Modified PCR-based assay for the differentiation of members of Anopheles fluviatilis complex in consequence of the discovery of a new cryptic species (species V).

BACKGROUND: Anopheles fluviatilis is a species-complex comprising of four cryptic species provisionally designated as species S, T, U and V. Earlier, a 28S-rDNA based allele-specific polymerase chain reaction (ASPCR) assay was developed for the differentiation of the then known three members of the An. fluviatilis complex, i.e., species S, T, and U. This assay was modified in consequence of the discovery of a new cryptic member, species V, in the Fluviatilis Complex to include identification of new species. METHODS: In the modified procedure, the ASPCR assay was performed first, followed by restriction digestion of PCR product with an enzyme BamH I, which cleaves specifically PCR amplicon of species V and the resultant PCR-RFLP products can differentiate all the four cryptic members of the complex. Morphologically identified An. fluviatilis samples were subjected to sibling species identification by modified PCR-based assay and standard cytotaxonomy. The result of PCR-based assay was validated through cytotaxonomy as well as DNA sequencing of some representative samples. RESULTS: The modified PCR-based assay differentiates all four sibling species. The result of modified PCR-based assay tested on field samples was in agreement with results of cytotaxonomy as well as DNA sequencing of representative samples. CONCLUSIONS: The modified PCR-based assay unambiguously differentiates all four known members of the An. fluviatilis species complex. This assay will be useful in studies related to bionomics of members of the Fluviatilis Complex in their role in malaria transmission.

Biology and bionomics of malaria vectors in India: existing information and what more needs to be known for strategizing elimination of malaria.

India has committed to eliminate malaria by 2030. The national framework for malaria elimination released by the Government of India plans to achieve this goal through strategic planning in a phased manner. Since vector control is a major component of disease management and vector elimination, it requires a thorough understanding of the biology and bionomics of malaria vectors exhibiting definite distribution patterns in diverse ecosystems in the country. Although a wealth of information is available on these aspects, lesser-known data are on biting time and rhythm, and the magnitude of outdoor transmission by the vectors which are crucial for effective implementation of the key vector control interventions. Most of the data available for the vector species are at sensu lato level, while the major vectors are species complexes and their members distinctly differ in biological characters. Furthermore, the persistent use of insecticides in indoor residual spray and long-lasting insecticidal nets has resulted in widespread resistance in vectors and changes in their behaviour. In this document, challenges in vector control in the Indian context have been identified and possible solutions to overcome the problem are suggested. Adequate addressing of the issues raised would greatly help make a deep dent in malaria transmission and consequently result in disease elimination within the targeted time frame.

Characterization and monitoring of deltamethrin-resistance in Anopheles culicifacies in the presence of a long-lasting insecticide-treated net intervention.

BACKGROUND: Deltamethrin-impregnated, long-lasting insecticidal nets (LLINs) were distributed in the study area from November 2014 to January 2015 to evaluate their impact on malaria transmission in the presence of insecticide-resistant vectors. Studies were carried out in 16 selected clusters in Keshkal sub-district, Chhattisgarh State, India to monitor and characterize deltamethrin resistance in Anopheles culicifacies sensu lato. RESULTS: Deltamethrin susceptibility of An. culicifacies decreased in a post-LLIN survey compared to a pre-LLIN survey and was not significant (p > 0.05) while, the knockdown values showed significant increase (p < 0.05). Pre-exposure to piperonyl butoxide, triphenyl phosphate showed synergism against deltamethrin (p < 0.001). Biochemical assays showed significantly (p < 0.05) elevated monooxygenases in 3 of 5 clusters in post-LLIN survey-I that increased to 10 of 11 clusters in post-LLIN survey-II, while esterases were found significantly elevated in all clusters and both enzymes were involved in conferring pyrethroid resistance, not discounting the involvement of kdr (L1014L/S) gene that was heterozygous and at low frequency (4-5%). CONCLUSION: This field study, in a tribal district of India, after distribution of deltamethrin-impregnated LLINs showed decrease in deltamethrin susceptibility in An. culicifacies, a major vector of malaria in this study area and in India. Results indicated development of resistance as imminent with the increase in insecticide selection pressure. There is an urgent need to develop new vector control tools, with insecticide classes having novel mechanisms of resistance, to avoid or delay the onset of resistance. Regular insecticide resistance monitoring and mechanistic studies should be the priority for the malaria control programmes to suggest strategies for insecticide resistance management. The global commitment to eliminate malaria by 2030 needs various efforts that include development of combination vector control products and interventions and few are becoming available.

