Vectorial capacity and genetic diversity of Anopheles annularis (Diptera: Culicidae) mosquitoes in Odisha, India from 2009 to 2011.

Anopheles annularis is one of the major vectors of malaria in Odisha, India. The present study was undertaken to determine the vectorial capacity and assess the genetic diversity of An. annularis collected from different endemic regions of Odisha. Mosquitoes were collected from thirteen endemic districts using standard entomological collection methods from 2009 to 2011. Sibling species of An. annularis were identified by PCR-RFLP and sequencing of D3 region of 28S ribosomal DNA (rDNA) region. Plasmodium falciparum (Pf) sporozoite rate and human blood fed percentage (HBF) were estimated by multiplex PCR using Pf and human specific primers. Genetic diversity of An. annularis was estimated by ISSR markers. Out of 1647 An. annularis collected, 1353 (82.15%) were collected by mechanical aspirators and 294 (17.85%) by light trap. 49 (2.97%) were positive for human blood and 18 (1.09%) were positive for Pf sporozoite. PCR-RFLP and sequencing analyses detected only An annularis A in the study areas. Overall genetic differentiation among An. annularis populations was moderate (FST=0.048) and showed significant correlation between genetic distance and geographic distance (r=0.882; P<0.05). Angul population proved to be genetically unique and was highly divergent FST>0.110) from other populations, suggesting low gene flow between them. The study indicated that only An. annularis A was found in Odisha with potential vectorial capacity that can play a major role in malaria transmission. ISSR markers proved to be useful molecular tools to evaluate genetic variability in An. annularis populations.

Genetic structure and Wolbachia genotyping in naturally occurring populations of Aedes albopictus across contiguous landscapes of Orissa, India.

BACKGROUND: Aedes albopictus has recently been implicated as a major vector in the emergence of dengue and chikungunya in several parts of India, like Orissa, which is gradually gaining endemicity for arboviral diseases. Ae. albopictus is further known to be naturally infected with Wolbachia (maternally inherited bacterium), which causes cytoplasmic incompatibility (CI) in mosquitoes leading to sperm-egg incompatibility inducing the death of embryo. Knowledge of genetic diversity of Ae. albopictus, along with revealing the type of Wolbachia infection in Ae. albopictus is important to explore the genetic and biological characteristics of Ae. albopictus, prior to exploring the uses of CI-based vector control strategies. In this study, we assessed the population genetic structure and the pattern of Wolbachia infection in Ae. albopictus mosquitoes of Orissa. METHODS AND RESULTS: Ae. albopictus mosquitoes were collected from 15 districts representing the four physiographical regions of Orissa from 2010-2012, analyzed for genetic variability at seven microsatellite loci and genotyped for Wolbachia strain detection using wsp gene primers. Most microsatellite markers were successfully amplified and were polymorphic, showing moderate genetic structure among all geographic populations (FST = 0.088). Genetic diversity was high (FST = 0.168) in Coastal Plains populations when compared with other populations, which was also evident from cluster analyses that showed most Coastal Plains populations consisted of a separate genetic cluster. Genotyping analyses revealed that Wolbachia-infected Ae. albopictus field populations of Orissa were mostly superinfected with wAlbA and wAlbB strains. Wolbachia superinfection was more pronounced in the Coastal Plain populations. CONCLUSION: High genetic structure and Wolbachia superinfection, observed in the Coastal Plain populations of Orissa suggested it to be genetically and biologically more unique than other populations, and hence could influence their vectorial attributes. Such high genetic diversity observed among Coastal Plains populations could be attributed to multiple introductions of Ae. albopictus in this region.

Genetic characterization of E2 region of Chikungunya virus circulating in Odisha, Eastern India from 2010 to 2011.

