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Malaria is a major public health problem in north-east India mainly transmitted by Anopheles baimai and An. minimus while Anopheles nivipes plays an important supportive role. The genetic diversity of An. nivipes in north-east India was investigated by employing two mitochondrial DNA markers namely NADH dehydrogenase 5 (ND5) and cytochrome oxidase sub unit II (COII). High genetic diversity in An. nivipes was observed by the detection of 16 haplotypes among 30 sequences of ND5 gene and 29 haplotypes among 29 COII sequences. Anopheles nivipes of north-east India was significantly differentiated genetically with that of neighboring South-east Asian An. nivipes as revealed by pairwise F ST values of 0.127 (p < 0.01) and 0.044 (p < 0.001) for ND5 and COII genes, respectively, suggesting geographical barriers to gene flow in this species between the two geographical areas resulting in significant population structuring.

Mosquitoes are the vectors of several life threatening diseases like dengue, malaria, Japanese encephalitis and lymphatic filariasis, which are widely present in the north-eastern states of India. Investigations on five local plants of north-east India, selected on the basis of their use by indigenous communities as fish poison, were carried out to study their mosquito larvicidal potential against Anopheles stephensi (malaria vector), Stegomyia aegypti (dengue vector) and Culex quinquefasciatus (lymphatic filariasis vector) mosquitoes. Crude Petroleum ether extracts of the roots of three plants viz. Derris elliptica, Linostoma decandrum and Croton tiglium were found to have remarkable larvicidal activity; D. elliptica extract was the most effective and with LC50 value of 0.307 μg/ml its activity was superior to propoxur, the standard synthetic larvicide. Half-life of larvicidal activity of D. elliptica and L. decandrum extracts ranged from 2-4 days.

We validated the feasibility of using Plasmodium falciparum, the human malaria parasite, DNA present in wild caught vector mosquitoes for the characterization of chloroquine resistance status. House frequenting mosquitoes belonging to Anopheles minimus complex were collected from human dwellings in a malaria endemic area of Assam, Northeast India and DNA was extracted from the head-thorax region of individual mosquitoes. Anopheles minimus complex mosquitoes were identified to species level and screened for the presence of Plasmodium sp. using molecular tools. Nested PCR-RFLP method was used for genotyping of P. falciparum based on K76T mutation in the chloroquine resistance transporter (pfcrt) gene. Three of the 27 wild caught An. minimus mosquitoes were harbouring P. falciparum sporozoites (positivity 11.1%) and all 3 were had 76T mutation in the pfcrt gene, indicating chloroquine resistance. The approach of characterizing antimalarial resistance of malaria parasite in vector mosquitoes can potentially be used as a surveillance tool for monitoring transmission of antimalarial drug resistant parasite strains in the community.

Anopheles minimus is one of the major vectors for transmission of malaria disease in north eastern (NE) region of India. The minimus species complex of Minimus subgroup of Myzomyia series of anophelines were studied in malaria affected states--Assam and Arunachal Pradesh (AP) of NE India. Ribosomal DNA markers--second internal transcribed spacer (ITS2) and third domain (D3) of 28S gene were used to characterize An. minimus species. Sequence homogeneity was observed in D3 sequences of An.minimus specimens throughout both the states. However, a transversion in ITS2 sequence of single specimen collected from Assam-Meghalaya border areas illustrates possibility of intra population polymorphism in ITS2 sequence within the geographical region.