Entomological determinants of malaria transmission in an epidemic prone area of District Nuh (Haryana state), India.

BACKGROUND & OBJECTIVES: Entomological investigations were carried out in highly malarious villages under Ujina PHC of District Nuh (Haryana state) which is an epidemic prone area in northwestern region of India. The study was aimed to have an in-depth understanding of the entomological parameters influencing malaria transmission in the study area. METHODS: The seasonal prevalence and biological attributes of vector mosquitoes were investigated during 2015 and 2016. Indoor resting vector mosquitoes were collected from human dwellings/cattle sheds and morphologically identified. Anopheles culicifacies were categorized to sibling species by species-specific inversions in polytene chromosomes and An. stephensi to ecological races on the basis of ridge number on egg float. The blood meal source analysis and incrimination studies of vectors were done by counter-current immunoelectrophoresis and enzyme-linked immunosorbent assay, respectively. Insecticide susceptibility test on vectors was performed as per WHO guidelines. RESULTS: Seasonal abundance of An. culicifacies and An. stephensi in the study area showed variation; the peak densities of both the vectors were observed during monsoon months which correlated well with the average monthly rainfall data. Though both vectors were found to be primarily zoophagic, the human blood index of An. culicifacies (HBI = 0.17) was significantly higher than that of An. stephensi (HBI= 0.02). Analysis of sibling species composition of An. culicifacies population showed that it comprised almost of sibling species A (>98%) which is an established malaria vector. Anopheles culicifacies was incriminated for Plasmodium vivax and P. falciparum circumsporozoite (CS) antigen during monsoon months in 2015 and 2016. Assessment of insecticide susceptibility status of malaria vectors against 0.5% deltamethrin revealed that An. culicifacies is more susceptible (95% mortality) than An. stephensi (85% mortality). INTERPRETATION & CONCLUSION: The results suggest that An. culicifacies (species A) is playing a major role in malaria transmission in the study area and is almost susceptible to deltamethrin. Timely two rounds of indoor residual spray of synthetic pyrethroid with proper dosage and good coverage would be helpful in reducing vector population and consequently the malaria incidence. In addition, personal protection measures by the community would supplement the major intervention tool (IRS) in decreasing the man-vector contact.

Malaria transmission in Tripura: Disease distribution & determinants.

BACKGROUND & OBJECTIVES: Malaria is a major public health problem in Tripura and focal disease outbreaks are of frequent occurrence. The state is co-endemic for both Plasmodium falciparum and P. vivax and transmission is perennial and persistent. The present study was aimed to review data on disease distribution to prioritize high-risk districts, and to study seasonal prevalence of disease vectors and their bionomical characteristics to help formulate vector species-specific interventions for malaria control. METHODS: Data on malaria morbidity in the State were reviewed retrospectively (2008-2012) for understanding disease distribution and transmission dynamics. Cross-sectional mass blood surveys were conducted in malaria endemic villages of South Tripura district to ascertain the prevalence of malaria and proportions of parasite species. Mosquito collections were made in human dwellings of malaria endemic villages aiming at vector incrimination and to study relative abundance, resting and feeding preferences, and their present susceptibility status to DDT. RESULTS: The study showed that malaria was widely prevalent and P. falciparum was the predominant infection (>90%), the remaining were P. vivax cases. The disease distribution, however, was uneven with large concentration of cases in districts of South Tripura and Dhalai coinciding with vast forest cover and tribal populations. Both Anopheles minimus s.s. and An. baimaii were recorded to be prevalent and observed to be highly anthropophagic and susceptible to DDT. Of these, An. minimus was incriminated (sporozoite infection rate 4.92%), and its bionomical characteristics revealed this species to be largely indoor resting and endophagic. INTERPRETATION & CONCLUSIONS: For effective control of malaria in the state, it is recommended that diseases surveillance should be robust, and vector control interventions including DDT spray coverage, mass distribution of insecticide-treated nets/ long-lasting insecticidal nets should be intensified prioritizing population groups most at risk to avert impending disease outbreaks and spread of drug-resistant malaria.

Comparative susceptibilities of species T and U of the Anopheles fluviatilis complex to Plasmodium vinckei petteri sporogony.