Chikungunya virus (CHIKV) infection has caught attention yet again as it rages around the globe affecting millions of people. The virus caused epidemic outbreaks affecting more than 15,000 people in Odisha, Eastern India since 2010. In this study, complete genetic characterization of E2 gene of CHIKV circulating in Odisha from 2010 to 2011 was performed by virus isolation, RT-PCR, molecular phylogenetics and bioinformatics methods. Phylogenetic analyses revealed the circulation of Indian Ocean Lineage (IOL) strains of ECSA genotype of CHIKV in Odisha. Several mutations were detected in the E2 gene, viz. E2-R82G, E2-L210Q, E2-I211T, E2-V229I and E2-S375T which had various adaptive roles during the evolution of CHIKV. The CHIKV E2 peptide 57KTDDSHD6³ was predicted to be the most probable T-cell epitope and peptide 84FVRTSAPCT9² predicted to be the common T and B cell epitope having high antigenicity. The amino acid positions 356-379 and 365-385 were predicted to be transmembrane helical domains and indicated E2 protein anchorage in intracellular membranes for effective interaction with the host receptors. Positive selection pressure was observed in five specific sites, 210, 211, 318, 375, and 377 which were observed to be fixed advantageously in most viral isolates. Structural modeling revealed that E2 gene of CHIKV was composed of 3 domains and the major adaptive mutations were detected in domain B, which can modulate binding of CHIKV to host cells, while the transmembrane domain in domain C and the epitopes were located in domain A, which was found to be most conserved. This is the first report from Eastern India demonstrating a predictive approach to the genetic variations, epitopic regions and the transmembrane helices of the E2 region. The results of this study, combined with other published observations, will expand our knowledge about the E2 region of CHIKV which can be exploited to develop control measures against CHIKV.

Anopheles culicifacies sibling species in Odisha, eastern India: First appearance of Anopheles culicifacies E and its vectorial role in malaria transmission.

OBJECTIVE: To identify the Anopheles culicifacies sibling species complex and study their vectorial role in malaria endemic regions of Odisha. METHODS: Mosquitoes were collected from 6 malaria endemic districts using standard entomological collection methods. An. culicifacies sibling species were identified by multiplex polymerase chain reaction (PCR) using cytochrome oxidase subunit II (COII) region of mitochondrial DNA. Plasmodium falciparum (Pf) sporozoite rate and human blood fed percentage (HBF) were estimated by PCR using Pf- and human-specific primers. Sequencing and phylogenetic analysis were performed to confirm the type of sibling species of An. culicifacies found in Odisha. RESULTS: Multiplex PCR detected An. culicifacies sibling species A, B, C, D and E in the malaria endemic regions of Odisha. An. culicifacies E was detected for the first time in Odisha, which was further confirmed by molecular phylogenetics. Highest sporozoite rate and HBF percentage were observed in An. culicifacies E in comparison with other sibling species. An. culicifacies E collected from Nawarangapur, Nuapara and Keonjhar district showed high HBF percentage and sporozoite rates. CONCLUSION: An. culicifacies B was the most abundant species, followed by An. culicifacies C and E. High sporozoite rate and HBF of An. culicifacies E indicated that it plays an important role in malaria transmission in Odisha. Appropriate control measures against An. culicifacies E at an early stage are needed to prevent further malaria transmission in Odisha.

Molecular investigations of dengue virus during outbreaks in Orissa state, Eastern India from 2010 to 2011.

Dengue is one of the most important arboviral diseases in India. Orissa state in Eastern India reported the first dengue outbreak in 2010, followed by extensive outbreaks in 2011, affecting large number of people. Detailed entomological, serological and phylogenetic investigations were performed in mosquitoes and patients serum collected from dengue virus (DENV) affected areas of Orissa. The combination of DENV specific IgM capture-ELISA and reverse-transcription PCR (RT-PCR) detected high DENV positivity in serum samples. DENV was detected in mosquitoes reared from field caught pupae by RT-PCR, which confirmed the vertical transmission of DENV that may have an important role in the recurrence of dengue outbreaks. Phylogenetic analyses revealed the circulation of Indian lineage of DENV-2 (genotype-IV) and DENV-3 (genotype-III) in vectors and patients serum in Orissa from 2010 to 2011, DENV-2 being the prevailing serotype. Selection analyses within the C-prM region showed that the emergence of DENV-2 and DENV-3 in Orissa was constrained by purifying selection which suggested the role of ecological factors like mosquito density and behavior in the recurrent outbreaks. Aedes albopictus was found to be the most abundant vector in the areas surveyed, followed by Aedes aegypti. Indoor breeding spots (earthen pots) were most abundant, with high pupal productivity (38.50) and contributed maximum Aedes species in the affected areas. The DENV infection rate estimated by maximum likelihood estimate (MLE) was high for indoor breeding Aedes (4.87; 95% CI: 1.82, 10.78) in comparison to outdoor breeding Aedes (1.55; 95% CI: 0.09, 7.55). The high MLE in Ae. albopictus (4.72; 95% CI: 1.94, 9.80) in comparison to Ae. aegypti (1.55; 95% CI: 0.09, 7.54) indicated that Ae. albopictus was the main DENV vector responsible for the outbreaks. The results indicated the circulation of two virulent serotypes of DENV in Orissa, mainly by Ae. albopictus with the implication for implementation of intradomecile vector control measures to prevent the spread of dengue.

