A total of 101 entomological specimens recovered from human cadavers were processed and studied. Analysis of the data indicated that about 95% of these specimens were maggots of flies. Maggots of the blowfly Chrysomya (Family: Calliphoridae) especially Ch. rufifacis and Ch. megacephala were predominantly found in 77 cases (76.2%) while larvae of several other flies of the genera Sarcophaga, Calliphora, Lucilia and hermetia were also recovered. It was notable that Musca domestica or other related flies were not found in all these specimens. The age of these larvae was useful in the determination of the minimum time lapsed after death. However, more biological studies on animal carcases should be conducted for more accurate determinations. Methods of collection, preservation and despatching of specimens were also discussed.
A nationwide screening program searching for microbial control agents of mosquitos was initiated in Malaysia in 1986. A total of 725 samples were collected and 2,394 bacterial colonies were isolated and screened for larvicidal activity. From such screening, 20 Bacillus thuringiensis, 6 B. sphaericus, 1 Clostridium bifermentans and 2 Pseudomonas pseudomallei larvicidal isolates were obtained. Of these, a new B. thuringiensis named as subspecies malaysianensis was found, while the C. bifermentans was also a new anaerobe individualized as serovar malaysia. It was concluded that this screening program was highly successful.
A strain of Clostridium bifermentans individualized as serovar malaysia (C.b.m.) according to its specific H antigen is toxic to mosquito and blackfly larvae when given orally. The toxicity occurs in sporulated cells which contain, in addition to spores, proteinic parasporal inclusion bodies and feather-like appendages; the amino acid content of the inclusion bodies is similar to that of Bacillus thuringiensis serovar israelensis (B.t.i.) and B. sphaericus crystals. The toxicity to Anopheles stephensi is as high as that of B.t.i. and the best strains of B. sphaericus. Culex pipiens is somewhat less susceptible, and Aedes aegypti much less. Pure parasporal inclusion bodies, isolated by ultracentrifugation on sucrose gradients, are highly toxic to mosquito larvae. The larvicidal power is destroyed by heating at 80 degrees C or by treatment with 50 mM NaOH. It is preserved by freeze-drying. The innocuity to mice of the sporulated cells is shown by different routes of administration: force-feeding, percutaneous, subcutaneous, intraperitoneal or intravenous injections. The potential for the biological control of mosquito and blackfly larvae is suggested.
A screening program searching for indigenous microbial control agents of mosquitos in Malaysia is initiated since 1987 and to date at least 20 isolates of mosquitocidal Bacillus thuringiensis serotypes have been obtained. Preliminary field evaluation of several isolates indicated that they are highly effective in the control of medically important mosquito species. For operational purposes, there is an urgent need to produce this agent utilizing cheap and locally available wastes through fermentation biotechnology. Fermentation studies in shake-flasks containing standard nutrient broth and soya bean waste, respectively, indicate that it takes about 37 hours for a Malaysian isolate of B. thuringiensis serotype H-14 to mature. In the grated coconut waste, fishmeal and rice bran, the bacteria took 28 hours, 26 hours and 126 hours respectively to mature. The endotoxin was harvested from the standard nutrient broth at 55 hours and at 50 hours from soybean, grated coconut waste and fishmeal. The endotoxin could only be harvested 150 hours after inoculation from rice bran medium. However, no bacterial growth was detected in palm oil effluent. In terms of endotoxin and biomass production, fishmeal appears to be a suitable medium. Variations in the pH of the fermenting media were also noted.
Rapid enzyme microassays for the detection of resistance due to organophosphate and carbamate in individual field-collected strains of Culex quinquefasciatus adults were conducted. These tests allowed accurate differentiation by eye, on the basis of color changes of susceptible and resistant individuals. Two separate tests were conducted for the biochemical assays. In the insensitive acetylcholinesterase (AChE) test, acetylthiocholine iodide (ACTH) and 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) were used as substrate and coupling agent respectively. The resulting yellow chromophore indicated AChE activity. Test results showed that the color intensity decreased as increasing concentrations of propoxur were added, thereby confirming the susceptibility of the enzyme to inhibitor. Assay of non-specific esterase however, indicated elevated levels which were correlated with degree of malathion resistance. Electrophoretic data revealed the presence of 2 esterase bands in all strains. It was concluded that such a pattern was not contributory to malathion resistance in adults.