Anopheles fluviatilis James is an important malaria vector in Indian subcontinent. An. fluviatilis exists as a complex of three sibling species, of which two species, T and U, have been colonized so far. Attempts were made to study the comparative susceptibility of species T and U of the An. fluviatilis complex to rodent malaria parasite Plasmodium vinckei petteri by using Anopheles stephensi Liston as calibrator for variable infectivity in different isolates. An. stephensi, which was used as control, became readily infected, with 60-65% mosquitoes carrying developing oocysts, whereas in species T and species U, approximately 50 and 63%, respectively, of mosquitoes carried oocyts. An. fluviatilis species T was found comparatively less susceptible to P. v. petteri sporogonic development compared with species U. Moreover, significantly lesser sporozoites rate (11%) was observed in species T compared with 31% in species U. Species T and species U are not considered as malaria vectors in India in the field. However, in the laboratory, both these species are able to support the malaria sporogony.

The complexities of malaria disease manifestations with a focus on asymptomatic malaria.

Malaria is a serious parasitic disease in the developing world, causing high morbidity and mortality. The pathogenesis of malaria is complex, and the clinical presentation of disease ranges from severe and complicated, to mild and uncomplicated, to asymptomatic malaria. Despite a wealth of studies on the clinical severity of disease, asymptomatic malaria infections are still poorly understood. Asymptomatic malaria remains a challenge for malaria control programs as it significantly influences transmission dynamics. A thorough understanding of the interaction between hosts and parasites in the development of different clinical outcomes is required. In this review, the problems and obstacles to the study and control of asymptomatic malaria are discussed. The human and parasite factors associated with differential clinical outcomes are described and the management and treatment strategies for the control of the disease are outlined. Further, the crucial gaps in the knowledge of asymptomatic malaria that should be the focus of future research towards development of more effective malaria control strategies are highlighted.

Malaria persistence in Kumaon foothills of District Nainital, Uttarakhand, India.

Clinic data depicted a high incidence of malaria in a forest ecotype in the Kumaon foothills of District Nainital, Uttarakhand, India. A study was therefore conducted to determine the risk factors associated with the transmission of malaria from 2002 to 2004. The man-hour densities of Anopheles culicifacies and An. fluviatilis varied from 2 to 139 and 1 to 69, respectively. The sporozoite rate of 0.24% was recorded in An. culicifacies. Sibling species investigation revealed prevalence of species B (53.8%) and C (46.2%) of An. culicifacies and of species T (100%) of An. fluviatilis complexes. The slide positivity rate and slide falciparum rate were 50.4% and 28.3%, respectively. The infant parasite rate was 42.9% and the enlarged spleen rate among the children was 25.95%. The findings revealed the persistence of malaria in the area mainly due to indigenous transmission through the malaria vector An. culicifacies and the inadequacy of intervention measures. Transmission could be curtailed by indoor residual application of malathion.

Genetic structure of Plasmodium falciparum field isolates in eastern and north-eastern India.

BACKGROUND: Molecular techniques have facilitated the studies on genetic diversity of Plasmodium species particularly from field isolates collected directly from patients. The msp-1 and msp-2 are highly polymorphic markers and the large allelic polymorphism has been reported in the block 2 of the msp-1 gene and the central repetitive domain (block3) of the msp-2 gene. Families differing in nucleotide sequences and in number of repetitive sequences (length variation) were used for genotyping purposes. As limited reports are available on the genetic diversity existing among Plasmodium falciparum population of India, this report evaluates the extent of genetic diversity in the field isolates of P. falciparum in eastern and north-eastern regions of India. METHODS: A study was designed to assess the diversity of msp-1 and msp-2 among the field isolates from India using allele specific nested PCR assays and sequence analysis. Field isolates were collected from five sites distributed in three states namely, Assam, West Bengal and Orissa. RESULTS: P. falciparum isolates of the study sites are highly diverse in respect of length as well as sequence motifs with prevalence of all the reported allelic families of msp-1 and msp-2. Prevalence of identical allelic composition as well as high level of sequence identity of alleles suggest a considerable amount of gene flow between the P. falciparum populations of different states. A comparatively higher proportion of multiclonal isolates as well as multiplicity of infection (MOI) was observed among isolates of highly malarious districts Karbi Anglong (Assam) and Sundergarh (Orissa). In all the five sites, R033 family of msp-1 was observed to be monomorphic with an allele size of 150/160 bp. The observed 80-90% sequence identity of Indian isolates with data of other regions suggests that Indian P. falciparum population is a mixture of different strains. CONCLUSION: The present study shows that the field isolates of eastern and north-eastern regions of India are highly diverse in respect of msp-1 (block 2) and msp-2 (central repeat region, block 3). As expected Indian isolates present a picture of diversity closer to southeast Asia, Papua New Guinea and Latin American countries, regions with low to meso-endemicity of malaria in comparison to African regions of hyper- to holo-endemicity.

