Candidate screening of blast resistance donors for rice breeding.

Rice blast is one of the most serious diseases in the world. The use of resistant cultivars is the most preferred means to control this disease. Resistance often breaks down due to emergence of new races; hence identification of novel resistance donors is indispensable. In this study, a panel of 80 released varieties from National Rice Research Institute, Cuttack was genotyped with 36 molecular markers that were linked to 36 different blast resistance genes, to investigate the varietal genetic diversity and molecular marker-trait association with blast resistance. The polymorphism information content of 36 loci varied from 0.11 to 0.37 with an average of 0.34. The cluster analysis and population structure categorized the 80 National Rice Research Institute released varieties (NRVs) into three major genetic groups. The principal co-ordinate analysis displays the distribution of resistant and moderately resistant NRVs into different groups. Analysis of molecular variance result demonstrated maximum (97%) diversity within populations and minimum (3%) diversity between populations. Among tested markers, two markers (RM7364 and pi21_79-3) corresponding to the blast resistance genes (Pi56(t) and pi21) were significantly associated and explained a phenotypic variance of 4.9 to 5.1% with the blast resistance. These associated genes could be introgressed through marker-assisted to develop durable blast resistant rice varieties. The selected resistant NRVs could be good donors for the blast resistance in rice crop improvement research.

Mechanism of plant mediated methane emission in tropical lowland rice.

Methane (CH4) is predominantly produced in lowland rice soil, but its emission from soil to atmosphere primarily depends on passage/conduit or capillary pore spaces present in rice plants. The gas transport mechanism through aerenchyma pore spaces of rice cultivars was studied to explore the plant mediated CH4 emission. Seven rice cultivars, based on the life cycle duration (LCD), were tested in tropical eastern India. Three LCD groups were, (a) Kalinga 1 and CR Dhan 204 (LCD: 110-120 days); (b) Lalat, Pooja and CR 1014 (LCD: 130-150 days); and (c) Durga and Varshadhan (LCD: 160-170 days). Rate of CH4emission, root exudates, root oxidase activities and shoot aerenchyma pore spaces were analyzed to study the mechanism of plant mediated emission from rice. Aerenchyma pore space was quantified in the hypothesis that it regulates the CH4 transportation from soil to atmosphere. The ratio of pore space area to total space was lowest in Kalinga 1 cultivar (0.29) and highest was in Varshadhan (0.43). Significant variations in the methane emission were observed among the cultivars with an average emission rate ranged from 0.86 mg m-2 h-1 to 4.96 mg m-2 h-1. The CH4 emission rates were lowest in short duration cultivars followed by medium and long duration ones. The greenhouse gas intensity considering average CH4 emission rate per unit grain yield was also lowest (0.35) in Kalinga 1 and relatively less in short and medium duration cultivars. Root exudation was higher at panicle initiation (PI) than maximum tillering (MT) stage. Lowest exudation was noticed in (197.2 mg C plant-1 day-1) Kalinga 1 and highest in Varsadhan (231.7 mg C plant-1 day-1). So we can say, the rate of CH4 emission was controlled by aerenchyma orientation, root exudation and biomass production rate which are the key specific traits of a cultivar. Identified traits were closely associated with duration and adaptability to cultivars grown in specific ecology. Therefore, there is possibility to breed rice cultivars depending on ecology, duration and having less CH4 emission potential, which could be effectively used in greenhouse gas mitigation strategies.

Humid thermal ratio as a tool to assess mango thrips dynamics under subtropical climatic condition.

Weather factors play an important role in occurrence of thrips on mango. Keeping this in view, the present investigation was set out to assess the thrips population dynamics using humid thermal index, based on data sets from 22 fixed plot mango orchards in and around Lucknow. Results revealed that the highest thrips population of 3.36/panicle was recorded in Kakori (Fixed plot -I) orchard, which was followed by 2.4 and 2.06 at CISH III block and Kanar (Fixed II) respectively during the year 2013, whereas corresponding values were 4.05, 3.08 and 2.50 at CISH Block III, CISH Bolck II and Allupur respectively during 2014. The frequency distribution explained that the thrips population of <2 /panicle was widely distributed with highest frequency level. The humid thermal ratio ranged from 1.44 to 2.27 and 1.20 to 2.34 during 2013 and 2014 respectively across standard meteorological weeks. The peak thrips incidence was 6.18 /panicle during 2013 and 4.67/panicle during 2014, the corresponding values of humid thermal ratio were 1.47 and 2.05 respectively. The positive correlation was found between humid thermal ratio and thrips population dynamics during 2013 (r = 0.52**) and 2014 (r = 0.72**). Pooled data showed significant and positive correlation between humid thermal ratio and thrips population. Pooled analysis had explained up to 94 per cent of variation with exponential model (Thrips population = 0.007e2.778HTR, R2 = 0.94**) and suggested that this index might be used in understanding the mango thrips population dynamics under subtropical environmental condition.

