Anaplasmosis and ehrlichiosis are of serious health concern worldwide for animals and humans. In the present study, we report the occurrence of Anaplasma platys and Ehrlichia canis in dogs and Rhipicephalus sanguineus sensu lato (s.l.) ticks from Peninsular Malaysia using a nested polymerase chain reaction assay based on amplification of the 16S rRNA gene. Anaplasma platys was detected from dogs and ticks with prevalence rates of 3.3% (8/240) and 2.9% (4/140), respectively. On the other hand, 12.9% (31/240) of the dogs and 0.7% (1/140) of the ticks were tested positive for E. canis. Additionally, co-infections of A. platys and E. canis with Babesia or Hepatozoon protozoa were also noted in this study. Double infection (E. canis + B. gibsoni) was observed in tick, whereas triple infections (E. canis + A. platys + B. vogeli and E. canis + A. platys + H. canis) were found in dogs. This study represents the first evidence of A. platys DNA in R. sanguineus s.l. in Malaysia.
Vector-borne infections are persistent public health threats worldwide. In recent years, a number of mosquito-borne viruses have emerged or re-emerged to cause major disease outbreaks. Other vector-borne pathogens, however, remain understudied and much neglected especially in the developing regions of the world including Southeast Asia. In this study, the brown dog tick Rhipicephalus sanguineus sensu lato, cat louse Felicola subrostratus, and cat fleas Ctenocephalides felis and Ctenocephalides orientis collected from free-ranging cats and dogs in Malaysia were molecularly screened for the presence of Bartonella and Rickettsia bacteria, and Dipylidium tapeworm. Our results showed the presence of Bartonella clarridgeiea, Bartonella henselae (lineage Marseille and lineage Houston-1), and Rickettsia sp. in C. felis. We also detected Rickettsia asembonensis in C. orientis and R. sanguineus s.l. Additionally, this study provides the first documentation on a potentially new species of Dipylidium infecting F. subrostratus and C. felis. Our results highlight the role of ectoparasites from free-ranging animals including cats and dogs, in harboring multiple transmissible pathogens.
Although the microbiome of blood-feeding insects serves an integral role in host physiology, both beneficial and pathogenic, little is known of the microbial community of black flies. An investigation, therefore, was undertaken to identify culturable bacteria from one of Malaysia's most common black flies, Simulium tani Takaoka and Davies, using 16S rDNA sequencing, and then evaluate the isolates for antibiotic resistance and virulence genes. A total of 20 isolates representing 11 bacterial species in four genera were found. Five isolates showed β-hemolysis on Columbia agar, and virulence genes were found in three of these isolates. Some degree of resistance to six of the 12 tested antibiotics was found among the isolates. The baseline data from this study suggest rich opportunities for comparative studies exploring the diversity and roles of the microbiome of S. tani and other Southeast Asian black flies.
The biodiversity of black flies (Diptera: Simuliidae), which are biting insects of medical and veterinary importance, is strikingly high in Southeast Asian countries, such as Indonesia, Malaysia, Philippines and Thailand. In 2013, we began to explore the fauna of black flies in Vietnam, which has so far been poorly studied. In this monograph, the wealth of the biodiversity of black flies in Vietnam is also confirmed on the basis of the results of our recent investigations, though limited to five provinces in the country. Morphotaxonomic studies of black flies obtained from Sapa, Lao Cai Province, northern Vietnam, in 2014 and Nghe An Province, northern Vietnam, in 2015, and reexaminations of black flies collected from Tam Dao, Vinh Phuc Province, northern Vietnam, in 2013, Thua Thien Hue Province, central Vietnam, in 2014, and Lam Dong Province, southern Vietnam, in 2014, were conducted. A total of 22 species are described as new, including one in the newly recorded subgenus Montisimulium Rubtsov, and three species are recognized as new records from Vietnam. This investigation brings the number of species of black flies known in Vietnam to 70, all of which are assigned to the genus Simulium Latreille, and are placed in four subgenera (25 in Gomphostilbia Enderlein, one in Montisimulium, seven in Nevermannia Enderlein, and 37 in Simulium Latreille s. str.). The numbers of species-groups recognized include seven in Gomphostilbia, three in Nevermannia and nine in Simulium, indicating a high diversity of putative phylogenetic lineages. New species include S. (G.) sanchayense sp. nov. (= the species formerly regarded as S. (G.) brinchangense Takaoka, Sofian-Azirun & Hashim), S. (S.) lowi sp. nov. (= the species formerly regarded as S. (S.) brevipar Takaoka & Davies), S. (S.) fuscicoxae sp. nov. [= the species formerly regarded as S. (S.) rufibasis Brunetti (in part)], S. (S.) suoivangense sp. nov. [= morphoform 'b' of the S. (S.) tani Takaoka & Davies (complex)]. Newly recorded species are S. (G.) parahiyangum Takaoka & Sigit, S. (N.) maeaiense Takaoka & Srisuka, and S. (S.) doipuiense Takaoka & Choochote (complex) [= the species formerly regarded as S. (S.) rufibasis Brunetti (in part)]. The substitute name, S. (S.) huense, is given for the species that was described under the name of S. (S.) cavum from southern Vietnam. A redescription of the female, male, pupa and larva of S. (G.) asakoae Takaoka & Davies is presented, and the female and larva of S. (G.) hongthaii Takaoka, Sofian-Azirun & Ya'cob are described for the first time. Keys to 10 subgenera in the Oriental Region and all 70 species recorded from Vietnam are provided for females, males, pupae and mature larvae. As investigations extend nationwide in all the provinces in Vietnam, more new species and records are expected to be discovered. It is hoped that this monograph will be useful as a baseline taxonomic reference for future studies of black flies in Vietnam and neighbouring countries.