Identification of all members of the anopheles culicifacies complex using allele-specific polymerase chain reaction assays.

Anopheles culicifacies, a complex of five isomorphic sibling species, is a major vector of malaria in India and neighboring countries. The five species are provisionally designated as species A, B, C, D, and E. Polytene chromosome examination has been the only method available that differentiates four members of this complex in areas where species E is not prevalent. However, this technique requires the mosquitoes to be in the half-gravid stage and thus limits its application to only about one fourth to one third of the total adult collection and excludes immature stages completely. For species E, both polytene chromosome examination and mitotic chromosome examination of F1 males are required. A polymerase chain reaction (PCR) assay based on the D3 domain (D3-PCR) of 28S rDNA and a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay involving ITS2 of rDNA are available for the discrimination of the members of the An. culicifacies complex. However, both these can only differentiate species A and D from species B, C, and E. We report here two allele-specific PCR assays (AD-PCR and BCE-PCR) using sequence differences in the mitochondrial cytochrome oxidase II (CO II) subunit. The AD-PCR assay distinguishes species A and D, whereas the BCE-PCR assay distinguishes species B, C, and E. Thus, with a combination of two PCR assays, namely the D3-PCR/ITS2-RsaI assay, followed by either the AD-PCR or the BCE-PCR assay, it is possible to identify individual specimens of any of the species of this complex. This assay system is the first, and the best available at present to distinguish all sibling species and especially to discriminate non-vector, species B from all the vector species, A, C, D, and E, of the An. culicifacies complex. Until another DNA-based method involving fewer steps is developed, this assay system can be used in all malaria epidemiologic studies where An. culicifacies is prevalent.

Epidemiology of malaria transmission in forest and plain ecotype villages in Sundargarh District, Orissa, India.

A study of the epidemiology of malaria transmission was undertaken in 13 tribal villages located in forest and plain areas of Sundargarh District of Orissa state, India, from January 2001 to December 2003. In forest areas, intense transmission of malaria is attributed to the highly anthropophagic vector Anopheles fluviatilis sibling species S and is complemented by A. culicifacies sibling species C. In plain areas, A. culicifacies sibling species C is responsible for malaria transmission. The entomological inoculation rate in the forest and plain areas was 0.311 and 0.014 infective bites/person/night, respectively, during 2003. Malaria transmission is perennial both in forest and plain areas but is markedly low in the plain area compared with the forest area. Plasmodium falciparum accounted for 85.0% of the total malaria cases during the study period. In forest and plain areas, the number of P. falciparum cases per 1000 population per year was 284.1 and 31.2, respectively, whereas the parasite rate was 14.0% and 1.7%, respectively. In forest areas, clinical malaria occurs more frequently in children aged 0-5 years and declines gradually with increasing age. The study showed that villages in forest and plain areas separated by short geographical distances have distinct epidemiology of malaria transmission.

Comparative assessment on the prevalence of mutations in the Plasmodium falciparum drug-resistant genes in two different ecotypes of Odisha state, India.

Considering malaria as a local and focal disease, epidemiological understanding of different ecotypes of malaria can help in devising novel control measures. One of the major hurdles in malaria control lies on the evolution and dispersal of the drug-resistant malaria parasite, Plasmodium falciparum. We herewith present data on genetic variation at the Single Nucleotide Polymorphism (SNP) level in four different genes of P. falciparum (Pfcrt, Pfmdr1, Pfdhfr, and Pfdhps) that confer resistance to different antimalarials in two different eco-epidemiological settings, i.e. Hilly-Forest (HF) and Riverine-Plain (RP), in a high malaria endemic district of Odisha state, India. Greater frequency of antimalarial resistance conferring SNPs and haplotypes was observed in all four genes in P. falciparum, and Pfdhps was the most variable gene among the four. No significant genetic differentiation could be observed in isolates from HF and RP ecotypes. Twelve novel, hitherto unreported nucleotide mutations could be observed in the Pfmdr1 and Pfdhps genes. While the Pfdhps gene presented highest haplotype diversity, the Pfcrt gene displayed the highest nucleotide diversity. When the data on all the four genes were complied, the isolates from HF ecotype were found to harbour higher average nucleotide diversity than those coming from RP ecotype. High and positive Tajima's D values were obtained for the Pfcrt and Pfdhfr genes in isolates from both the HF and RP ecotypes, with statistically significant deviation from neutrality in the RP ecotype. Different patterns of Linkage Disequilibrium (LD) among SNPs located in different drug-resistant genes were found in the isolates collected from HF and RP ecotypes. Whereas in the HF ecotype, SNPs in the Pfmdr1 and Pfdhfr were significantly associated, in the RP ecotype, SNPs located in Pfcrt were associated with Pfmdr1, Pfdhfr and Pfdhps. These findings provide a baseline understanding on how different micro eco-epidemiological settings influence evolution and spread of different drug resistance alleles. Our findings further suggest that drug resistance to chloroquine and sulfadoxine-pyrimethamine is approaching fixation level, which requires urgent attention of malaria control programme in India.