Anopheles (Cellia) maculatus is considered a group of at least nine formally named species. Faced with the difficulty of correct morphological identification due to overlapping characters, several member species of the An. maculatus group are known to play important role in malaria transmission in the Oriental region. Current assemblage, distribution and vectorial importance of the member species within the Maculatus group is far from clear in the north-eastern region of India. Our study encompassing 410 individuals, collected from 67 geo-referenced spots across the eight north-east Indian states, identified the presence of 6 member species of the Maculatus group using the molecular tools. Anopheles dravidicus and Anopheles rampae were documented for the first time in this part of India with latter forming the new country record. While Anopheles pseudowillmori (59.5%) and An. maculatus (32%) were widely available species in most of the north-eastern states, restricted distribution of Anopheles willmori to Nagaland and that of Anopheles sawadwongporni and An. rampae to Mizoram state was noted. None of the species was found positive for human malaria parasite. While no intraspecific differences existed in the sequences of second internal transcribed spacer (ITS2) region of ribosomal DNA (r-DNA) of the member species of the Maculatus group within north-east India, few differences were detected in the sequences of An. dravidicus, An. maculatus and An. pseudowillmori from north-east India with species from the neighbouring countries., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Anopheles philippinensis-nivipes species of mosquitoes are considered secondary malaria vectors in the north-east India. However, difficulty in separating An. philippinensis and An. nivipes accurately on the basis of morphological identification, due to the overlapping female wing characters, has led to the paucity of information on their spatial distribution and vectorial importance, essential for understanding malaria disease epidemiology in areas under their influence. In view of this a study was undertaken to generate authentic information on distribution of An. philippinensis and An. nivipes in the north-east India using molecular tools. Between the two, An. nivipes was recorded as the dominant species in Assam, and Nagaland states whereas An. philippinensis was found predominant in the state of Mizoram and Arunachal Pradesh. Species differentiation based on female wing characters proved wrong in about 74% specimens of An. philippinensis but found accurate in case of An. nivipes. No intra-specific variation in second internal transcribed spacer (ITS2) region of ribosomal DNA(r-DNA) in either An. nivipes or An. philippinensis from different geographical areas of north-east India was noted., (Copyright © 2012. Published by Elsevier B.V.)

Background: Anopheles baimaii is a primary vector of human malaria in the forest settings of Southeast Asia including the north-eastern region of India. Here, the genetic population structure and the basic population genetic parameters of An. baimaii in north-east India were estimated using DNA sequences of the mitochondrial cytochrome oxidase sub unit II (COII) gene., Methods: Anopheles baimaii were collected from 26 geo-referenced locations across the seven north-east Indian states and the COII gene was sequenced from 176 individuals across these sites. Fifty-seven COII sequences of An. baimaii from six locations in Bangladesh, Myanmar and Thailand from a previous study were added to this dataset. Altogether, 233 sequences were grouped into eight population groups, to facilitate analyses of genetic diversity, population structure and population history., Results: A star-shaped median joining haplotype network, unimodal mismatch distribution and significantly negative neutrality tests indicated population expansion in An. baimaii with the start of expansion estimated to be ~0.243 million years before present (MYBP) in north-east India. The populations of An. baimaii from north-east India had the highest haplotype and nucleotide diversity with all other populations having a subset of this diversity, likely as the result of range expansion from north-east India. The north-east Indian populations were genetically distinct from those in Bangladesh, Myanmar and Thailand, indicating that mountains, such as the Arakan mountain range between north-east India and Myanmar, are a significant barrier to gene flow. Within north-east India, there was no genetic differentiation among populations with the exception of the Central 2 population in the Barail hills area that was significantly differentiated from other populations., Conclusions: The high genetic distinctiveness of the Central 2 population in the Barail hills area of the north-east India should be confirmed and its epidemiological significance further investigated. The lack of genetic population structure in the other north-east Indian populations likely reflects large population sizes of An. baimaii that, historically, were able to disperse through continuous forest habitats in the north-east India. Additional markers and analytical approaches are required to determine if recent deforestation is now preventing ongoing gene flow. Until such information is acquired, An. baimaii in north-east India should be treated as a single unit for the implementation of vector control measures.

Entomological survey was conducted to know the breeding habitat preference of the forest breeder malaria vector Anopheles baimaii, known earlier as An. dirus species D in the northeastern region of India. Breeding potential of the vector in forest areas was found to be high in water stored in jungle pool (69.84%) followed by elephant footprints with clear water (39.13%) and with turbid water (26.19%), whereas in forest fringe areas, the vector breeding was more prominent in elephant footprints: 65.11% in clear water and 62.5% in turbid water. Although other habitats had shown only low breeding of the vector, all types of habitats were positively correlated with malaria occurrence. Cattle hoof marks (r = 0.998) and elephant footprint (turbid; r = 0.999) explained nearly the same amount of variance. It was observed that deforestation as well as elephant habitat-type destruction had engendered man-elephant conflicts intensively in fringe areas. Seasonal abundance pattern of this vector was found to vary in forest and forest fringe areas in relation to different habitats. Seasonal abundance of An. baimaii was significantly different in different habitats. The Tukey post hoc comparisons indicated that the abundance of An. baimaii in different habitats was significantly higher (P < 0.05) in monsoon season than that of premonsoon and postmonsoon seasons. No significant difference was observed between premonsoon and postmonsoon seasons. The findings therefore will eventually help to predict transmission of malaria in targeted area and in formulating an improved malaria control program in the northeastern region of India.