Aedes albopictus was bioassayed to determine resistance development to malathion (OP). Two methods were applied, including WHO larval bioassay to determine the susceptibility to lethal concentration (LC), and adult bioassay to determine lethal time (LT). Larvae from colonies that had undergone selection pressure with malathion to yield 50% mortality were further subjected to selection for subsequent 10 generations. Selection of Ae. albopictus with malathion could relatively induce a consistent resistance ratio of 1.0 throughout 10 generations. It was noted that Ae. albopictus larvae showed less susceptibility to malathion compared to adults. The susceptibility test of adult mosquitoes to diagnostic dosage of 5.0% malathion-impregnated paper showed a variety of susceptibility to malathion when compared to the susceptible strain. Bioassay results indicated that the LT50 values of malathion-selected Ae. albopictus ranged between 11.5 - 58.8 minutes for ten consecutive generations. Biochemical enzyme studies indicated that there was a significant difference (p < 0.05) in esterase level in malathion-selected mosquitoes compared to non-selected control. Electrophoretic patterns of non-specific esterases at different life stages in malathion-selected Ae. albopictus suggested that non-specific esterases do not play a role in resistance of malathion-selected Ae. albopictus.
This study was conducted to monitor the susceptibility status of Aedes aegypti (Linnaeus) larvae in the Sunda Islands of Indonesia against various organophosphates and organochlorines. Larval bioassay was performed in accordance with the World Health Organization standard protocol. Field-collected and reference strains of Ae. aegypti larvae were tested against diagnostic doses of eight larvicides belonging to organophosphates and organochlorines, namely bromophos (0.050 mg/liter), chlopyrifos (0.002 mg/liter), fenitrothion (0.020 mg/liter), fenthion (0.025 mg/liter), malathion (0.125 mg/liter), temephos (0.012 mg/liter), DDT (0.012 mg/liter), and dieldrin (0.025 mg/liter). Mortality rates of larvae were recorded at 24-h posttreatment. This study showed that Ae. aegypti larvae from Padang, Samarinda, Manggarai Barat, and South Central Timor were susceptible to both fenitrothion and dieldrin (mortality rates ≥ 98%). About 6 out of 10 field strains of Ae. aegypti larvae were resistant (<80% mortality rates) against fenthion, whereas Ae. aegypti larvae from Kuningan, Samarinda, Sumba, and South Central Timor exhibited some degrees of resistance (mortality rates 80-98%). All field-collected Ae. aegypti larvae were resistant against diagnostic doses of chlorpyrifos, malathion, temephos, and DDT with mortality rates ranging from 0 to 74.67%. Continued insecticide susceptibility studies are essential to identify the efficacy of insecticides for an improved dengue vector control and to delay the development of insecticide resistance.
An entomological study was conducted in Tanjung Sepat, Selangor, Malaysia in May until September 2007 revealing five species of butterflies (all from family Nymphalidae) were attracted to pig carcasses placed in an oil palm plantation. Euploea mulciber (Cramer 1777), Hypolimnas bolina (Linnaeus 1758), Elymnias hypermnestra (Linnaeus 1763), Mycalesis mineus (Linnaeus 1758) and Ypthima baldus (Fabricius 1775) came to the carcasses at different stages of decomposition. From this study, we know that nymphalid butterflies are attracted to carcasses but their roles are most probably unimportant in post-mortem estimation.