Mosquitocidal strains of B. sphaericus serotype H-5a5b were shown for the first time to exhibit antagonistic activities against several human pathogens especially Salmonella. These strains of B. sphaericus also exhibited high larval toxicity against several mosquitoes.
A novel method for the control of Mansonia larvae was developed and tested. In this method, foliar absorption and translocation of a chemical insecticide, monocrotophos, a known systemic insecticide was studied in the Eicchornia plant. Acetone solution of the insecticide was painted onto leaves of the plant. At daily intervals, stems were severed and divided into equal sections which were introduced into bowls. Larvae of Aedes aegypti were tested for the presence of monocrotophos. It was found that translocation of the insecticide occurred at different rates in the stems and in some plants the chemical was also released into the surrounding water. Based on these results, 2 insecticides namely, monocrotophos and temephos were painted onto leaves of the host plant and their translocation to the root and water environment was examined by testing with Mansonia and Aedes aegypti larvae. The results again confirmed the translocation process and it was found that the insecticides were secreted into the surrounding water, thereby killing the larvae. However, in leaves painted with permethrin (synthetic pyrethroid) or flufenoxuron (chitin synthesis inhibitor), such a process was not detected. The potential of this new concept in Mansonia larval control is examined.
The ability to identify the occurrence of different resistance genotypes in field populations of mosquito is considered important for the purpose of optimising chemical control operations. The recent development of rapid microassays of enzymes responsible for resistance has provided a means for rapidly assessing the genetic background of target mosquito populations. This concept is the topic of investigation in this study. Non-specific esterase activity, which is responsible for the resistance to organophosphates in Malaysian Culex quinquefasciatus Say adults, was determined in 3 field populations from Kuala Lumpur City using rapid enzyme assay. The optical density results were used to estimate the genotypic frequencies of the populations. Subsequently, time-dependent changes in the various frequencies were determined. Such techniques allowed rapid assessment of resistance genotypes for decision-making and its possible use in insect control merits further investigation.
Comparative DDT-susceptibility status and glutathion s-transferase (GST) activity of Malaysian Anopheles maculatus, Culex quinquefasciatus and Aedes aegypti was investigated to ascertain the role of this enzyme in DDT resistance. The standardised WHO dose-mortality bioassay tests were used to determine DDT susceptibility in these mosquitos, whilst GST microassay (Brogdon and Barber, 1990) was conducted to measure the activity of this enzyme in mosquito homogenate. It appeared that DDT susceptibility status of Malaysian mosquitos was not correlated with GST activity.
A new mosquitocidal Bacillus thuringiensis subsp., jegathesan, has recently been isolated from Malaysia. Parasporal crystal inclusions were purified from this strain and bioassayed against fourth-instar larvae of Culex quinquefasciatus, Aedes aegypti, Aedes togoi, Aedes albopictus, Anopheles maculatus, and Mansonia uniformis. The 50% lethal concentration of crystal inclusions for each species was 0.34, 8.08, 0.34, 17.59, 3.91, and 120 ng/ml, respectively. These values show that parasporal inclusions from this new subspecies have mosquitocidal toxicity comparable to that of inclusions isolated from B. thuringiensis subsp. israelensis. Solubilized and chymotrypsin-activated parasporal inclusions possessed low-level hemolytic activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the crystals were composed of polypeptides of 77, 74, 72, 68, 55, 38, 35, 27, and 23 kDa. Analysis by Western blotting (immunoblotting) with polyclonal antisera raised against toxins purified from B. thuringiensis subsp. israelensis reveals that proteins in parasporal inclusions of subsp. jegathesan are distinct, because little cross-reactivity was shown. Analysis of the plasmid content of B. thuringiensis subsp. jegathesan indicates that the genes for toxin production may be located on 105- to 120-kb plasmids. Cry- clones that have been cured of these plasmids are nontoxic. Southern blot analysis of plasmid and chromosomal DNA from subsp. jegathesan showed little or low homology to the genes coding for CryIVA, CryIVB, and CryIVD from B. thuringiensis subsp. israelensis.
