POPULATION BIOLOGY/GENETICS
Isolation and Characterization of Polymorphic Microsatellite Markers from the Malaria Vector Anopheles fluviatilis Species T (Diptera: Culicidae) MANILA LATHER,1 DIVYA SHARMA,1 AMITA S. DANG,2 TRIDIBES ADAK,1 AND OM P. SINGH1,3
J. Med. Entomol. 52(3): 408–412 (2015); DOI: 10.1093/jme/tjv007
ABSTRACT Anopheles fluviatilis James is an important malaria vector in India, Pakistan, Nepal, and Iran. It has now been recognized as a complex of at least four sibling species—S, T, U, and V, among which species T is the most widely distributed species throughout India. The taxonomic status of these species is confusing owing to controversies prevailing in the literature. In addition, chromosomal inversion genotypes, which were considered species-diagnostic for An. fluviatilis species T, are unreliable due to the existence of polymorphism in some populations. To study the genetic diversity at population level, we isolated and characterized 20 microsatellite markers from microsatellite-enriched genomic DNA library of An. fluviatilis T, of which 18 were polymorphic while two were monomorphic. The number of alleles per locus among polymorphic markers ranged from 4 to 19, and values for observed and expected heterozygosities varied from 0.352 to 0.857 and from 0.575 to 0.933, respectively. Thirteen markers had cross-cryptic species transferability to species S and U of the Fluviatilis Complex. This study provides a promising genetic tool for the population genetic analyses of An. fluviatilis. KEY WORDS Anopheles fluviatilis T, malaria, microsatellite marker, population genetics
Anopheles fluviatilis sensu lato James is an important malaria vector in India, Pakistan, and Nepal (Rao 1984) belonging to the Minimus subgroup in the series Myzomyia. It is widely distributed in eastern Asia (Pakistan, Afghanistan, India, Nepal, and Bangladesh) and in parts of western Asia (Iran, Iraq, eastern and southern Saudi Arabia, Oman, Bahrain, and Russia; Rao 1984). Earlier record of presence of An. fluviatilis in Myanmar, Thailand, and southern China is doubtful and possibly is a case of misidentification as An. minimus (Harrison 1980, Chen et al. 2002, Singh et al. 2010). This taxon is composed of at least four isomorphic species, provisionally designated as S, T, U, and V identifiable based on fixed paracentric inversions present on polytene chromosome arm 2 and 3 (Subbarao et al. 1994, Nanda et al. 2013). Chromosomal karyotype 2 þ q1 þ r1 þ s1; 3 þ S is the standard arrangement for species S, 2q1 þ r1 þ S1; 3 þ S for species T, 2 þ q1r1 þ s1; 3 þ S for species U, and 2 þ q1 þ r1s1; 3S for species V (where numbers indicate chromosome numbers; þ q1, þ r1, þ s1, and þ S are the standard arrangment; and q1, r1, s1, and S are inverted arrangment). Marked differences in density, host-feeding
1
National Institute of Malaria Research (NIMR), Sector-8, Dwarka, New Delhi-110077, India. Maharshi Dayanand University (MDU), Rohtak-124001 (Haryana), India. 3 Corresponding author, e-mail:
[email protected]. 2
preferences, and role in malaria transmission exist among these members. Species S is predominantly anthropophagic, found mainly in hilly and forested areas, and an efficient malaria vector, whereas species T and U are primarily zoophagic (Nanda et al. 1996). However, reviews of old vector incrimination data suggest that An. fluviatilis s.l. is an important malaria vector throughout country (Nagpal and Sharma 1995). Because An. fluviatilis T is the only species widely distributed throughout India, it is suspected that it is an important malaria vector species. Laboratory feeding experiment has shown that species T is highly efficient malaria vector (Adak et al. 2005). Cytological evidences reveal existence of two types of species T populations, one with fixed chromosomal inversion karyotype, i.e., monomorphic for standard karyotype þ q1, and other is polymorphic, i.e., with karyotypes q1, þ q1, and q1/ þ q1 in Hardy–Weinberg equilibrium (where þ q1 is standard and q1 is inverted karyotype present on chromosome 2; Subbarao 1998, Singh et al. 2004). It is not clear whether these two populations are same or reproductively isolated. If same, then it is necessary to know the extent of gene flow between these two populations. The highly polymorphic microsatellite markers may provide a better understanding of population genetics of An. fluviatilis species T. No microsatellite marker has been previously reported for An. fluviatilis which can be substantial in establishing extent of gene flow. The present work describes the isolation and characterization of
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LATHER ET AL: MICROSATELLITES FROM Anopheles fluviatilis T
polymorphic microsatellite markers that can be used to study the genetic variation among populations of An. fluviatilis T.