Selected physiological and biochemical parameters were monitored at the vegetative and reproductive growth stages in potted Bougainvillea plants treated with five different concentrations of TRIA. Advanced flowering, flower bud number, and blooming rate increased significantly with 0.5 and 1.0 mg/L TRIA treatments. Similarly, photosynthetic rate, pigment content, quantum yield, and stomatal conductance increased significantly with 2.5, 1.0, and 5.0 mg/L TRIA treatments. Higher levels of N, P, and K, as well as increased total soluble solids (TSS) and higher sugar and protein contents, were recorded in treated plants. Furthermore, 46% more flowers, a 1.5-fold increase in bract weight, increased longevity, and 40% less leaf abscission were recorded following 2.5 mg/L TRIA treatment. Phenol and flavonoid contents, sucrose phosphate synthase (SPS), and antioxidant activities were also markedly increased with 2.5 and 1.0 mg/L TRIA treatments. However, ethylene production was significantly lower in the treated plants. Positive correlations were observed between leaf TSS and flowering time and flower number, between leaf sugar content and bract weight, and between net photosynthesis and bract growth and dry matter production. It can be concluded that the foliar spray of TRIA stimulates growth, enhances flowering, and improves the quality of potted Bougainvillea plants.
Two new species of black flies, Simulium (Comphostilbia) terengganuense sp. nov. and Simulium (Gomphostilbia) aziruni sp. nov. (Diptera: Simuliidae), are described on the basis of reared adult, pupal, and larval specimens collected from Peninsular Malaysia. Both species are placed in the batoense species-group within the subgenus Gomphostilbia, one of two dominant subgenera of the genus Simulium in Peninsular Malaysia as well as in the Oriental Region. Strikingly, three morphological characteristics that rarely occur in the subgenus Gomphostilbia are found in these two new species: the very narrow female frons and the mushroom-like pupal terminal hooks in S. (G.) terengganuense sp. nov. and the pupal gill composed of an inflated horn-like structure and eight slender filaments in S. (G.) aziruni sp. nov.
Simulium (Gomphostilbia) yvonneae sp. nov. is described based on adults, pupae, and mature larvae from Vietnam. This new species belongs to the Simulium duolongum subgroup in the S. batoense species-group of the subgenus Gomphostilbia Enderlein. It is distinguished by having a relatively larger number of male upper-eye facets in 16 vertical columns and 16 horizontal rows and a pupal gill with eight filaments arranged as 3+(1+2)+2 from dorsal to ventral, of which two filaments of the ventral pair are 1.8 times as long as the longest filament of the middle and dorsal triplets. Morphological comparisons are made to distinguish this new species from all 22 related species. The genetic distinctiveness of this new species in the S. duolongum subgroup is also presented based on the DNA barcoding COI gene.
A new simuliid species, Simulium kalimantanense sp. nov., is described on the basis of females, males, pupae, and mature larvae from East Kalimantan, Indonesia, and is assigned to the Simulium banauense species-group of Simulium (Gomphostilbia). This new species has close similarities to S alienigenum Takaoka from the Philippines, in many characters including the adult antennal color pattern and pupal gill with four long filaments arranged in two pairs each bearing a long stalk, but is distinguished from the latter in the female by the longer sensory vesicle and in the pupa by the gill with an elongate common basal stalk. Simulium kalimantanense sp. nov. is the first member of the S. banauense group in Borneo, and marks the most southerly distribution of the group. Keys to identify 19 Bornean species of the subgenus Gomphostilbia are provided.
Two new black fly species, Simulium (Gomphostilbia) brinchangense and S. (G.) tanahrataense, are described on the basis of reared adult females, males, pupae and larvae from Cameron's Highlands, Peninsular Malaysia. These new species are assigned to the asakoae species-group within Simulium (Gomphostilbia) and taxonomic notes are given to distinguish each new species from six known species in Malaysia. Revised keys to identify all 21 species including 13 species from other countries are provided for females, males, pupae and mature larvae. The species diversity of the asakoae species-group in Cameron's Highlands is briefly noted.
Simulium (Comphostilbia) izuae sp. nov. is described from female, male, pupal, and larval specimens collected from Cameron's Highlands, Peninsular Malaysia. This new species is placed in the asakoae species-group of the subgenus Gomphostilbia. The pupa of this new species is characterized by the gill with eight long filaments arranged as (3 + 3) + 2 filaments, of which the ventral pair of filaments is borne on a stalk that is always shorter than the common basal stalk. Taxonomic notes to distinguish this new species from five other Malaysian species and 12 other species of the asakoae species-group from other countries are given. Keys to identify all 18 species of the asakoae species-group are also provided for females, males, pupae, and mature larvae.
Simulium (Gomphostilbia) sofiani sp. nov. is described on the basis of reared adult female, male, pupal and larval specimens collected from Cameron Highlands, Pahang state, Malaysia. This new species is placed in the ceylonicum species-group within the subgenus Gomphostilbia and is easily distinguished from all the related known species by the combination of the following characteristics: an elongate sensory vesicle and yellow hair tuft on the stem vein of the wing in the female, the greater number of large upper-eye facets (15 or 16 vertical columns and 15 or 16 horizontal rows) and almost entirely darkened hind basitarsus in the male, and the gill bearing a long common basal stalk and 8 filaments arranged as [(1+2)+(1+2)] +2 filaments from dorsal to ventral in the pupa.