Larvivorous fish in wells target the malaria vector sibling species of the Anopheles culicifacies complex in villages in Karnataka, India.

Malaria was a major problem in a sericulture area of Karnataka, south India, where Anopheles culicifacies s.l. and A. fluviatilis s.l. were considered to be the main vectors. Sibling species complexes of these two species were analysed in three ecologically different villages. Among A. culicifacies, only sibling species A and B were found. In Puram, a village with 22 wells, species A predominated; species B predominated in a village with four wells and a stream, and in a village with a stream and no wells. Poecilia reticulata fish were introduced into all wells and streams in the villages, and after one year no vectors were found in Puram, and all, or nearly all, A. culicifacies were species B in the other two villages. All A. fluviatilis belonged to the sibling species T. Blood meal analysis indicated that a few of the A. culicifacies collected had fed on humans while all the A. fluviatilis had fed on bovines. Before the introduction of fish, the annual parasite incidence for malaria was high in Puram, but much lower in the other two villages. From 1998 (over one year after release of fish) until 2003, no malaria cases were detected in the three villages.

Comparative susceptibility of three important malaria vectors Anopheles stephensi, Anopheles fluviatilis, and Anopheles sundaicus to Plasmodium vivax.

The 3 laboratory-colonized malaria vectors, i.e., Anopheles stephensi, An. sundaicus, and An. fluviatilis, were studied for their comparative susceptibility to Plasmodium vivax sporogony. There was no significant difference in oocyst and sporozoite recruitment by these 3 species, whereas the geometric mean (GM) of the oocyst number per midgut was significantly lower in An. fluviatilis as compared with that in the other 2 species. There was no difference in the GM of oocyst between An. stephensi and An. sundaicus. Adaptability to laboratory conditions and susceptibility to plasmodial infection suggest that An. fluviatilis and An. sundaicus can also be used as a vector model for vector-parasite interaction studies.

Relative Abundance and Plasmodium Infection Rates of Malaria Vectors in and around Jabalpur, a Malaria Endemic Region in Madhya Pradesh State, Central India.

BACKGROUND: This study was undertaken in two Primary Health Centers (PHCs) of malaria endemic district Jabalpur in Madhya Pradesh (Central India). METHODS: In this study we had investigated the relative frequencies of the different anopheline species collected within the study areas by using indoor resting catches, CDC light trap and human landing methods. Sibling species of malaria vectors were identified by cytogenetic and molecular techniques. The role of each vector and its sibling species in the transmission of the different Plasmodium species was ascertained by using sporozoite ELISA. RESULTS: A total of 52,857 specimens comprising of 17 anopheline species were collected by three different methods (39,964 by indoor resting collections, 1059 by human landing and 11,834 by CDC light trap). Anopheles culicifacies was most predominant species in all collections (55, 71 and 32% in indoor resting, human landing and light trap collections respectively) followed by An. subpictus and An. annularis. All five sibling species of An. culicifacies viz. species A, B, C, D and E were found while only species T and S of An. fluviatilis were collected. The overall sporozoite rate in An. culicifacies and An. fluviatilis were 0.42% (0.25% for P. falciparum and 0.17% for P. vivax) and 0.90% (0.45% for P. falciparum and 0.45% for P. vivax) respectively. An. culicifacies and An. fluviatilis were found harbouring both P. vivax variants VK-210 and VK-247, and P. falciparum. An. culicifacies sibling species C and D were incriminated as vectors during most part of the year while sibling species T of An. fluviatilis was identified as potential vector in monsoon and post monsoon season. CONCLUSIONS: An. culicifacies species C (59%) was the most abundant species followed by An. culicifacies D (24%), B (8.7%), E (6.7%) and A (1.5%). Among An. fluviatilis sibling species, species T was common (99%) and only few specimens of S were found. Our study provides crucial information on the prevalence of An. culicifacies and An. fluviatilis sibling species and their potential in malaria transmission which will assist in developing strategic control measures against these vectors.

