Xil International Conference on ‘Vector an Vector Borne Diseases —Challengesin 21" Century: Their Global Impact and stratepic Management Population Genetic Structure of Malaria Vector Anopheles stephensi in North-Western India Vipin’, Sharma Vinita’, Mukesh’ and Arti Prasad’ riage Insite of India, Chandrabani Post Box No. 11, Dehradun, Utarathand, India *Department of Zoology, University College of Science, Mohanlal Sukhadia University, Udaipur, Rajasthan, India E-mail: 'vipinsharma_24@yahoo.com ABSTRACT We studied five population of Anopheles stephens n North-west India to pers ther fo conto! ‘molaria at vector lev Trronah transgenic mosquitoes, having paras nhibiting genes, and detecting the spread of insecticide resistance gene The Bayesion analysis of five populations using STRUCTU mined three clusters viz, cluster 1 (hunjtunu), cluster {(Nogour, alore and Joipur) and cluster 3 (Udorpur). shun wos found to be isolated and unique in its genet tmakeup from the remaining populations of Anopheles stephens. Facto spondence analysis and first generati migrant tests also corroborated the fini suring analysis of population assignment. The Anopheles stepher population from shunjunun came : S whch can be used for introduction of transgenic mosquitoes, Wi parasitic inhibiting genes, 05 2 part Stusies for controling the malaria at vector level. The Anopheles stepher comaations {rom eipur, Nogaur ond Jjore showed more connectivity in the frm of mare migration between them shot | tested for the spread of ins we genes on priority basis ifthe conditions of insecticide resistance arises that region, Keywords: Arovol ils, Bayesian Analysis, FatoralCorespondence Anclysiy Malaria Control renee? INTRODUCTION “Anopheles stephensi Liston, 1901 is the vector of malaria on Indian subcontinent (Subbarao et 1987). It is one of the main vectors of malaria in the state of Rajasthan (Joshi er al., 2005). development of resistance against insecticides in mosquitoes has hampered the control of malar Vector level. The genetic modification of mosquitoes to reduce the vector competence holds promising alternative to control the malaria (Moreira etal 2002). "To achieve that transgenic mosquitoes with parasitic inhibiting genes can be released in imalatia affected regions. For this the knowledge of proper gene flow and population ge structure in the populations of mosquitoes in that region is essential. Keeping this in mind, a pioneer study on gene flow and genetic structure in Anopheles step populations along and across the Aravalli Hills in North-west India was done by us during i 3009 (Vipin ef af, 2010). The Aravali hills starts from Delhi and running mainly through iisecting the state of Rajasthan diagonally into two regions namely the north-west and the s¢ aaeeeere, ond in Dungarpur district of Gujarat state. The study was conducted assumin, parbarity of Aravali hills asa reason for uneven distribution of Anopheles stephens and so as mi aaa i North-west India. Anopheles sephensi mosquitoes were collected from three places © se iF the Avavalli Hills ie. from Jalore (25°21' N, 72°37) Nagaur 27°12! N, 7344 E Jhunjhunu (28°08 ‘N, 75°24" E). On the east of the hills, mosquitoes were collected from Ud (24°35" N, 73°43E) and Jaipur (26°55'N, 75°49" E), while no mosquitoes were found at Bhilwar ‘hari Sokvadia Unversity, Udaipor, R__ Population Genet Sructure of Malaria Vector Anopheles stephens/in Nodh-Wester Indi - sar. The populations of Anopheles stephensi were genotyped using nine loci (F10, H2ii, E7T, E12, GI1, Bl, DST and A10) developed by Veradi et al. 22002). The results showed that the level of e flow along both sides of Aravalli Hills (Nm = 0.92 and 0.14; west vs. east of Aravalli Hills, sectively) was low as compared to across the Hills (Nm = 2.25) (Vipin et al., 2010). This was ther supported by the positive correlation (r = 0.0149 and 0.157, respective to Fsr and Rsx) ween genetic differentiations and geographic distances irrespective of the hills, which indicated 1 the geographical distance was the main reason for genetic differentiation and gene flow than Aravalli Hills (Vipin et al., 2010). However, it is still not clear that which population should be checked on priority basis for the ead of insecticide resistance genes and which population should be