Temephos is the World Health Organization (WHO) recommended larvicide and is still being utilized worldwide to control larvae of dengue vectors; Aedes aegypti and Aedes albopictus. The efficacy of a commercial temephos product; Temebate® to exterminate the local populations of Ae. albopictus larvae originated from different land use particularly dengue-risk and dengue-free housing localities as well as agrarian localities including oil palm plantations, rubber estates and paddy fields was assessed to verify its bioefficacy in these localities. Field populations of Ae. albopictus larvae were attained via a larval survey at each study locality. Each Ae. albopictus larval population was subjected to a 24-h larval bioassay using Temebate® at operational dosage of 1 mg/L. Almost all Ae. albopictus larval populations demonstrated mortalities between 7.00% and 100.00% by the end of the first 4 h of Temebate® exposure with the resistance ratios between 0.94 and 8.33. After 24 h of Temebate® exposure, all sixteen Ae. albopictus larval populations exhibited increased mortalities with ten of them showing 100% mortalities. These results confirmed the relevance of Temebate® to be continuously used by the residents of these localities as their control efforts against dengue vectors. Nevertheless, Temebate® application by consumers in dengue-risk localities need to be carefully monitored to prevent further development of temephos resistance among Ae. albopictus populations and substantiated with other vector control approaches.
Species of the Simulium (Simulium) melanopus species-group in Sabah are taxonomically revised by examining type specimens of S. (S.) crassimanum S. (S.) laterale, and S. (S.) nigripilosum, all described from females by Edwards in 1933, and newly collected samples from the vicinity of Mt. Kinabalu. The females of these three species are redescribed, and their males and pupae are described for the first time based on adults reared from pupae. Simulium (S.) liewi Takaoka, 2007 and S. (S.) kinabaluense Smart & Clifford, 1969 are synonymized with S. (S.) crassimanum and S. (S.) laterale, respectively. Simulium (S.) cheedhangi Takaoka, Sofian-Azirun & Ya'cob, 2015 is newly recorded from Sabah. Two new related species, S. (S.) lardizabalae and S. (S.) timpohonense, are described from males reared from pupae. Keys to identify eight species of the S. melanopus species-group in Sabah are provided for females, males, pupae and mature larvae.
A species of Simulium in the Simulium melanopus species-group of the subgenus Simulium (formerly misidentified as S. laterale Edwards from Sabah and Sarawak, Malaysia) is suspected to have dimorphic male scutal color patterns linked with different numbers of upper-eye facets. This study aimed to confirm whether or not these two forms of adult males represent a single species.
A preliminary study on the vertical dispersal of Aedes populations in high-rise apartments was carried out in Presint 9, Putrajaya, Malaysia. Ovitraps were placed indoors within four blocks of high-rise apartments from the ground floors (0.0 - 3.0 m) until up to the tenth floors (28.1 - 30.0 m). Aedes aegypti was the dominant species found in the ovitraps (87.85%), while Aedes albopictus was found in lower numbers. From total number of larvae collected (650), 40.92% of these larvae were obtained from the fourth block; Block D. The peak density of Aedes sp. was observed at level 6 (16.1 - 18.0 m), while Ae. aegypti was found until the tenth floor (28.1 - 30.0 m). In contrast, Ae. albopictus was found only up to the sixth floor (16.1 - 18.0 m). A poor correlation of the mean number of Aedes larvae collected with the level of high-rise apartments occupied (N=40; ρ=-0.349) was also observed which indicated the possibility of lesser Aedes populations to be found at higher level of high-rise apartments. Therefore, larger scale studies are strongly recommended to examine the vertical dispersal of Aedes mosquitoes.
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.
Flea-borne pathogens were screened from 100 individual cat fleas using a PCR approach, of which 38 % were infected with at least one bacterium. Overall, 28 % of the flea samples were positive for Bartonella as inferred from ITS DNA region. Of these, 25 % (7/28) were identified as Bartonella clarridgeiae, 42.9 % (12/28) as Bartonella henselae consisted of two different strains, and 32.1 % (9/28) as Bartonella koehlerae, which was detected for the first time in Malaysia. Sequencing of gltA amplicons detected Rickettsia DNA in 14 % of cat flea samples, all of them identified as Rickettsia asembonensis (100 %). None of the flea samples were positive for Mycoplasma DNA in 16S rRNA gene detection. Four fleas were co-infected with Bartonella and Rickettsia DNAs. Statistical analyses reveal no significant association between bacterial infection and mtDNA diversity of the cat flea. Nevertheless, in all types of pathogen infections, infected populations demonstrated lower nucleotide and haplotype diversities compared to uninfected populations. Moreover, lower haplotype numbers were observed in infected populations.