the mammalian host.
The toxic effects of Abate (temephos) on mosquito larvae and non-target organisms were studied in the rice-field and in the laboratory. In the laboratory tests, Culex tritaeniorhychus larvae and cladoceran zooplanktons (predominantly Diaphanosoma and Moinodaphnia species) were found to be highly susceptible to Abate with LC50 values of 0.27 and less than 0.10 parts per billion respectively. Other non-target species in decreasing degree of susceptibility to Abate were copepods (Tropodiaptomus spp.), Aplocheilus panchax and Tubifex worms. In field study, Abate at concentrations 60, 100 and 200 gm hectare-1 is effective in maintaining the rice-fields free of Anopheles and Culex mosquitoes for at least 2 days. No mortality was observed for Aplocheilus panchax and Tubifex worms at the above concentrations; nevertheless, populations of cladoceran zooplanktons and copepods were reduced up to seven days posttreatment.
A total of 37 species of mosquitoes from seven genera were collected in six villages in the Bengkoka Peninsula, Sabah State, during two visits in 1981 in connection with studies on malaria and filariasis. Fifty-five per cent of the total mosquitoes collected were Mansonia. An. collessi constituted a new record of the species from Sabah. An. balabacensis was found to be naturally infected with sporozoites. Ma. bonneae was found to be naturally infected with Brugia, probably B. malayi. Parous rates of An. balabacensis and Ma. bonneae were very high with consequent high probability of survival ideally suiting transmission of malaria and filariasis.
The possible role of pigs as arbovirus maintenance hosts and their importance as amplifier hosts was studied. Blood samples from 464 pigs of all ages collected in 1962 and 1964 were tested against 10 arboviruses. Antibodies to Japanese encephalitis and Getah viruses were particularly prevalent and their calculated monthly infection rates were 19-5% and 13-3% respectively. In 1969, 447 pigs were bled monthly throughout the year and the infection rates for Japanese encephalitis virus were calculated in pigs during the first year of life. Infection rates were not uniform throughout the year; the rate increases as the pig grew older and there was a marked seasonal increase in the infection rate in the period from November to January. This coincided with the seasonal major population peak of Culex tritaeniorhynchus following intense breeding of this mosquito prior to rice planting. It is suggested that, in Sarawak, the pig acts as a maintenance host of Japanese encephalitis in a cycle involving C. gelidus mosquitoes and also acts as an important amplifier host towards the end of the year in a cycle involving C. tritaeniorhynchus. It is further suggested that Getah virus is maintained in a similar cycle between C. tritaeniorhynchus and pigs.
Thirty isolations of Tembusu virus and four of Sindbis virus were obtained from approximately 280 000 mosquitoes collected between October 1968 and February 1970 in Sarawak, particularly from K. Tijirak, a Land Dyak village 19 miles South of Kuching. Twenty-two isolations of Tembusu virus and two of Sindbis virus were from Culex tritaeniorhynchus; two of Tembusu virus and two of Sindbis virus came from Culex gelidus. Tembusu virus was active throughout the year at K. Tijirak, the highest infection rates in C. tritaeniorhynchus being in January-March and May-August, when the C. tritaeniorhynchus population was declining and ageing. These results confirm that C. tritaeniorhynchus is the principal arthopod host of Tembusu virus in Sarawak. Antibody studies suggest that birds, particularly domestic fowl, are probably vertebrate maintenance hosts of Tembusu and Sindbis viruses in Sarawak.
A nationwide investigation was performed to detect the presence of 1014 mutation(s) in voltage gated sodium channel (kdr) gene of Culex quinquefasciatus from 14 residential areas across 13 states and a federal territory in Malaysia. Molecular genotyping of kdr mutation was performed via a modified three tubes allele-specific-polymerase chain reaction (AS-PCR) and direct sequencing of kdr gene. Based on the results of AS-PCR, homozygous susceptible (SS) genotype was found in nine out of 14 populations with 38 individuals from a total sample size of 140. Heterozygous (RS) genotype was most predominant (99 individuals) and distributed across all study sites. Homozygous resistance (RR) genotype was detected in Perak (one individual) and Selangor (two individuals). The resistance kdr allele frequencies ranged from 0.1 to 0.55, with the highest being detected in Cx. quinquefasciatus population from Selangor. This study has documented the first field-evolved instance of 1014F mutation in Malaysian mosquitoes and the findings of this study could be utilized in the implementation of strategic measures in vector control programs in Malaysia.