targeted if the need arises to ease transgenic mosquitoes to control the malaria at vector level. Therefore, we further analyzed allelic data of of nine loci, to know the population genetic structure in that region to get ‘onal information with our earlier study for preparing better malaria control strategies lizing mosquitoes having vector competent genes and to get the information on spread of seticide resistance genes. We used Factorials Correspondence Analysis (FCA) and Bayesian Sed Marko Chain Monte Carlo method to get the information on population structuring and sub ‘ucturing. We studied the migration between the individuals of five Anopheles stephensi mosquito pulations using first generation migrant test. ATERIALS AND METHODS udy area, sample collection, DNA isolation, PCR amplification of microsatellite and genotyping te described in the previous study (Vipin er al, 2010). Genotyping data was further analyzed for stecting genetic diversity using GENALEX 6 (Peakall and Smouse, 2006), clustering between spulations using STRUCTURE 2.33 (Pritchard et al, 2000a) and GENETIX 4.05 (Belkhir etal. 104). Clustering method implemented in STRUCTURE 2.3.3 (Pritchard et al. 2000a) using imixed model with burning length 40000 and run length 60000 and setting the k from 2 to 8 was sed to infer population structure in five Anopheles stephensi mosquito populations. The value of Ak ae calculated using STRUCTURE HARVESTER (Earl ef al, 2012). The analysis of first sneration migrants in five different populations was done by GENECLASS 2 (Piry et al., 2004) ESULTS iENETIC DIVERSITY ‘he allele frequencies and allelic patterns in five Anopheles stephensi populations over 9 loci are resented in figure 1 and table 1, respectively. The numbers of locally common alleles with requeney less than 25% were ail, however, with a frequency less than 30% were found in all five ‘opulations (Table 1):The numbers of alleles (6.8) and expected heterozygosity (0.59) in five Inophelesstephensi populations were come out 10 be almost same in concordance with earlier study. Jumbers of different alleles with a frequency equal and greater than 5% were found in all five vopulations of Anopheles stephensi (Table 1). Unbiased expected heterozygosity came out to be ‘igher than Hi,(Table 1), Effective numbers of alleles differed among all populations (Table 1). The talues of Shannon’s Informative Index (I) and numbers of private alleles in all populations are siven in table 1. The values of standard errors are shown in bold in Table 1 97\ector and Vector Borne Diseases-—Challenges in 21" Century: Their Global Impact and Strategic Management Figure 1: Allele Frequency Distribution in Five Anopheles stephensi Populations ‘Table 1: Mean Allelic Patterns Across Populations: Mean Values Population Jhuajhuaw Nagaur Jalore Jaipur | Udaipur Na 73561425 | 3.6671500_| 6.000 1302 | 7.2221.722 | 7.333 1.667 Na Freq. >= 5% 43330687 | 3.4440.801 [4.220.760 | 4.222 0830 | 4.000.645 Ne 4.093 1.097 | 3.0140925 [3.597 0.812 | 4.0051.204 | 4.219 0.894 I 14430204 | 1.0900246 | 1.3090.232 | _1.26400.289 | 1.430.215 ‘No, Private Alleles 1.440.669 | 1.000373 _|1.44400.444 | 1.44405503 | 1.778 0.722, ‘No. [Comm Alleles (=: 10,000-0.000 | 0.000 0.000 | 0.000 0.000 | ~0.000-0.000 | 0.000 0.000 ‘No. LComm Alleles (=: 0.8890.351__|~ 0.778 0364 | 0.5560.242 | ~0.8890.309 | 11110309 He. 0.66000.051 | 0.509 0.093 | 0.614002 | 0.545.0.100 | 0.678 0.082 ‘UHe 0.67 0.052 | 0.5230.095 | 0.633 0.094 | 0.3590.102 | 0.6930.053, Na=No. of Different Alleles Na (Freq >=5%)=No. of Different Alleles with a Frequency >=5% Ne=No. of Effective Alleles=1 / (Sum pi*2) * Sum (pi * Ln (pi) ‘. of Alleles Unique to a Single Population I=Shannon's Information Index: No. Private Alleles=: No. LComm Alleles (<=25%)=No. of Locally Common Alleles (Freq. or Fewer Populations No. LComm Alleles (<=50%) or Fewer Populations 5%) Found in 25% ‘0. of Locally Common Alleles (Freq. >=5%) Found in 50% He=Expected Heterozygosity=1-Sum pi ~2 UHe=Unbiased Expected Heterozygosity=(2N / (2N-1)) * He POPULATION STRUCTURE The ad hoc quantity (Ak) came out to be 3 for five Anopheles stephensi populations. More than 9! percent individuals from Jhunjhunu and Udaipur were grouped in cluster 1 and cluster respectively, however, individuals from Jaipur, Nagaur