Molecular investigations of chikungunya virus during outbreaks in Orissa, Eastern India in 2010.

Chikungunya virus (CHIKV), an arthritogenic alphavirus, is transmitted to humans by mosquitoes of genus Aedes, mainly Aedes aegypti and Aedes albopictus. The resurgence of CHIKV in different parts of India is a point of major public health concern. In 2010, chikungunya outbreaks with high epidemic magnitude were recorded in coastal areas of Orissa, Eastern India, affecting more than 15,000 people coupled with severe arthralgia and prolonged morbidites. Detailed entomological, serological and molecular investigation of this unprecendented outbreak was carried out by collecting and studying 1359 mosquito samples belonging to A. albopictus, A. aegypti, A. vittatus, A. edwardsii and Culex species and 220 patients serum from the affected areas. In this study, CHIKV specific IgM capture-ELISA and reverse-transcription PCR (RT-PCR) were done to detect recent infection of CHIKV in serum samples and adult mosquitoes collected from the affected areas. The high maximum likelihood estimate (MLE) (15.2) in A. albopictus mosquitoes indicated that it was the principal vector involved in transmission of CHIKV in Orissa. Phylogenetic analysis revealed that the CHIKV strains involved in the outbreak belonged to the Indian Ocean Lineage (IOL) group within the East, Central and South African (ECSA) genotype. Genetic characterization of envelope glycoprotein (E1 and E2) genes revealed that all the CHIKV isolates from Orissa had the E1-A226V mutation that enhances viral dissemination and transmissibility by A. albopictus mosquitoes along with E2-L210Q and E2-I211T mutations, which play an epistatic role with E1-A226V mutation in adaptation of CHIKV to A. albopictus by increasing its midgut infectivity, thereby favoring its vectorial capacity. Our results showed the involvement of A. albopictus vector in the recent outbreaks in Orissa and circulation of IOL strains of ECSA genotype of CHIKV with E1-A226V, E2-L210Q and E2-I211T mutations in vectors and patients serum.

Development and evaluation of a single-step multiplex PCR to differentiate the aquatic stages of morphologically similar Aedes (subgenus: Stegomyia) species.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

The mosquitocidal activity of methanolic extracts of Lantana cramera root and Anacardium occidentale leaf: role of glutathione S-transferase in insecticide resistance.

Larvicidal activity of methanolic plant extracts of Lantana cramera (P1) root and Anacardium occidentale (P2) leaf was investigated against the larvae of the three mosquito species (Culex quinquefasciatus, Anopheles stephensi, and Aedes aegypti reared in the laboratory), and the respective glutathione S-transferase (GST) activity was analyzed as an index of protection against the extracts. The LC50 (extract concentration that shows 50% mortality) values of P1 extract for An. stephensi, Ae. aegypti, and Cx. quinquefasciatus were 132.55, 27.82, and 11.68 ppm, respectively, whereas those of P2 extract were 56.81, 912, and 10.79 ppm, respectively. In general, in the untreated groups, the level of GST activity was significantly higher in Ae. aegypti in comparison with An. stephesi and Cx. quinquefasciatus. However, the enzyme activity failed to show any response when treated with either of the plant extracts in Ae. aegypti. However, an increase in the GST activity was recorded in extract-treated larvae of both An. stephensi and Cx. quinquefasciatus. The results of the current study suggest that both the plant extracts show species-specific mosquitocidal potential. Induction of GST activities in survived An. stephensi and Cx. quinquefasciatus larvae suggests the role of this enzyme in conferring resistance to the plant extracts.