Status of DDT and pyrethroid resistance in Indian Aedes albopictus and absence of knockdown resistance (kdr) mutation.

BACKGROUND & OBJECTIVES: Aedes albopictus is one of the vectors for dengue and chikungunya and emergence of pyrethroid resistance in this species could be of a major concern in controlling the vector. This study reports insecticide susceptibility status of Ae. albopictus to DDT and pyrethroids in some Indian populations and status of presence of knockdown resistance (kdr) mutations. METHODS: Three to four day old adult female Ae. albopictus collected from Delhi, Gurgaon (Haryana), Hardwar (Uttarakhand), Guwahati (Assam) and Kottayam (Kerala) were bio-assayed with DDT (4%), permethrin (0.75%) and deltamethrin (0.05%) impregnated papers using WHO standard susceptibility test kit. Mosquitoes were PCRgenotyped for F1534C kdr-mutation in the voltage-gated sodium channel (VGSC) gene. DDT and pyrethroid resistant individuals were sequenced for partial domain II, III and IV of VGSC targeting residues S989, I1011, V1016, F1534 and D1794 where kdr mutations are reported in Ae. aegypti. RESULTS: Adult bioassays revealed varying degree of resistance against DDT among five populations of Ae. albopictus with corrected mortalities ranging between 61 and 92%. Kerala and Delhi populations showed incipient resistance against permethrin and deltamethrin respectively. All other populations were susceptible for both the synthetic pyrethroids. None of the kdr mutations was detected in any of DDT, deltamethrin and permethrin resistant individuals. INTERPRETATION & CONCLUSION: Ae. albopictus has developed resistance against DDT and there is emergence of incipient resistance against pyrethroids in some populations. So far, there is no evidence of presence of knockdown resistance (kdr) mutation in Ae. albopictus.

Gut microbes influence fitness and malaria transmission potential of Asian malaria vector Anopheles stephensi.

The midgut of parasite transmitting vector, Anopheles stephensi is a physiologically dynamic ecological niche of resident microbes. The gut resident microbes of anisomorphic and physiologically variable male and female A. stephensi mosquitoes were different (Rani et al., 2009). To understand the possible interaction of gut microbes and mosquito host, we examined the contribution of the microbe community on the fitness of the adult mosquitoes and their ability to permit development of the malaria parasite. A. stephensi mosquitoes were fed with antibiotic to sterilize their gut to study longevity, blood meal digestion, egg laying and maturation capacity, and consequently ability to support malaria parasite development. The sterilization of gut imparted reduction in longevity by a median of 5 days in male and 2 days in female mosquitoes. Similarly, the sterilization also diminished the reproductive potential probably due to increased rate of the resorption of follicles in ovaries coupled with abated blood meal digestion in gut-sterilized females. Additionally, gut sterilization also led to increased susceptibility to oocyst development upon feeding on malaria infected blood. The susceptibility to malaria parasite introduced upon gut sterilization of A. stephensi was restored completely upon re-colonization of gut by native microbes. The information provided in the study provides insights into the role of the gut-resident microbial community in various life events of the mosquito that may be used to develop alternate malaria control strategies, such as paratransgenesis.

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.

Salivary gland transcriptome analysis in response to sugar feeding in malaria vector Anopheles stephensi.

In this study, we analyzed a small scale transcriptome of salivary glands in sugar fed female mosquitoes. Thirty five percent of the transcripts could not be assigned a function. Some of them may code for salivary gland specific products involved in sugar feeding. We identified and characterized two new putative cDNAs encoding a sugar transporter and a cAMP generating DAPIT (Diabetes-Associated proteins in insulin sensitive tissues). Down regulation of these two cDNAs in response to blood feeding suggest that both AsST and AsDAPIT salivary genes may specifically be involved in the facilitation of sugar metabolism and energy production. The inability to absorb or digest sugar may cause organ failure, improper functioning of nervous system, behavioral disorder and death. Further functional characterization of theses putative transcripts is under investigation to examine their role in the mosquito salivary glands.