The Anopheles philippinensis and An. nivipes mosquitoes that form part of the An. annularis species group are morphologically very similar and difficult to differentiate as adults. In consequence, researchers generally refer to all field-collected individuals of these species simply as the An. philippinensis-nivipes species complex. Although this species complex is understood to play a role in the transmission of parasites causing human malaria in north-eastern India, the identity of the exact species involved, and the relative importance of the local An. philippinensis and An. nivipes, are far from clear. To settle this issue, house-frequenting female adults of the An. philippinensis-nivipes species complex were collected from 23 localities in the six north-eastern states, identified to species (using an allele-specific PCR to explore the insects' ribosomal-DNA internal-transcribed-spacer-2 sequences) and checked for Plasmodium DNA (using a nested PCR based on the 18S subunit of the parasite's ribosomal DNA). Of the 337 females of the An. philippinensis-nivipes species complex that were investigated, 275 were identified as An. nivipes and 62 as An. philippinensis. Malarial infection was detected in the heads/thoraces of just two specimens, with P. falciparum DNA detected in one An. nivipes from Nagaland state and one An. nivipes from Assam state. These results provide unambiguous evidence of the presence of both An. philippinensis and An. nivipes in the north-eastern region of India and the involvement of An. nivipes in transmitting P. falciparum in this area.

The identity and distribution of the prevalent member species of the Anopheles dirus complex mosquitoes in the north-eastern region of India was investigated in a cross-sectional study. We altogether collected 267 individuals of An. dirus s.l. from 27 forested/forest fringed locations spread across the seven north-eastern states, identified the species using a ribosomal DNA (r-DNA) second internal transcribed spacer (ITS2) based allele specific polymerase chain reaction (ASPCR) method and sequenced the ITS2 locus in a sub set of mosquitoes. An. baimaii was identified as the main, almost exclusive (266/267), species of the Dirus complex throughout the north-east India with no intraspecific variation in the 479 base pair long ITS2 sequences in 59 of the 60 individuals sequenced. Ribosomal DNA of one individual from Assam state did not amplify in the ASPCR, possessed 786 base pair long ITS2 sequence and showed 99.7% similarity with the sequence of An. dirus species D (An. baimaii) from Yunnan province of China, later referred to as species X of the Dirus complex. These observations suggest the presence of another, possibly the new, species of the Dirus complex, sympatric with An. baimaii, in Assam warranting investigations on its distribution, biology and role in human malaria transmission in north-east India., (Copyright 2010 Elsevier B.V. All rights reserved.)

The sibling species composition of the Anopheles minimus and Anopheles dirus complexes is poorly known in the highly malarious north-eastern region of India where these two vector taxa are accountable for most of the malaria transmission among 30.7 million inhabitants. Prevalent members of these two complexes in this part of India were identified using sequences for the second internal transcribed spacer (ITS2) of ribosomal DNA. Anopheles baimaii (species D) of the An. dirus complex and An. minimus s.s. (species A) of the An. minimus complex were detected in Arunachal Pradesh, Assam, Meghalaya and Nagaland states. No intraspecific variation was observed in the ITS2 sequence (479bp) of An. baimaii whereas a single substitution was detected in the ITS2 sequence (372bp) of An. minimus from Nagaland state.

Anopheles (Cellia) philippinensis Ludlow and Anopheles (Cellia) nivipes (Theobald) are two closely related, morphologically very similar, mosquito species in the Annularis group, which play a supportive role in malaria transmission in north-east India. We amplified and performed sequence analysis for the internal transcribed spacer 2 (ITS2) locus of ribosomal DNA (rDNA) gene of morphologically confirmed specimens of these two species from the states of Assam and Nagaland. An. philippinensis and An. nivipes shared 85.2% sequence similarity and no intra-species variation was found in the nucleotide sequences of the two species. Overall, sequence data of the ITS2 marker revealed that both these species from north-east India differed by as much as have been reported from specimens of eastern Thailand.

The risk of acquiring malaria transmitted by Anopheles baimaii Sallum & Peyton, 2005, formerly known as An. dirus species D (Sallum et al., 2005) (Diptera: Culicidae), at different hours of the night in a forest-fringed village of Assam, North-east India was assessed through all-night mosquito landing catches during 1995-2000. An estimated overall mean biting rate of 36.1 bites/person/night (95% CI = 26.2-45.8), a sporozoite rate of 1.9% (95% CI = 1.1-2.9%) and a parous rate of 58.7% (95% CI = 55.3-62.0%) were recorded. Parous and sporozoite-positive females tended to be caught mainly before midnight. The effective entomological inoculation rate was the highest (0.249 positive bites/person/night) from 21.00 to 24.00 hours, suggesting that the second quartile of the night is the most risky period for malaria transmission by An. baimaii. Considering that approximately 21% of mean inoculations take place before 21.00 hours, it appears that there is a need for appropriate protective measures during the pre-bed time period to supplement the impact of insecticide-treated nets against An. baimaii in north-east India.