Differentiation of members of the Anopheles fluviatilis species complex by an allele-specific polymerase chain reaction based on 28S ribosomal DNA sequences.

Anopheles fluviatilis, one of the major vectors of malaria in India, is a complex of at least three cryptic species provisionally designated as species S, T, and U. Identification of the cryptic species of An. fluviatilis complex is of paramount importance in disease control program due to contrasting differences in their vectorial efficiency, preference for feeding on humans, and resting behavior. Species S, T, and U are morphologically indistinguishable at any stage of their life cycle and can be identified only by the examination of species-specific fixed inversions in the polytene chromosomes. We report an allele-specific polymerase chain reaction assay for the differentiation of members of An. fluviatilis complex, which is based on differences in nucleotide sequences in D3 domain of 28S ribosomal DNA. The assay was evaluated against chromosomally examined individuals from different localities with different sympatric associations and was found to differentiate unambiguously all the members of the complex.

Morphotaxonomic studies to identify the members of the Anopheles subpictus Grassi (Diptera: Culicidae) species complex in riverine villages of District Sonepat, Haryana State, India.

Anopheles subpictus was reported to be a complex of four sibling species provisionally designated as species A, B, C and D. Present studies in District Sonepat, Haryana have revealed the sympatricity of species A, C and D based on the reported distinct morphotaxanomical identification characters in different life stages of the mosquito. Studies on field collected adult mosquitoes and isofemale progeny did not show variations in the prevalence of sibling species and also showed a possibility of use of a single reported morphological character in a given life stage for the identification of the members of this complex.

A review of malaria transmission dynamics in forest ecosystems.

Malaria continues to be a major health problem in more than 100 endemic countries located primarily in tropical and sub-tropical regions around the world. Malaria transmission is a dynamic process and involves many interlinked factors, from uncontrollable natural environmental conditions to man-made disturbances to nature. Almost half of the population at risk of malaria lives in forest areas. Forests are hot beds of malaria transmission as they provide conditions such as vegetation cover, temperature, rainfall and humidity conditions that are conducive to distribution and survival of malaria vectors. Forests often lack infrastructure and harbor tribes with distinct genetic traits, socio-cultural beliefs and practices that greatly influence malaria transmission dynamics. Here we summarize the various topographical, entomological, parasitological, human ecological and socio-economic factors, which are crucial and shape malaria transmission in forested areas. An in-depth understanding and synthesis of the intricate relationship of these parameters in achieving better malaria control in various types of forest ecosystems is emphasized.

Population cytogenetic and molecular evidence for existence of a new species in Anopheles fluviatilis complex (Diptera: Culicidae).

Anopheles fluviatilis James, an important malaria vector in the Oriental region has been established as a complex of at least three cryptic species which vary in their biological characteristics and malaria transmission potential. The sibling species S, T and U of Fluviatilis Complex can be identified by examination of species-specific fixed inversions in the polytene chromosomes and can also be differentiated by an allele-specific PCR assay based on differences in the D3 region of 28S ribosomal DNA (rDNA) of these species. Here we report a new An. fluviatilis population from villages under Laksar Community Health Centre, District Haridwar (Uttarakhand state), India which differs from the three sibling species of Fluviatilis Complex by two fixed paracentric inversions, s(1) and S in polytene chromosome arms 2 and 3 respectively. Longitudinal study carried out in study villages showed that the new cytotype was sympatric with species T and U in all the collections and no inversion heterozygotes were observed between them. Thus presence of two fixed paracentric inversions in polytene chromosomes with total absence of inversion heterozygotes demonstrates reproductive isolation which unequivocally establishes this cytological variant as a new species, provisionally designated as species V in the Fluviatilis Complex. Analysis of DNA sequences of D3 domain of 28S rDNA and ITS 2 region has also shown that species V is distinctly different from species S, T and U. With the discovery of new species in the Fluviatilis Complex, in-depth studies are required to know its distribution pattern and biological characteristics and to ascertain its role in malaria transmission.