Effect of anti-mosquito midgut antibodies on development of malaria parasite, Plasmodium vivax and fecundity in vector mosquito Anopheles culicifacies (Diptera: culicidae).

The effect of anti-mosquito-midgut antibodies on the development of the malaria parasite, P. vivax was studied by feeding the vector mosquito, An. culicifacies with infected blood supplemented with serum from immunized rabbits. In order to get antisera, rabbits were immunized with midgut proteins of three siblings species of Anopheles culicifacies, reported to exhibit differential vectorial capacity. The mosquitoes that ingested anti-midgut antibodies along with infectious parasites had significantly fewer oocysts compared to the control group of mosquitoes. The immunized rabbits generated high titer of antibodies. Their cross reactivity amongst various tissues of the same species and with other sibling species was also determined. Immunogenic polypeptides expressed in the midgut of glucose or blood fed An. culicifacies sibling species were identified by Western blotting. One immunogenic polypeptide of 62 kDa was exclusively present in the midgut of species A. Similarly, three polypeptides of 97, 94 and 58 kDa and one polypeptide of 23 kDa were present exclusively in species B and C respectively. Immunoelectron microscopy revealed the localization of these antigens on baso-lateral membrane and microvilli. The effects of anti-mosquito midgut antibodies on fecundity, longevity, mortality and engorgement of mosquitoes were studied. Fecundity was also reduced significantly. These observations open an avenue for research toward the development of a vector-based malaria parasite transmission-blocking vaccine.

Laboratory colonization of Anopheles fluviatilis species T and U.

The populations of Anopheles fluviatilis James (1902), a foothill vector, collected from village Tilpuri of district Udham Singh Nagar and from villages Auspur, Ismailpur, and Durgapur of district Hardwar were maintained at National Institute of Malaria Research Insectory at 28 +/- 1 degrees C and 80-85% RH. Anopheles fluviatilis sensu lato was identified for two sibling species T and U. A total of 94% of the females of both species T and U oviposited by day 4 after the blood meal. Maximum hatching, that is, 80 and 62% of the eggs of species T and U, was observed on the second and third day, respectively. For species T, mortality in second and third instars was recorded to be 144 +/- 9 (N = 1,600) and 48 +/- 6 (N = 1,200), whereas in species U, it was 196 +/- 13 (N = 1,400) and 70 +/- 8 (N = 1,000), respectively. Mortalities in second instars of species T and U were significantly higher than third instars (P = 0.05). The female and male ratio in pupal stage of species T and U was found to be 53:47 and 58:42, respectively.

Genetic diversity in merozoite surface protein-1 and 2 among Plasmodium falciparum isolates from malarious districts of tribal dominant state of Jharkhand, India.

INTRODUCTION: The genetic make-up of malaria parasite is potent for understanding the parasite virulence, designing antimalarial vaccine and evaluating the impact of malaria control measures. There is a paucity of information on genetic structure of Plasmodium falciparum in Jharkhand, India where malaria is rampant and this study aimed to establish molecular characterization of P. falciparum field isolates from Jharkhand measured with two highly polymorphic genetic markers, i.e. the merozoite surface proteins (MSPs) 1 and 2. METHODS: The genetic diversity of P. falciparum population from low transmission area, Ranchi, Bokaro and Hazaribagh and highly malarious area, Latehar and Palamau districts of Jharkhand were evaluated by polymerase chain reaction-sequencing analyzing msp-1 and msp-2 genes to explore the genetic structure of parasite from this understudied region. RESULTS: A total of 134 P. falciparum isolates were analyzed by polymorphic regions of msp-1 and msp-2 and classified according to prevalence of allelic families. The majority of patients from all the five sites had mean monoclonal infections of 67·1 and 60·4% of P. falciparum for msp-1 and msp-2, respectively, whereas, mean multiple genotypes of 32·8 and 39·5% for msp-1 and msp-2, respectively. Interestingly, we observed higher multiclonal infection in low transmission area as compared to highly malarious area in the case of msp-1 genotypes, whereas in msp-2 higher multiclonal infection was observed in highly malarious area compared to low transmission area. The overall multiplicities of infection of msp-1 and msp-2 were 1·38 and 1·39, respectively. CONCLUSION: This is the first report on molecular characterization of P. falciparum field isolates from Jharkhand. The genetic diversity and allelic distribution found in this study is somewhat similar to other reports from India and Southeast Asian countries. However, P. falciparum infection can be highly complex and diverse in these disease-endemic regions of Jharkhand, suggesting continual genetic mixing that could have significant implications for the use of antimalarial drugs and vaccines.