and Jalore were grouped in cluster (Table 2). Individuals from Nagaur, Jalore and Jaipur were found to be admixed (Figure 2). proportion of membership of each population in each of the three clusters in five Anopheles steph populations is given in table 2. The expected heterozygosity between individuals in cluster 1, 2 98Population Genetic Structure of Malaria Vector Anopheles stephensi in North-Western India were found to be 0.73, 0.65 and 0.75, respectively. The allele frequency divergence between cluster and 2 was 0.11 which was less than between cluster 2 and 3. Factorials Correspondence Analysis CA) implemented in GENETIX also showed the formation of three clusters (Figure 3). Cluster 1 4s formed with individuals from Jhunjhunu showing one individual from Nagaur. Cluster 2 was tmed by individuals from Jalore, Nagaur and Jaipur. Cluster 3 was formed by the individuals >m Udaipur having some sympatricity with cluster 2. The level of gene flow was more in cluster 2 57) than between cluster I vs 2 (Nm=1.42) and cluster 2 os 3 (Nm=1.50), Table 2: Proportion of Membership of each Population in each of Three Clusters Given Population Inferred Clusters Total no of Assigned | Total no of un-Assigned 1 2. 3 Individuals Individuals (unjhunu (28) 0.027 (0) [0.030 (0) 0.944 (27) 27 T agaur 30) 0.202 (2) [0.738 (18) | 0.059 (0) 20 10 lore (30) 0.425(2) [oat a) | 0.43406) 3 2 ipur 30) 0.027 (0) [0.867.(26) | 0.106 (1) z 3 daipur GO) 0.932 (26) [0.054(0) | 0.014 (0) 26 4 Cluster | dead al Figure 2; Population Genetic Structure in Anopheles Stephensi Populations Revealed three Clusters by bayesian based Marko Chain Monte Carlo Method Implemented in Structure 2.3.2 eer pbatbeeusse ie bee Figure 3: Factorials Correspondence Analysis (FCA) of Anopheles stephensi Mosquitoes from Five Populations using GENETIX 4.05 Software Showed the Presence of Three Clusters‘esa and Vester Borne Diseases—Chalenges in 21 Cenuy: Ther Global Impact and Svatege Management The results of first generation migrant test, implemented in GENECLASS 2, also supported earlier two analyses. In which Jhunjhunu and Udaipur populations were found to having only one and two individuals with first generation migrant probability below 0.01, respectively. In Nagaur, Jalore and Jaipur 3, 3 and 2 individuals were found to be with first generation migrant probabi ity below 0.01, respectively. Analysis of molecular variance (AMOVA) showed 14.78% variation among three clusters and 30.4% variation among individuals within clusters, DISCUSSION Distribution of alleles varied significantly among five populations (Figure 1). The individuals were evenly distributed among populations as the Shannon’s information index values for all populations were higher than zero. The environment of Anopheles tephensi distribution in Aravalli Hills range seems to be not much different as the numbers of locally common alleles (with a Frequency 50% or less) is more or less the same in all five populations except the population of Jalore (Table 1) because these alleles represents the presence of local adaptations. of cerain genotypes. Presence of unique alleles (Table 1) in all five populations add further support to the previous study by proving again the mutations as the basis for genetic differentiation, Tn the earlier Stuy a significant differentiation was found in genetic diversity between east and west of Aravalli Hills. In this study18 % variation among populations and 82% variation within populations way found in overall analysis by molecular variance (Figure 4; table 3). As mentioned in results 14.78% variation was found among three clusters and 30.4 % variation among individuals. An overall gene, flow among all individuals in all ive populations was found to be 1.16, while it was mere as7 among jadividuals from Jaipur, Nagaur and Jalore. Moreover, the clustering of five slnopheles stephens: populations into three groups by STRUCTURE and GENETIX also indicates mone gene flow in Jaipur, Nagaur and Jalore populations. In FCA analysis the compression of axis extremes ive. the spacing of samples along an axis may not be the effect of true differences in species Somposition but the two factors, like more number first generation migrants in the populations of Nagaur, Jalore and Jaipur and average distances