Rickettsioses are emerging zoonotic diseases reported worldwide. In spite of the serological evidence of spotted fever group rickettsioses in febrile patients in Malaysia, limited studies have been conducted to identify the animal reservoirs and vectors of rickettsioses. This study investigated the presence of rickettsiae in the tissue homogenates of 95 wild rats and 589 animal ectoparasites. Using PCR assays targeting the citrate synthase gene (gltA), rickettsial DNA was detected in the tissue homogenates of 13 (13.7%) wild rats. Sequence analysis of the gltA amplicons showed 98.6-100% similarity with those of Rickettsia honei/R. conorii/R. raoultii (Rickettsiales: Rickettsiaceae). Sequence analysis of outer membrane protein A gene (ompA) identified Rickettsia sp. TCM1 strain from two rats. No rickettsia was detected from Laelaps mites, Rhipicephalus sanguineus and Haemaphysalis bispinosa ticks, and Felicola subrostratus lice in this study. R. felis was identified from 32.2% of 177 Ctenocephalides felis fleas. Sequence analysis of the gltA amplicons revealed two genotypes of R. felis (Rf31 and RF2125) in the fleas. As wild rats and cat fleas play an important role in the enzoonotic maintenance of rickettsiae, control of rodent and flea populations may be able to reduce transmission of rickettsioses in the local setting.
The present study explored the intraspecific genetic diversity, dispersal patterns and phylogeographic relationships of Culex quinquefasciatus Say (Diptera: Culicidae) in Malaysia using reference data available in GenBank in order to reveal this species' phylogenetic relationships. A statistical parsimony network of 70 taxa aligned as 624 characters of the cytochrome c oxidase subunit I (COI) gene and 685 characters of the cytochrome c oxidase subunit II (COII) gene revealed three haplotypes (A1-A3) and four haplotypes (B1-B4), respectively. The concatenated sequences of both COI and COII genes with a total of 1309 characters revealed seven haplotypes (AB1-AB7). Analysis using tcs indicated that haplotype AB1 was the common ancestor and the most widespread haplotype in Malaysia. The genetic distance based on concatenated sequences of both COI and COII genes ranged from 0.00076 to 0.00229. Sequence alignment of Cx. quinquefasciatus from Malaysia and other countries revealed four haplotypes (AA1-AA4) by the COI gene and nine haplotypes (BB1-BB9) by the COII gene. Phylogenetic analyses demonstrated that Malaysian Cx. quinquefasciatus share the same genetic lineage as East African and Asian Cx. quinquefasciatus. This study has inferred the genetic lineages, dispersal patterns and hypothetical ancestral genotypes of Cx. quinquefasciatus.
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
Studies of blackfly vectors of Onchocerca dewittei japonica Uni, Bain & Takaoka (Spirurida: Onchocercidae), a parasite of wild boar implicated in the aetiology of zoonotic onchocerciasis in Japan, and six other zoonotic Onchocerca species of this country are reviewed. Molecular identification of infective larvae found in wild-caught female blackflies showed that Simulium bidentatum (Shiraki) (Diptera: Simuliidae) is a natural vector of O. dewittei japonica, and also Onchocerca sp. sensu Fukuda et al., another parasite of wild boar. Inoculation experiments demonstrated that Simulium arakawae Matsumura and four other Simulium species are putative vectors. Similarly, S. arakawae, S. bidentatum and Simulium oitanum (Shiraki) are putative vectors of Onchocerca eberhardi Uni & Bain and Onchocerca skrjabini Rukhlyadev, parasites of sika deer. Morphometric studies of infective larvae indicated that Onchocerca lienalis Stiles, a bovine species, is transmitted by S. arakawae, Simulium daisense (Takahasi) and Simulium kyushuense Takaoka, and that Onchocerca sp. sensu Takaoka & Bain, another bovine species, is transmitted by S. arakawae, S. bidentatum, S. daisense and S. oitanum. Prosimulium sp. (Diptera: Simuliidae) and Simulium japonicum Matsumura are suspected vectors of Onchocerca suzukii Yagi, Bain & Shoho and O. skrjabini [Twinnia japonensis Rubtsov (Diptera: Simuliidae) may also transmit the latter], parasites of Japanese serow, following detection of the parasites' DNA genes in wild-caught blackflies.