Expression profile of prophenoloxidase-encoding (acppo6) gene of Plasmodium vivax-refractory strain of Anopheles culicifacies.

Anopheles culicifacies is the main vector for transmission of Plasmodium vivax malaria in the Indian subcontinent. A strain of An. culicifacies isolated from its natural niche displayed complete refractoriness to P. vivax by melanotic encapsulation of ookinetes. Prophenoloxidases are key components of the phenoloxidase cascade that leads to recognition and melanization of invading organisms. We isolated and cloned prophenoloxidase-encoding acppo6 gene of An. culicifacies and analyzed its expression profile under various regimens of immune challenge. The acppo6 was differentially expressed during various stages of larval development. The acppo6 transcription was also up-regulated in response to bacteria and Plasmodium vinckei petteri challenge. The transcript levels of the acppo6 gene were higher in naive adult refractory female mosquitoes as compared with female susceptible mosquitoes. Furthermore, the induction of acppo6 in the susceptible strain upon Plasmodium infection was negligible as compared with that of the refractory strain. The observation is suggestive of the role of acppo6 in effectuating a melanotic response in Plasmodium-incompetent naturally occurring refractory An. culicifacies strain.

Molecular evidence of misidentification of Anopheles minimus as Anopheles fluviatilis in Assam (India).

Anophelesminimus s.l. and Anophelesfluviatilis s.l., two closely related taxa, are reported vectors of malaria in Assam state of India. We determined the DNA sequences of morphologically identified A. minimus s.l. and A. fluviatilis s.l. collected from the Kamrup district in Assam, for two rDNA loci-internal transcribed spacer 2 (ITS2) and D3 domain of 28S rDNA (28S-D3). Analysis of rDNA data revealed that the sequences of both the morphologically identified A. minimus s.l. and A. fluviatilis s.l. from Assam are identical, homologous to the sequences of A. minimus s.s. (former species A) and different from that of all the reported members of the Fluviatilis Complex (species S, T and U). This indicates that A. fluviatilis s.l. being reported in Kamrup district, Assam, in low density, mostly during January to April, is actually a hypermelanic and seasonal variant of A. minimus. It was also found that the banding pattern on chromosome arm 2 (which bears species-diagnostic inversions for the Fluviatilis Complex) of A. minimus and of A. fluviatilis s.l. from Assam is homosequential with A. fluviatilis species U suggesting that probably previously described A. fluviatilis U from Assam were also A. minimus.

Role of CR1 Knops polymorphism in the pathophysiology of malaria: Indian scenario.

BACKGROUND & OBJECTIVES: Plasmodium falciparum is the leading cause of mortality and causes cerebral malaria associated with sequestration caused by cytoadherence of the trophozoite and schizont-infected erythrocytes to the endothelial cells of the deep vascular beds in the brain. Pathophysiology of malaria is complicated by rosetting. Rosetting is a process of binding of uninfected erythrocytes to the erythrocytes infected with mature asexual parasites and is controlled by expression of complement receptor 1 (CR1) on RBC surface. Various polymorphic forms of CR1 are known including molecular weight polymorphism, red blood cell expression levels/density polymorphism and Knops (KN) polymorphism. The Knops blood group includes several allelic pairs; Knops a and b (Kna and Knb), McCoy a and b (McCa, McCb), Swain-Langley (Sla), and Villien (Vil). Knops phenotype Sl (a-) has been found to rosette less effectively than Sl (a+) and hence suggested to be more protective. P. falciparum cases have not reduced much as compared to the reduction in the total number of malaria cases in the past few years. In addition, P. falciparum is the leading cause for all mortality and most of the morbidity in India. We, therefore, investigated the role of CR1 Knops polymorphism in the pathophysiology of malaria in Indian population. METHODS: A case control approach was used for this study. CAPS (Cleaved amplified polymorphic sequence) methodology was adopted. A total of 100 normal individuals (free from any ailment) and 100 individuals suffering from P. falciparum infection (uncomplicated malaria) were recruited for this study. RESULTS: We found that in Indian population (normal individuals and P. falciparum-infected individuals), only the wild type allele is present. INTERPRETATION & CONCLUSION: We concluded that the process of rosetting in the Indian context could be occurring independently of the effect of Knops polymorphism and in part could be controlled by other polymorphisms of the CR1 gene (density and structural polymorphism).

