[Show abstract][Hide abstract] ABSTRACT: To develop a single-step multiplex PCR to differentiate the aquatic stages of Aedes aegypti, Aedes albopictus and Aedes vittatus collected from different breeding spots in arbovirus endemic/epidemic areas and to detect the most abundant species by the multiplex PCR.
Aquatic stages of different mosquito species were sampled by inspecting artificial and natural breeding sites in domestic and peridomestic areas. DNA was isolated from different stages of the three Aedes species. Using novel primers based on 18S rDNA sequence, a single-step multiplex PCR was developed to clearly distinguish the three Aedes species. It was then evaluated in the aquatic stages of Aedes species collected from different areas.
A total of 1150 aquatic stages were collected from 294 breeding spots, of which 156 contained Aedes species. Discarded tires were the major breeding spots of Aedes species. The aquatic stages were clustered into 230 pools; Ae. albopictus was detected in the largest number of pools, followed by Ae. aegypti and Ae. vittatus.
The Multiplex PCR clearly differentiated the aquatic stages of the three Aedes species and detected that Ae. albopictus was most profuse in different breeding spots surveyed, hence indicating to be the main vector in this region. So control measures can be designed against Ae. albopictus at an early stage to prevent any arboviral outbreak. This method is a convenient tool for precise identification of Aedes vectors during entomological surveys in arbovirus endemic/epidemic areas where several species coexist.
Tropical Medicine & International Health 10/2011; 17(2):235-43. DOI:10.1111/j.1365-3156.2011.02899.x · 2.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Any biological study is only meaningful if the concerned organism is accurately identified; this is particularly important in vector-borne disease studies where correct and precise identification of the target species has medical and practical implications, such as in vector control. The Myzomyia series is divided into four groups including the Funestus group, which consists of five subgroups, i.e. Aconitus, Culicifacies, Funestus, Minimus, Rivulorum, and the Neocellia series, which is divided into three groups Annularis, Jamesii and Maculatus. Members of the Funestus group of Myzomyia and the Annularis group of the Neocellia series are difficult to identify because of the morphological overlap that exists within the groups. Therefore a multiplex polymerase chain reaction (PCR) assay was developed based on the sequence of the D3 region of 28S rDNA to distinguish between four members (An. fluviatilis, An. culicifacies, An. varuna and An. aconitus) of three subgroups (Minimus, Aconitus, Culicifacies) of the Funestus group of Myzomyia and three members (An. annularis, An. pallidus and An. philippinensis) of the Annularis group of the Neocellia series of the Anopheles subgenus Cellia, prevalent in Orissa, India. Polymorphism present on the D3 region of rDNA allowed the development of a species-specific primer that when combined with two universal primers lead to a simple and sensitive multiplex allele-specific polymerase chain reaction (AS-PCR) assay. This assay can be applied as an unbiased confirmatory method for the identification of morphological variants, imperfectly preserved specimens and life stages for which taxonomic keys do not allow a definitive species determination. Finally, phylogenetic relationships between the members of the two series were determined using D3 sequence data. The phylogenetic relationships inferred from maximum parsimony and the neighbour joining analysis separated two distinct monophyletic clades, one consisting of species of Myzomyia and other of species of the Neocellia series. The molecular phylogeny obtained in this work matches with that of the classical morphological taxonomy reasonably well, with proper species arrangements.