The diversity of ectoparasites in Southeast Asia and flea-host associations remain largely understudied. We explore specialization and interaction patterns of fleas infesting non-volant small mammals in Bornean rainforests, using material from a field survey carried out in two montane localities in northwestern Borneo (Sabah, Malaysia) and from a literature database of all available interactions in both lowland and montane forests. A total of 234 flea individuals collected during our field survey resulted in an interaction network of eight flea species on seven live-captured small mammal species. The interaction network from all compiled studies currently includes 15 flea species and 16 small mammal species. Host specificity and niche partitioning of fleas infesting diurnal treeshrews and squirrels were low, with little difference in specialization among taxa, but host specificity in lowland forests was found to be higher than in montane forests. By contrast, Sigmactenus alticola (Siphonaptera: Leptopsyllidae) exhibited low host specificity by infesting various montane and lowland nocturnal rats. However, this species exhibited low niche partitioning as it was the only commonly recorded flea from rats on Borneo. Overall complementary specialization was of intermediate intensity for both networks and differed significantly from random association; this has important implications for specific interactions that are also relevant to the potential spread of vector-borne diseases.
The Old World screwworm fly (OWS), Chrysomya bezziana Villeneuve (Diptera: Calliphoridae), is a myiasis-causing blowfly of major concern for both animals and humans. Surveillance traps are used in several countries for early detection of incursions and to monitor control strategies. Examination of surveillance trap catches is time-consuming and is complicated by the presence of morphologically similar flies that are difficult to differentiate from Ch. bezziana, especially when the condition of specimens is poor. A molecular-based method to confirm or refute the presence of Ch. bezziana in trap catches would greatly simplify monitoring programmes. A species-specific real-time polymerase chain reaction (PCR) assay was designed to target the ribosomal DNA internal transcribed spacer 1 (rDNA ITS1) of Ch. bezziana. The assay uses both species-specific primers and an OWS-specific Taqman((R)) MGB probe. Specificity was confirmed against morphologically similar and related Chrysomya and Cochliomyia species. An optimal extraction protocol was developed to process trap catches of up to 1000 flies and the assay is sensitive enough to detect one Ch. bezziana in a sample of 1000 non-target species. Blind testing of 29 trap catches from Australia and Malaysia detected Ch. bezziana with 100% accuracy. The probability of detecting OWS in a trap catch of 50 000 flies when the OWS population prevalence is low (one in 1000 flies) is 63.6% for one extraction. For three extractions (3000 flies), the probability of detection increases to 95.5%. The real-time PCR assay, used in conjunction with morphology, will greatly increase screening capabilities in surveillance areas where OWS prevalence is low.
This study examined the fecundity, oviposition, nymphal development and longevity of field-collected samples of the tropical bedbug, Cimex hemipterus (Fabricius) (Hemiptera: Cimicidae). Under environmental conditions of 26+/-2 degrees C, 70 +/- 5% relative humidity and a 12-h photoperiod, with bloodmeals provided by a human host, six strains of tropical bedbug had a fecundity of up to 50 eggs per lifetime, over 11-14 oviposition cycles. Increased feeding frequency improved fecundity. After feeding and mating, adult females normally took 2-3 days to produce a first batch of eggs. The oviposition period lasted 2-7 days before cessation of the oviposition cycle. The egg incubation period usually lasted 5-7 days before the emergence of first instars. The nymphs underwent five stadia (the first four of which each took 3-4 days, whereas the last took 4-5 days) before becoming adults at a sex ratio of 1 : 1. More than five bloodmeals were required by the nymphs to ensure a successful moult. Unmated adults lived significantly longer than mated adults (P < 0.05). Unmated females lived up to almost 7 months, but the longevity of mated males and females did not differ significantly (P > 0.05).