Anopheles philippinensis-nivipes complex mosquitoes, captured in outdoor human landing catches and light traps in human dwellings from four different sites in Assam state and adjoining areas, were examined (n=1670) for the presence of circumsporozoite antigen (CSA) through enzyme linked immunosorbent assays (ELISA), using species specific capture monoclonal antibodies, of Plasmodium falciparum and two Plasmodium vivax polymorphs (Pv 210 and VK 247). In ELISA, 28 pools were found positive for CSA that accounted for minimum sporozoite rate of 1.7% (95% CI 1.11-2.41). Twenty five percent (7/28) of the positive pools were reactive for P. falciparum and between the two polymorphs of P. vivax, VK 247 was predominant with 77% (20/26) of all P. vivax positive pools. Results were suggestive of most likely involvement of Anopheles philippinensis-nivipes complex mosquitoes in malaria transmission in north-east India.

To examine the distribution pattern of Anopheles philippinensis and An. nivipes, mosquitoes belonging to An. philippinensis/nivipes group were collected from five sites in north-east Indian state of Assam and its adjoining areas and identified. Presence of both An. philippinensis and An. nivipes, with predominance of the latter taxon, was recorded with conformity on the basis of pupal paddle characteristics. Based on adult wing characters, 91.4% specimens were identified as An. philippinensis and 8.6% as An. nivipes with gradually increasing proportion of An. nivipes in westwardly direction. An. philippinensis/ nivipes was noted to be mid night biter, mainly exophagic and exclusively exophilic. Of the two species, An. philippinensis appears to be mostly anthropophilic and available in broken forest areas whereas An. nivipes seems to be mostly zoophilic and generally found in ecotone zones in plains areas.

In north-eastern India, Anopheles minimus, An. dirus and An. fluviatilis are considered the three major vectors of the parasites causing human malaria. The role in transmission of the other Anopheles species present in this region is not, however, very clear. To examine the vectorial role of the more common anopheline mosquitoes, the heads and thoraces of 4126 female Anopheles belonging to 16 species (collected using miniature light traps set in human dwellings in a foothill village in the Jorhat district of Assam state) were tested, in ELISA, for the circumsporozoite proteins (CSP) of Plasmodium falciparum or the VK-210 and VK-247 polymorphs of P. vivax. Sixty-five pools of head-thorax homogenates, representing 10 different species of Anopheles, were found reactive, giving an overall minimum prevalence of infection (MPI) of 1.58%, with a 95% confidence interval (CI) of 1.21%-2.0%. Among the CSP-reactive pools of mosquitoes, 31% were positive only for P. falciparum, 45% only for P. vivax VK 247, 6% only for P. vivax VK 210, and 18% for both P. falciparum and P. vivax VK 247. The results indicate that not only the proven vector, An. minimus s.l. (MPI = 0.71%), but also several species of Anopheles previously considered unimportant in the epidemiology of malaria, especially An. aconitus (MPI = 3.95%), An. annularis (MPI = 5.8%), the An. hyrcanus group (MPI = 0.48%), An. kochi (MPI = 1.28%), the An. philippinensis-nivipes complex (MPI = 0.94%), and An. vagus (MPI = 3.87%), are important vectors in the foothills of Assam.

Larval ecology of Anopheles dirus, the main vector of forest malaria in north-eastern region of India, was studied in relation to physico-chemical characteristics of its breeding habitats in a rain forest area of Assam, India. Shady stream side pools, positive for the breeding of An. dirus, had significantly higher amounts of total hardness (P < 0.024) in comparison to negative pools of similar type. When compared with An. dirus negative breeding habitats, in hot-wet as well as in cool-dry seasons, An. dirus positive shady ground pools showed higher mean values of total alkalinity, hardness and chloride content, whereas lower pH, dissolved oxygen along with higher total alkalinity and hardness were possessed by An. dirus positive stream side pools. Specificity of breeding habitats of An. dirus in relation to its ecology has been discussed.