between individuals in cluster 2 wae lower (0.65) than cluster 1 (0.73) and 3 (0.75), seems sufficient enough to overrule the this assumption, The “fasons for clustering ean be hypothesized because of connectivity between these three populations dlue to access of easy passages through low height Aravalli Hills, which we have tied re explain through an imaginary extrapolation of results of STRUCTURE and GENETIX overlaid on topography of the region using a modified map from Das (2010; Figure 5). So far as the height of SECO aoe Anopheles stephensi is concerned it has been reported in higher numbers at the height of 600 meters and in maximum at the height of 380 meters, while immature were confined to *p0- 345 meters from the state of Uttarakhand (Devi and Jauhari, 2008). Other mosquito species like Cy, mtaeniovionchus, an Asian Japanese Encephalitis virus vector, has been collected from the height of ‘more than 100 meters in India and China (Ritchie and Rochester, 2001). Cx, anuliosirs wos prhorted at the height of 310 meters in New South Wales, Australia (Ritchie and Rochester, 2001). Therefore, based on the average range of height of occurrence (300-600 meters) and their clustering Pe CktaPolated the possible route of migration of Anopheles stephensi mosquitoes in the Aravallt hills region (Figure 5). The 690 km stretch of Atavalli Hills is not continuous throughout its range Put discontinuous and may provide easy passages for mosquitoes to migrate through, It appears from the map that mosquitoes from Jaipur region can easily pass through these discontineces stretches of low heights to the regions of Nagaur and Jalore. Bit the values of F.,, between Jaipur ys Nagaur and Jaipur vs Jalore were 0.20 and 0.21, respectively, which are mose than Fen, value between Udaipur vs Jalore (0.14) and Jaipur vs Jhunjhunu (0.17) which indicate the height of 100)Population Genetic Structure of Malaria Vector Anopheles stephens! in North-Western india he structuring is genetic but it could have onl tation. Therefore, the possible route, of mi; vothesized, which represents the low land valli Hills range. ly happen through migration over time and igration for mosquitoes could have been the same as areas in comparison to the average height of entire Figure 4: Percentages of Molecular Variance in Five Anopheles stephensi Populations ‘Table 3: Summary of AMOVA in Five Anopheles stephensi Populations Source rig 3S MS Est. Var, % gPops 4 28130 70.330 2.030 13% in Pops 135, 1368-600 338 9.439 a2 i 19) 1649.920 T1468 100% igure 5: Extrapolation of Three Clusters in Anopheles stephensi Populations Overlaid on Topography of the Aravalli Hills Region using Modified Map from Das, 2010, 404Vector and Vector Bore Diseases —Challenges in 21" Century: Ther Global Impact and Strategic Management As the purpose of this study was to look for connectivity and gene flow between Anoph stephensi populations for control of malaria at vector level by having an idea on possibility of spr. of insecticide resistance genes and to detect the possibilities of spread of a transgene in transge mosquitoes making them incapable of harboring the malaria parasites. The prerequisite to carry any such experiment always require an isolated population to avoid any harmful and irreversi results. In this context the population of Jaunjunun came out to be completely isolated from res the four populations which can prove to be a good experimental population to carry out such mo studies. Further the Anopheles stephensi population of Jaipur, Nagaur and Jalore shall be checked spread of insecticide resistance, if it happens in near future in that area, on priority basis becauss their more connectivity with each other. REFERENCES [1] Das, Sanat Kumar (2012) Natural vs Anthropogenic Background Aerosol Contribution to the Radiation Budget « Indian Thar Desert, Atmospheric Aerosols-Regional Characteristics-Chemistry and Physics, Hayder Abdul-Ras Ed), ISBN: 978:953-51-0728-6, InTech, Available from: htp:/www.intechopen.com/books/atmespherie-seros regional-characteristics-chemistry-and-physicv/natural-vs-anthropogenic-background-aerosol-contribution-to-the ‘adiation-budget-over-indian-thar-de /ldx.doi.org/L0.5772048722 [2] Devi, N. Pemola, Jauhari, R. K. 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