Molecular identification and phylogeny of Myzomyia and Neocellia series of Anopheles subgenus Cellia (Diptera: Culicidae).

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.

Analysis of the phylogenetic relationship of Anopheles species, subgenus Cellia (Diptera: Culicidae) and using it to define the relationship of morphologically similar species.

Studies on the relationship of various vectors and non-vectors of malaria from the evolutionary point of view are important. Use of molecular methods to define phylogeny helps to understand the interrelationship among the members of the anophelines and elucidate the ambiguity that has arisen from improper classification. It could also help to design molecular markers for species differentiation, particularly in those which pose difficulty when classified, based on morphological features. In the present study, the phylogenetic relationships among the species of the anophelines of subgenus Cellia are inferred from the mitochondrial genes COI and COII, the ribosomal RNA gene, in particular the D3 region, and Internal Transcribed Spacer 2 (ITS2) region. The molecular phylogeny obtained in this work matches with that of the classical morphological taxonomy reasonably well, and was useful in properly defining species positions and resolving the ambiguity that normally arises due to morphological taxonomy. The correct arrangement of the various anopheline taxa as per the traditional morphological character-based classification of anophelines was there when we considered the D3 region of 28S rRNA gene and ITS2 region of rDNA. However, the arrangement of the taxa did not match with that of the morphological classification in some aspects, when we considered the COI and COII region of mitochondrial DNA. It may have been due to the variable degree of the rate of evolution of the different genes within the organism. Thus, a proper selection of those particular genes that evolve at the rate that is reflected at the species differentiation level, could help to construct the correct phylogenetic relationship among the anophelines and could be used to correlate with the grouping pattern done from the morphological perspective.

A unique methodology for detecting the spread of chloroquine-resistant strains of Plasmodium falciparum, in previously unreported areas, by analyzing anophelines of malaria endemic zones of Orissa, India.

Generally, clinical data is referred to study drug-resistance patterns of Plasmodium falciparum in an area. This is only possible after a clear manifestation of drug-resistance parasites inside the human host, and thereafter detection by healthcare persons. The detection of spread of drug-resistant P. falciparum in a population, before any pathological symptoms detected in humans is possible by analyzing the anopheline vectors, transmitting malaria. In the present study we implemented a new strategy to detect the spread of chloroquine-resistant (CQR) strains of P. falciparum by the major malaria vectors prevalent in selected endemic regions of Orissa, India. We screened P. falciparum positive vectors by using polymerase chain reaction (PCR)-based assay and thereafter detected K76T mutation in the Pfcrt gene, the chloroquine-resistance marker, of parasites present within the vectors. This study showed higher transmission rate of chloroquine-resistant P. falciparum parasites by Anopheles culicifacies and Anopheles fluviatilis. This study will help in assigning chloroquine-resistant P. falciparum sporozoite transmission potential of malaria vectors and suggest that by adopting the mentioned methodologies, we can detect the spreading of the drug-resistant P. falciparum in its transmission. This approach of studying the anophelines during regular vector collection and epidemiological analysis will give the knowledge of chloroquine-resistance pattern of P. falciparum of an area and help in devising effective malaria control strategy.

The development and evaluation of a single step multiplex PCR for simultaneous detection of Anopheles annularis group mosquitoes, human host preference and Plasmodium falciparum sporozoite presence.

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.

Multiplex PCR assay for the detection of Anopheles fluviatilis species complex, human host preference, and Plasmodium falciparum sporozoite presence, using a unique mosquito processing method.

A multiplex PCR assay has been developed for detection of Anopheles fluviatilis cryptic species, their human host preference, and Plasmodium falciparum presence in the mosquito. PCR conditions were optimized using primer sets specific for A. fluviatilis cryptic species, Homo sapiens, and P. falciparum and evaluated with field-collected mosquitoes. A unique mosquito processing method was used for screening P. falciparum carrying capacity and human host preference of A. fluviatilis mosquitoes in first-round multiplex PCR. The vectorial status of the mosquito for P. falciparum parasite was confirmed in second-round PCR. Of the 121 collected mosquitoes, 92 were of S type, 26 of T type, and 3 were of other types. Human host preference was dominant in S type, of which 4% were P. falciparum sporozoite positive. This assay and processing method can also be used to evaluate vector competence of other anophelines.