Among Oriental anopheline mosquitoes (Diptera: Culicidae), several major vectors of forest malaria belong to the group of Anopheles (Cellia) leucosphyrus Dönitz. We have morphologically examined representative material (> 8000 specimens from seven countries) for taxonomic revision of the Leucosphyrus Group. Six new species are here described from adult, pupal and larval stages (with illustrations of immature stages) and formally named as follows: An. latens n. sp. (= An. leucosphyrus species A of Baimai et al., 1988b), An. cracens n. sp., An. scanloni n. sp., An. baimaii n. sp. (formerly An. dirus species B, C, D, respectively), An. mirans n. sp. and An. recens n. sp. Additionally, An. elegans (James) is redescribed and placed in the complex of An. dirus Peyton & Harrison (comprising An. baimaii, An. cracens, An. dirus, An. elegans, An. nemophilous Peyton and Ramalingam, An. scanloni and An. takasagoensis Morishita) of the Leucosphyrus Subgroup, together with An. baisasi Colless and the An. leucosphyrus complex (comprising An. balabacensis Baisas, An. introlatus Baisas, An. latens and An. leucosphyrus). Hence, the former Elegans Subgroup is renamed the Hackeri Subgroup (comprising An. hackeri Edwards, An. pujutensis Colless, An. recens and An. sulawesi Waktoedi). Distribution data and bionomics of the newly defined species are given, based on new material and published records, with discussion of morphological characters for species distinction and implications for ecology and vector roles of such species. Now these and other members of the Leucosphyrus Group are identifiable, it should be possible to clarify the medical importance and distribution of each species. Those already regarded as vectors of human malaria are: An. baimaii[Bangladesh, China (Yunnan), India (Andamans, Assam, Meghalaya, West Bengal), Myanmar, Thailand]; An. latens[Borneo (where it also transmits Bancroftian filariasis), peninsular Malaysia, Thailand]; probably An. cracens (Sumatra, peninsular Malaysia, Thailand); presumably An. scanloni (Thailand); perhaps An. elegans (the Western Ghat form of An. dirus, restricted to peninsular India); but apparently not An. recens (Sumatra) nor An. mirans[Sri Lanka and south-west India (Karnataka, Kerala, Tamil Nadu)], which is a natural vector of simian malarias. Together with typical An. balabacensis, An. dirus and An. leucosphyrus, therefore, the Leucosphyrus Group includes about seven important vectors of forest malaria, plus at least a dozen species of no known medical importance, with differential specific distributions collectively spanning > 5000 km from India to the Philippines.
A multi-locus approach was used to examine the DNA sequences of 10 nominal species of blackfly in the Simulium subgenus Gomphostilbia (Diptera: Simuliidae) in Malaysia. Molecular data were acquired from partial DNA sequences of the mitochondria-encoded cytochrome c oxidase subunit I (COI), 12S rRNA and 16S rRNA genes, and the nuclear-encoded 18S rRNA and 28S rRNA genes. No single gene, nor the concatenated gene set, resolved all species or all relationships. However, all morphologically established species were supported by at least one gene. The multi-locus sequence analysis revealed two distinct evolutionary lineages, conforming to the morphotaxonomically recognized Simulium asakoae and Simulium ceylonicum species groups.
The possible insecticide resistance mechanisms of four Malaysian field-collected strains of the German cockroach, Blattella germanica (Linnaeus) (Dictyoptera: Blattellidae), were characterized with biochemical assays and native polyacrylamide gel electrophoresis (PAGE). Elevated esterase activity (at low to moderate frequency) and altered acetylcholinesterase (low frequency) were detected in all field strains, while elevated glutathione S-transferase levels were present in only two strains. Seven esterase bands were separated by native PAGE; a greater intensity occurred in three bands in the resistant strains compared to the susceptible strain. Inhibition studies using specific inhibitors on polyacrylamide gels suggested that the slowest of these three esterases is a cholinesterase, while the other two are carboxylesterases with a preference for beta- over alpha-naphthyl acetate.
A morphological and molecular analysis was undertaken with the objective of identifying markers for geographical populations of Old World screwworm flies, Chrysomya bezziana Villeneuve (Diptera: Calliphoridae). The morphological analysis involved 192 adult flies from 14 countries, and the molecular analysis involved 45 larvae or adults from 14 populations in 11 countries. Principal components and cluster analysis of 10 morphological characters indicated that flies from Papua New Guinea (PNG) were a distinct group and most similar to flies from nearby Asian islands (Java, Sabah). There was poor resolution of other geographical regions, but some support for clustering of flies from Africa or India. Cladistic analysis of mitochondrial DNA sequences gave strong support for recognizing two races of Old World screwworm, one from sub-Saharan Africa and the other from the Gulf region and Asia. This latter race could be further divided into two lineages, i.e. one from mainland Asia (from Iraq to the Malay Peninsula) and the other from two islands of PNG.
The Anopheles dirus Peyton & Harrison complex of mosquitoes (Diptera: Culicidae) comprises seven known species, including important malaria vectors in Southeast Asia. Specific identification of each species of the complex, which cannot be distinguished using morphological characters, is crucial for understanding vector ecology and implementing effective control measures. Derived from individual random amplified polymorphic DNA (RAPD) markers, sequence characterized amplified regions (SCAR) were analysed for the design of specific paired-primers. Combination of six SCAR primers resulted in the development of a simple, robust, single multiplex PCR able to identify three important malaria vectors among the four most common species (A, B, C, D) of the complex: species A from several Southeast Asian countries, species B from Perlis, Malaysia, and species C and D from Thailand.