To estimate the vectorial capacity of Anopheles dirus, the main vector of forest malaria in the northeastern region of India, in order to gain an understanding of entomological factors related to malaria transmission in forest-fringe areas of Assam, India, an isolated village in the tropical rain forest-fringed area in the district of Dibrugarh, Assam, under the influence of An. dirus alone was studied. Data on various entomological variables required for computation of the vectorial capacity were generated in each month from June 1999 to May 2000 in the field using standard techniques. Malaria prevalence was also studied during the same period in the study village and correlated with the estimated vectorial capacity of An. dirus. Vectorial capacity of An. dirus was highest, 0.779 for Plasmodium vivax (Pv) and 0.649 for Plasmodium falciparum (Pf), during the hot-monsoon season (June-September) and decreased to 0.08 (Pv) and 0.07(Pf) in the temperate postmonsoon season (October-November) before attaining zero values in the cool-dry season (December-February). With increasing temperature in the temperate premonsoon season (March-May), vectorial capacity recorded was 0.119 and 0.82 for Pv and Pf, respectively. Significant positive correlation was seen between the estimated vectorial capacity of An. dirus and the number of new Pf (r = 0.86, p < 0.001) and Pv (r = 0.69, p < 0.02) cases in the study village in different months. Thus, this study highlights the pattern of malaria transmission by An. dirus in a forest-fringe area of Assam that begins in March, peaks in July/August, subsides by November, and remains interrupted between December and February. Measures for controlling malaria in forest-fringe areas should be scheduled accordingly.

Seasonal abundance of Anopheles dirus (s.l.) and malaria prevalence in an isolated forest fringed village was monitored at monthly intervals during August 1995 to July 1996. An. dirus was the only vector species detected during the study period. Its density pattern showed distinct seasonality with the peak occurring in the month of July and very low number during cool dry months. Positive correlation (r = 0.721) was found between the density of An. dirus and the amount of rainfall occurring two weeks prior to the collections. Overall sporozoite rate of 1.6% and parous rate of 64.7% were found in the study. Malaria transmission closely followed the density pattern of An. dirus and was seasonal with slide positivity rate and P. falciparum percentage of 47 and 83% respectively. Mean malaria prevalence was higher (p < 0.05) in females.

Longitudinal surveys at monthly frequency were carried out during 1995-1996 in a forest fringed village of district Dibrugarh, Assam to decipher the breeding and day resting habitats of Anopheles dirus. It regularly bred in small, shallow, rain filled, transient, shady or partly shady puddles/ground pools in the rainy months and in the perinneal streams in the adjoining forest of the village during dry months. In pools, the degree of interspecific association (0.238 +/- 0.174) and index of association (0.428) of An. dirus breeding was highest with Aedes caecus. An. dirus was completely exophilic and rested during day time in the forest mostly on tree trunks upto the height of 4-5 feet from the ground in dark moist niches avoiding direct sun light.

The feeding behavior of Anopheles dirus, the forest breeding, major malaria vector in northeast India was studied. The analysis of blood meals collected from this mosquito revealed that the species was highly anthropophilic in nature, the anthropophilic index being 90.5. The results of bait collection on human and cattle bait also confirmed its biting preference for human hosts. The species was observed to land on human bait throughout the night, showing prominent biting time at 20.00-21.00, 23.00-24.00 and 02.00-03.00 hours during the study period.

Summary: A study was conducted to evaluate the preventive efficacy of insecticide-treated mosquito nets (ITMNs) and mosquito repellent (MR) in a malaria-endemic foothill area of Assam, India, with forest ecosystem. During the first year, a survey was conducted in four demarcated sectors (A–D) to observe the malaria endemicity and vector prevalence patterns before implementing intervention measures. All four sectors were endemic for malaria. The prevalence of established malaria vectors such as Anopheles dirus, A. minimus and A. philippinensis was observed. During the second year, intervention measures were implemented in the four sectors as follows: A, ITMN + MR; B, ITMN; C, MR; D, no intervention. The most effective intervention was in sector A, followed by sectors B and C. Sectors A and B exhibited significantly higher (P < 0.001) malaria protective efficacy during both the first and second years of intervention compared with sector D. The total vector population in the three intervention sectors decreased significantly compared with that of the non-intervention one. Information–education–communication activities motivated the residents to participate actively in the intervention programme. The finding could be an effective model for containment of high malaria morbidity in inaccessible forest fringe areas of the northeastern region of India. [ABSTRACT FROM AUTHOR]