Alleles -308A and -1031C in the TNF-alpha gene promoter do not increase the risk but associated with circulating levels of TNF-alpha and clinical features of vivax malaria in Indian patients.

The biological significance of TNF promoter polymorphism and infectious disease association prompted us to investigate whether TNF-alpha -308 G/A and -1031 T/C promoter polymorphisms are associated with Plasmodium vivax infection, cellular TNF-alpha level and possibly with clinical symptoms by employing PCR-RFLP methods. An overall significant elevation of serum TNF-alpha, IL-6 content (p=0.0002, p=0.002, respectively), whereas highly significant depletion of IL-10 content (p=0.0001) was observed in vivax patients. In addition, TNF-alpha concentration in patients with and without fever were found to be significant (p=0.0001, p=0.0004, respectively). The genotypic distribution for -308 G/A and -1031 T/C positions were found non significant, but it was clinically potent to observe statistically significant distribution of genotypes (p=0.032) in patients with and without fever. Furthermore, the TNF-alpha level in TNF1 and TNF2 genotype for -308 position was significantly higher (p=0.010, p=0.006 respectively). In case of -1031 position TNF-alpha level was significant in ancestral (TT) genotype (p=0.0007) in patients compared to healthy subjects and significantly higher in rare (CC) genotype (p=0.021) as compared to ancestral genotype. In addition, the two polymorphisms 308G/A and -1031T/C were in highly significant LD (D'=0.7992, r(2)=0.6005, p=0.0001) in the patients as well as it is interesting to report that the distribution of novel 308A: 1031C alleles associated haplotypes are nearly the same in patients (0.2610) and in healthy subjects (0.2636). In view of present observation of promoter polymorphism with TNF-alpha level and other clinical parameters of vivax infection, we suggest that evaluation of TNF level and its polymorphisms in the promoter region may be considered to be reliable molecular and immunological markers, possess promising rational for diagnostic potential and immunotherapeutic interventions in clinical vivax malaria. Genetic variation in the promoter region is of biological significance and may play important roles in host defense mechanisms against vivax infection by enhancing cell-mediated immunity and stimulating the protective immunological cascade.

Decreased glutathione-S-transferase activity: diagnostic and protective role in vivax malaria.

OBJECTIVES: The study was undertaken to establish data on the comparative status of antioxidant enzyme GST activity, levels of lipid peroxidation and catalase activity during pathology of Plasmodium vivax malaria in Indian population. We investigated whether serum and plasma glutathione-S-transferase activity in vivax patients are unique to the disease or act as one of the important antioxidant marker for diagnostic potential and candidate for chemoprevention. METHODS: We measured activity of antioxidant enzyme GST, levels of lipid peroxidation and catalase activity during vivax infection. RESULTS: Mean activity of antioxidant enzyme GST in patients serum and plasma were less (6.43 and 5.65 IU/L respectively) than healthy subjects (11.65 and 10.09 IU/L respectively). Lipid peroxidation level and catalase activity of patients (1.77 micromol/L and 29.64 U/mL) with vivax malaria were higher than those of healthy subjects (1.03 micromol/L and 10.87 U/mL). GST activity in serum and plasma was inversely correlated with age in case of vivax patient and were found significant (R2=0.1907 and 0.1605 and p<0.0007 and p<0.01). CONCLUSIONS: In view of the present findings we suggest that GST, lipid peroxidation and catalase evaluation may be considered to be reliable biochemical markers and possess promising rational for diagnostic and therapeutic potential in vivax malaria. Decreasing GST activity and elevated activity of lipid peroxidation and catalase may play important roles in host defence mechanisms against vivax infection by up-regulating oxidative defence mechanisms.