1. Seven types of water-insoluble adhesives were evaluated in sticky traps for collecting adults of Musca domestica L. and Chrysomya megacephala (Fabricius) or mosquito larvae (Aedes aegypti (L.) and Culex quinquefasciatus Say). 2. Adhesive viscosity affected the tackiness of the glues and this determined their trapping efficiency in air or water. 3. From the 'Hyvis' range of adhesives tested, 'Hyvis 200' was most effective for trapping adult flies. 4. With 24 h exposure to fourth instar Ae.aegypti larvae in tapwater, submerged plates coated with 'Hyvis 10', 'Hyvis 30' or 'Hyvis 200' formulations trapped the majority of larvae. In polluted water the highest rates of trapping were 17.3% of Ae.aegypti and 18.7% of Cx quinquefasciatus with 'Hyvis 200'. Floating traps were consistently less productive than submerged traps under laboratory conditions. 5. In a heavily polluted natural breeding-site of Cx quinquefasciatus, floating traps were more productive than submerged sticky traps with four of seven adhesives tested, the most efficient being 'Hyvis 200' (4.2 mosquitoes per hour) and Hyvis:polyethylene 90:10 (4.5/h). Despite the relative inefficiency of aquatic traps, emergent adults, pupae and second to fourth instars of larvae were collected quickly from the habitat.
An exophilic population of the vector mosquito Anopheles balabacensis Baisas was investigated in two mark-recapture studies (16.ix-13.x.1986 and 6-26.i.1987) at an inland, foothill village in Sabah, Malaysia. Wild female mosquitoes were intercepted as they came to feed on man or buffalo, given a bloodmeal, marked with fluorescent dust and released. The recapture rate was about 12%. A new method of analysis is proposed which uses cross-correlation and a time series model. The estimated survival per oviposition cycle was 0.48-0.54 and the oviposition cycle interval 2-3 days.
Studies on larval population densities and adult emergence rates of the Brugian filariasis vectors Mansonia bonneae Edwards and Ma. dives Schiner were conducted in freshwater swamp forest bordering the Sadong River, Serian District, Sarawak, East Malaysia, during 1984-85. Three species of aquatic host-plants in the Family Araceae were identified as supporting immature stages of the Ma. bonneae/dives complex. Proportions of positive plants were 4.7%, 6.5% and 3.4% with 6.4 +/- 2.6, 7.3 +/- 2.8 and 10.1 +/- 1.1 larvae per positive plant, respectively, for the plant species Homalomena cordata Schott, H. rostrata Griffiths and Hydrostemma motleyi (Hook. f.) Mabberley. These data indicate no significant preferences between the three types of host-plant. Detailed monitoring of the host-plant H. cordata revealed no significant monthly fluctuations in larval density per plant nor the proportion of positive plants. 11.6% of larvae were Ma. dives and 88.4% were Ma. bonneae. Mean daily yields of Ma. bonneae/dives adults per square metre of H. cordata vegetated water surface were 0.45 males plus 0.57 females during the wet season (December-February) compared with 0.2 males plus 0.31 females during the dry season (June-August). Thus output of adults per plant was approximately halved, and suitable breeding areas were further reduced, during the dry season. By extrapolation from these rates, a crude mean estimate for productivity of Ma. bonneae/dives females is 1.6 million per hectare per annum in swamp forest habitats vegetated with any of the host-plants studied.
The role of some adult flies (Diptera: Cyclorrhapha) as carriers of helminth parasites of man was studied at four sites in Malaysia: a refuse dump, where no helminth-positive flies were detected, and in three peri-domestic situations where four species of flies carried up to three types of nematodes. The dominant fly species Chrysomya megacephala (Fabricius) carried eggs of the roundworm Ascaris lumbricoides L., the pinworm Trichuris trichiura (L.) and hookworm on the adult external body surface and in the gut lumen, in association with Bukit Lanjan aborigines. Chrysomya rufifacies (Macquart) and Sarcophaga spp. also had Ascaris lumbricoides and Trichurus trichiura eggs in their gut contents. Human helminths were not recovered from Lispe leucospila (Wiedemann), Lucilia cuprina (Wiedemann) or the housefly Musca domestica L. In an urban slum area of Kuala Lumpur city, filariform larvae identified as the hookworm Necator americanus (Stiles) occurred in the intestines of the face-fly Musca sorbens Wiedemann (22 larvae per 100 flies) and of Chrysomya megacephala (4.5 larvae per 100 flies). This concentration of apparently infective N. americanus in M. sorbens, a fly which often breeds in faeces and browses on human skin, could have transmission potential.