Infection, genetics and evolution: journal of molecular epidemiology and evolutionary genetics in infectious diseases 10/2010; 10(7):931-9. DOI:10.1016/j.meegid.2010.05.016 · 3.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Anopheles annularis group mosquitoes, subgenus Cellia Theobald (Diptera: Culicidae), includes five recognized species: An. annularis Van der Wulp, An. nivipes Theobald, An. pallidus Theobald, An. philippinensis Ludlow and An. schueffneri Stanton. From these five, the three most common species found in Orissa were considered for this study because of their remarkable vectorial and behavioral variation and the important role they play in malaria transmission. To identify and understand their role in malaria transmission we developed a single multiplex PCR-based assay. This assay included the detection of human blood feeding habit and Plasmodium falciparum sporozoite presence. Of the 186 An. annularis mosquitoes collected, morphological character-based identification showed that 94 were An. annularis, 54 were An. philippinensis and 38 were An. pallidus. However, the multiplex PCR assay confirmed that 91 were An. annularis, 56 were An. philippinensis and 39 were An. pallidus individuals after adjustments were made for misidentified specimens in the morphological method. Anopheles annularis and An. philippinensis were found positive for human blood, and two samples of An. annularis species were positive for P. falciparum sporozoites. This one-step PCR-based method constitutes a very powerful tool in large surveys of anopheline populations.
Transactions of the Royal Society of Tropical Medicine and Hygiene 05/2009; 103(11):1146-52. DOI:10.1016/j.trstmh.2009.03.022 · 1.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Blood meals of 1,491 Anopheles fluviatilis sensu lato (s.l.), 1,690 An. culicifacies s.l., 719 An. annularis s.l., and 358 An. varuna sensu stricto were examined by gel diffusion method. The overall anthropophilic index (AI) was 78.9%, 1.6%, 3.2%, and 6.7% for An. fluviatilis, An. culicifacies, An. annularis, and An. varuna, respectively. Out of 4 anopheline species studied, only 0.2% of An. culicifacies blood meals contained blood from humans and cattle. Anopheles fluviatilis and An. culicifacies revealed seasonality in their anthropophilic index. An. fluviatilis showed a human forage ratio of more than 1, whereas An. culicifacies, An. annularis, and An. varuna had forage ratios of 2.6, 2.5, and 2.4, respectively, for bovine. There was a correlation between the Al of An. fluviatilis and the malaria slide positivity rate. This study suggests that the use of repellent, insecticide-treated nets will be effective for controlling biting mosquitoes inside houses in Orissa.
Journal of the American Mosquito Control Association 01/2007; 22(4):629-34. DOI:10.2987/8756-971X(2006)22[629:HFPOMV]2.0.CO;2 · 0.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Malaria has declined around Chilika Lake (85 degrees 20'E, 19 degrees 40'N) in Orissa State, India, from hyperendemicity in the 1930s to hypoendemicity during recent decades. Six decades ago, 21 spp. of Anopheles mosquitoes (Diptera: Culicidae) were recorded from this area, including the well known Indian malaria vectors An. culicifacies Giles, An. fluviatilis James, An. maculatus Theobald, An. stephensi Liston and An. sundaicus (Rodenwaldt), the last formerly regarded as the main vector locally. Surveys of Chilika area during 1995-96 found 8 spp. of culicine plus 14 spp. of anopheline mosquitoes, the latter comprising An. subpictus Grassi sensu lato, An. hyrcanus (Pallas) s.l., An. vagus Dönitz, An. annularis van der Wulp s.l., An. culicifacies Giles s.l., An. aconitus Dönitz, An. varuna Iyengar, An. barbirostris van der Wulp s.l., An. philippinensis Ludlow, An. ramsayi Covell, An. jeyporiensis James, An. pallidus Theobald, An. tessellatus Theobald and An. karwari James in decreasing order of abundance. Among indoor-resting female mosquitoes, the anthropophilic index was 4-7% and some species (An. culicifacies, An. subpictus, An. vagus) tended to enter houses for resting after blood-feeding outside. Females of potentially infective age (three-parous) were obtained for An. culicifacies (11%) and An. annularis (<2%), the more abundant established vector in this coastal area, but not for small samples of An. subpictus and An. vagus. Anophelines reported previously but not found in our survey were An. fluviatilis, An. jamesii Theobald, A. maculatus, An. splendidus Koidzumi, An. stephensi, An. theobaldi Giles and the former main vector An. sundaicus.
Medical and Veterinary Entomology 12/2000; 14(4):445-9. DOI:10.1046/j.1365-2915.2000.00265.x · 2.86 Impact Factor