The efficacy of the larvicidal and pupicidal agent (Agnique) MMF was evaluated against larvae of An. arabiensis and Culex (Diptera: Culicidae) under field conditions in Bahary Locality, Khartoum, Sudan. At an applied dosage of 0.25 ml/m2, MMF resulted in 89.4, 79.8 and 88.2% reductions in L3-L4 instars An. arabiensis and 63.5% in Culex larvae (all stages) 24 to 72 hours post-treatment. Pupae were completely eliminated (100%) within 24 hours posttreatment. The earlier instars (L1-L2) of An. arabiensis were more tolerant with a 62.5% reduction at 72 hours post-treatment compared to (L3-L4) instars and pupae. At 7-days post-treatment Agnique gave a 57.5% reduction in L1-L2 and 92.6% in L3-L4 instar larvae of An. arabiensis and 57.3% and 86.4% in Culex larvae and pupae, respectively. We conclude that Agnique can perform effectively against L3-L4 instars and pupae of An. arabiensis for only 1 week, and 3 to 4 days against L1-L2 instars of Culex spp.
Adult mosquito collections were conducted for 12 weeks in two residential areas in Kuala Lumpur. The CDC light traps were compared using dry ice and yeast as sources of carbon dioxide attractants for mosquitoes. The efficacy of the dry ice baited trap was significant over yeast generated CO2 trap. The predominant species obtained were Culex quinquefasciatus, Stegomyia albopicta and Armigeres subalbatus.
The susceptibility of Culex quinquefasciatus to chemical insecticides in two field sites in Kuala Lumpur was evaluated using the WHO standard susceptibility test. Less then 7 days old female mosquitos, reared from wild caught females were exposed to discriminating dosages of insecticides at recommended exposure periods. The larval bioassay were conducted using the multiple concentrations and the LC50 value was determined. The results indicated that cyfluthrin is the most effective among all the insecticides tested with LT50 value of 29.95 min and 28.59 min, for the strain from Ampang Hill and Pantai Dalam, respectively. It was surprisingly to note that both these field strains showed 0% mortality when tested against malathion and DDT. The LC50 value indicated that both strains were highly resistant to malathion with resistance ratio of 17,988 folds and 14,053 folds, respectively. This concludes that resistance at larval stages is extremely high compared to adult stages.
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.
Laboratory strain of the Malaysian Culex quinquefasciatus was susceptible to Wuchereria bancrofti. Thirty three percent of the Cx. quinquefasciatus that fed on W. bancrofti patient were infective after 12-14 days. There is a possibility for W. bancrofti to occur in the urban areas of the Malaysia in the near future.
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.
Mosquitoes in malaria endemic areas needs to be monitored constantly in order to detect demographic changes that could affect control measures. A 12-month mosquito population survey was conducted in several malaria endemic areas in Pos Lenjang, Kuala Lipis, Pahang, Malaysia. Collection of mosquitoes using a human landing catch technique was carried out indoors and outdoors for 12 hours from 7:00 PM to 7:00 AM for 42 nights. Anopheles maculatus Theobald (31.0%), Armigeres flavus Leicester (11.3%), Armigeres annulitarsis Leicester (11.0%), Culex vishnui Theobald (9.6%) and Aedes albopictus Skuse (7.0%) were the predominant species caught in the study area. The salivary gland and midgut of all anopheline mosquitoes were dissected to determine the presence of malaria parasites but none were positive. A high rate of human biting by An. maculatus was detected during December, but the rate was lower in January. Mosquito larvae were carried by the rapid current of the river downstream causing a decrease in the larvae population. Of the five predominant species, only Ar. annulitarsis exhibited a significant positive correlation in numbers with monthly rainfall (p < 0.05). An. maculatus biting activity peaked during 10:00 PM to 11:00 PM. Ae. albopictus, Ar. annulitarsis, and Ar. flavus exhibited similar activities which peaked during 7:00 PM to 8.00 PM.