Efficacy of Agnique MMF monomolecular surface film against Anopheles stephensi breeding in urban habitats in India.

Efficacy of Agnique MMF, a monomolecular film formulation, was tested against immatures of Anopheles stephensi, an urban malaria vector in India, in simulated and natural habitats. Simulated field trials carried out in cement tanks showed 100% inhibition of adult emergence for up to 1 wk at 0.4 ml/m2 and up to 3 wk at 1 ml/m2. A small-scale field trial in tanks and wells at 1 and 2 ml/m2 produced more than 75% reduction of late instars and 100% reduction of pupae on day 1. The reduction in pupae at 1 and 2 ml/m2 lasted up to 2 wk in tanks and 5 wk in wells. These results suggest that Agnique MMF could be used as one of the choices in an urban malaria control program.

Effectiveness of mosquito nets treated with a tablet formulation of deltamethrin for malaria control in a hyperendemic tribal area of Sundargarh District, Orissa, India.

A village-scale trial on the efficacy of mosquito nets treated with a tablet formulation of deltamethrin (K-OTAB) against malaria in comparison to untreated nets or no net was conducted in Sundargarh District of Orissa, India, which is characterized by perennial transmission with Plasmodium falciparum accounting for more than 80% of malaria cases. Three villages with similar topographical and epidemiological situations were selected and randomly assigned to 3 arms of the study: treated net, untreated net, and no net. Distribution of nets, based on a sleeping pattern survey, was carried out to cover 100% of the population in treated-net and untreated-net villages. Longitudinal and cross-sectional surveys were conducted to measure malaria incidence, prevalence, and splenomegaly. Malaria incidence was reduced by 64.3% in the village with treated nets, 45.2% in the village with plain nets, and 21.4% in the control village without nets. Comparison of malaria incidence data after 1 year of intervention showed significant difference between villages with treated net vs. untreated net (P < 0.05) and treated net vs. no net (P < 0.005). The incidence of clinical attack rate due to P. falciparum was significantly lower in the population using treated nets than in those using untreated nets and no nets. However, no age-specific protective efficacy of treated nets or untreated nets was observed. A significant reduction occurred in spleen rate and parasite rate in children aged 2-9 years using treated nets or untreated nets. An overall significant reduction was found in parasite rate in the total population using treated and untreated nets as compared to nonusers.

Isolation of a Plasmodium vivax refractory Anopheles culicifacies strain from India.

Anopheles culicifaciessensu lato comprises five sibling species. We report the isolation of an An. culicifacies species B strain which is completely refractory to Plasmodium vivax sporogonic development and partially refractory to P. falciparum. Parasite development in this strain is arrested by a melanotic encapsulation mechanism in the mid-gut. We compare the infectivity of this refractory strain and four other species B strains from different epidemiological zones of India with P. vivax in the laboratory.

On the conspecificity of Anopheles fluviatilis species S with Anopheles minimus species C.

Anopheles fluviatilis and An. minimus complexes,each comprising of at least three sibling species, are closely related and important malaria vectors in Oriental Region. Recently An. fluviatilis species S, which is a highly efficient malaria vector in India, has been made conspecific with An. minimus species C (senior synonym) on the basis of homology in 335 base pair nucleotide sequence of D3 domain of 28S ribosomal DNA (rDNA). We examined the conspecificity of these two nominal species by obtaining and analysing the DNA sequences of nuclear ribosomal loci internal transcribed spacer 2 (ITS2) and D2-D3 domain of 28S rDNA (28S-D2/D3) from those of An. fluviatilis S and An. minimus C. We found that the sequences of An. fluviatilis S are appreciably different from those of An. minimus C with pair-wise distance (Kimura-2-parametre model)of 3.6 and 0.7%for loci ITS2 and 28S-D2/D3, respectively. Pair-wise distance and phylogenetic analyses using ITS2 sequences of members of Minimus and Fluviatilis Complexes revealed that An. fluviatilis S is distantly related to An. minimus C as compared to any other members of the Fluviatilis Complex. These findings suggest that the two nominal species, An. fluviatilis S and An. minimus C, do not merit synonymy. The study also confirms that the reported species An. fluviatilis X is synonym with species S.