A case of true enteric myiasis in a 7-year-old girl is reported. Two larvae were obtained from the vomitus of the patient. After processing and identification, the larvae were found to be those of Hermetia illucens (Soldier Fly). This is the first case of true enteric myiasis due to these larvae in Malaysia.
A novel Bacillus thuringiensis strain highly toxic to mosquitoes was isolated from soil samples in Malaysia. This strain was shown to display a new subfraction of the H-28 flagellar antigen determining a new serovar H28a28c, which was designated serovar jegathesan. Bioassays indicated that Culex quinquefasciatus larvae are the most susceptible to this new isolate, whereas toxicity to Anopheles maculatus and Aedes aegypti larvae was 10 times lower. The potency of this new serotype is also comparable to most of the Malaysian B. thuringiensis H-14 isolates.
In an effort to develop a more effective technique in dispersing a microbial control agent, Bacillus thuringiensis (Bt), a truck-mounted ultra low volume (ULV) generator (Scorpion) was used to disperse B. thuringiensis israelensis (Bti) and Bti with malathion. Complete larval and adult mortalities for all tested mosquito species within the first 70-80 feet from the ULV generator were achieved. Beyond that distance less than 50% mortality was achieved as insufficient sprayed particles reached the area. A minimum of 10(3) Bti colony forming units per ml is required to cause 100% larval mortality. The sprayed Bti larvicidal toxins were persistent in the test water 7 days post ULV. The effectiveness of B. thuringiensis jegathesan (Btj), a new mosquitocidal Bt serotype was also evaluated. Similar mortality results as Bti were achieved except that the Btj toxins underwent degradation in the test water, since less than 50% less in larval mortality was observed in 7 days post ULV samples. This ULV method has the potential to disperse Bt and malathion effectively for a simultaneous control of mosquito adults and larvae.
Forensically important entomological specimens recovered from 95 forensic cases of human cadavers from April 1993 to May 1996 in Malaysia were identified and analysed. The results indicated that 73.7% of these specimens were Chrysomya species, occurring either as single or mixed infestations. Of these, the most prominent species were Ch megacephala (F.) and Ch rufifacies (Macquart). Other fly maggots recovered included Sarcophaga spp., Lucilia spp. and Hermetia spp., mostly occurring together with other calliphorine flies. A member of Muscidae fly, Ophyra spp. was also recovered for the first time.
Evaluation of the effectiveness of Bacillus thuringiensis ssp. israelensis (B.t.i.) against mosquito larvae dispersed by ultralow volume (ULV) spraying was conducted in simulated field trials. Effectiveness was measured using 3 different indicators: larval mortality, colony-forming unit enumeration, and droplet analysis. B.t.i. was dispersed with a ULV generator using 2 different flow rates: 0.3 and 0.5 liter/min on 2 different days. Based on the results of this study, it can be concluded that an output of 0.3 liter/min is effective for controlling Aedes aegypti. although a dosage of 0.5 liter/min can be used when high residual activity is desired. For Culex quinquefasciatus control, both dosages were effective but with low residual activity. For Anopheles maculatus control, only a discharge rate of 0.5 liter/min was effective with low residual activity. B.t.i. application at both dosages penetrated tires well, indicating that B.t.i. ULV application is an effective method for controlling container-inhabiting mosquitoes. Good coverage of target area and penetration were attributed to satisfactory droplet profiles.
The insecticide resistance status of 4 strains of adult male Blattella germanica, viz M (Malacca), E (England), F (restaurant) and K (cafeteria) against malathion and bendiocarb compared with a reference susceptible strain (S) was determined by using a modified WHO bioassay method. The results indicated that all the 4 strains were resistant to the insecticides albeit in different degrees. Resistance ratios for malathion ranged from 1.85-41.07-fold, whereas that of bendiocarb ranged from 1.68-4.83-fold. The biochemical microplate enzyme assays technique employed indicated that the resistance in M and E strains were attributed to acetylcholinesterase insensitivity. Multiple resistance was not detected in any of the 4 strains. Parameters of the identified resistance mechanism correlated well with the observed level of resistance. Agar gel electrophoresis showed that variations in esterase isoenzymes did not confer organophosphate and carbamate resistance to the 4 strains.