A small-scale trial was carried out in the Upper Kinabatangan district of Sabah, Malaysia, to determine the effect of using permethrin-impregnated bednets on malaria transmission. A total of 306 nylon bednets with cotton borders, impregnated at a dose estimated to have been 0.062 g permethrin/m2 of nylon netting, were distributed to 139 households in five villages. At the time of distributing bednets, mass drug administration with Fansidar plus primaquine was also administered to the human population to clear all parasitaemias due to Plasmodium falciparum Welch. In another village, for comparison, mass drug administration was the only intervention. After intervention measures in December 1984 and January 1985, the parasite rates in children declined in all villages during the first month, significantly more in the villages with impregnated bednets than in the control, thus proving that the nets had an impact on malaria. However, after about 2 months, parasite rates started to increase again. After 4-6 months, parasite rates in the villages with bednets approached the rate in the control village without nets. The increase in parasite rates was paralleled by a significant deterioration in the quality, physical condition and the degree of non-utilization of bednets. Entomological evaluation proved the efficacy of permethrin-impregnated nets for controlling Anopheles balabacensis Baisas and other anophelines. Bioassays (1 h exposure) of permethrin-impregnated bednets gave 100% mortality initially and 44-61% mortality after 85-106 days. Mosquito collections in treated bednets were significantly reduced for at least 217 days. The project failed to achieve prolonged suppression of malaria transmission for a combination of entomological, sociological and practical reasons which are discussed in relation to the objectives and implementation of future bednet studies.
Houseflies (Musca domestica L., Diptera: Muscidae) are cosmopolitan, colonizing, and eusynanthropic. Their distribution in the Malaysian archipelago provides an opportunity to study successive waves of colonization and extinction during the Pleistocene and Recent epochs. We scored single-strand conformation polymorphisms (SSCPs) at 16S2 and COII mitochondrial loci in 47 housefly samples from the Australian, Austro-Malayan, Indo-Malayan, Manchurian and Indo-Chinese subregions of Wallace's zoogeographical classification. We discuss the results in light of the Pleistocene vs. post-Pleistocene dispersal and faunal exchange in the Asia-Pacific area. Fourteen haplotypes were detected, of which 10 were confined to a single subregion. No haplotype was ubiquitous and only one was found in four subregions. Population diversity, HS, was greatest in the Indo-Malayan (0.36) and heterogeneous among subregions. The mean subregional diversity was 0.21 +/- 0.03, representing the probability that two randomly chosen flies, from any subregion, had different haplotypes. The hierarchical partition of diversity indicated restricted maternal gene flow among subregions (GRT = 0.60, Nm approximately 0.32). These results suggest long-standing genetic isolation of houseflies in the Malaysian archipelago and support the hypothesis that they dispersed widely during the Pleistocene. Haplotypes common among mainland populations but shared with island groups in low frequencies (<1%) indicate surprisingly little recent gene flow.
The fleas (Siphonaptera: Pygiopsyllidae) Farhangia quattuordecimdentata sp. n. and Farhangia sedecimdentata sp. n. are described from pygmy squirrels (Prosciurillus spp.), and Nestivalius sulawesiensis sp. n. from murine rodents, all from Sulawesi, Indonesia. Both new species of Farhangia were collected in Central Sulawesi (Sulawesi Tengah); F. quattuordecimdentata sp. n. was recovered mainly from P. murinus, whereas F. sedecimdentata sp. n. was recovered mainly from P. leucomus. These new species are compared with the two previously described species of Farhangia: F. celebensis (Ewing) from P. murinus in North Sulawesi (Sulawesi Utara) and F. sciuri (Ewing) from the tree squirrel Callosciurus prevosti in Sabah (Borneo). Nestivalius sulawesiensis sp. n. was collected from six species of endemic murine rodents in both North and Central Sulawesi. It is compared to the morphologically similar N. pomerantzi (Traub) from Mindanao, which parasitizes murine hosts that are endemic to the Philippines.