Materials and Methods DNA Isolation and Microsatellite-Enriched Library Construction. Microsatellites were isolated using the protocol by Glenn and Schable (2005). Total genomic DNA was extracted from a pool of An. fluviatilis T mosquitoes, originating from Haldwani, Uttarakhand (29.22 N, 79.52 E), and maintained in laboratory, using the DNeasy blood and tissue kit (Qiagen, Krefeld, Germany). Genomic DNA was digested with RsaI restriction enzyme (New England Biolabs, USA) at 37 C for 60 min, and double-stranded superSNX linkers (Glenn and Schable 2005) were ligated to resulting DNA fragments. Linker-ligated fragments were hybridized with biotin-labeled (GA)15 and (GT)15 probes and captured with streptavidincoated magnetic beads (NEB, USA). Enriched DNA fragments were amplified by a PCR using the superSNX-24 forward as a primer. The amplified fragments were cloned in pGEM-T Easy Vector System (Promega Corporation, Fitchburg, WI) and transformed into competent cells (DH5a strain of Escherichia coli). The competent cells were grown on agar plate in presence of X-gal and IPTG (Isopropyl-b-D-1thiolgalactopyranoside). White colonies were picked up and checked for the insert of proper size by colony PCR using universal M13/T7 primers. A total of 142 clones which had DNA insert of size ranging 300 bp–1.0 kb were selected and sequenced. Sequencing reaction was performed using BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) and electrophoresed in ABI 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA). Out of 142 clones, 118 were found positive for microsatellite repeats. PCR Amplification and Genotyping. Flanking primers were designed for 35 microsatellite loci using Primer 3.0 software (Rozen and Skaletsky 2000). Cold primers were checked for PCR amplification using DNA of two laboratory colonized populations of An. fluviatilis T originating from Haldwani and Rourkela (Odisha; 22.25 N, 84.88 E). Twenty primer pairs, which gave successful PCR amplification with single discrete band, were used to check polymorphism in An. fluviatilis species T individuals collected from Pauri Garhwal, Uttarakhand (29.80 N, 78.74 E). Identification of field mosquitoes for sibling species was done by a species-diagnostic allele-specific PCR (Singh et al. 2004). For DNA fragment analysis of some of the microsatellite markers, one primer of each primer-pair was labeled at 50 end with fluorescent dyes 6-FAM, NED, PET, or VIC, and PCR amplification was performed in a total volume of 15 ml containing 1 PCR buffer (10 mM Tris-HCl, 1.5 mM MgCl2), 200 mM of each dNTP, 0.25 mM forward and reverse primers, and 0.75 U of Taq polymerase (AmpliTaq Gold). PCR reactions were carried out with initial denaturation at 95 C for 3 min; followed by 35 cycles of 95 C for 30 s, 55 C
409
for 30 s, 72 C for 30 s; and a final extension at 72 C for 15 min. For fragment analysis of other markers, method developed by Schuelke et al. (2000) was used where one of the each microsatellite flanking primer pair was tailed with M13(-21) universal sequence (50 TGTAAAACGACGGCCAGT-30 ) at 50 end. In this method, an additional M13(-21) primer labeled with fluorescent dye (6-FAM or VIC) was used in a PCR reaction. The PCR reactions contained 1 PCR buffer (10 mM Tris-HCl, 1.5 mM MgCl2), 200 mM of each dNTP, 0.0625 mM of M13(-21)-tailed primer, 0.25 mM of untailed primer, 0.25 mM of fluorescent-labeled M13(-21) universal primer, and 0.75 U of Taq polymerase (AmpliTaq Gold). Reactions were performed with initial denaturation at 95 C for 3 min; 27 cycles at 95 C for 30 s, 55 C for 30 s, 72 C for 30 s; followed by 8 cycles at 95 C for 30 s, 53 C for 30 s, 72 C for 30 s; and a final extension at 72 C for 15 min. PCR products were checked for amplification on 2% agarose gel. Resulting PCR-amplified products were run on the ABI 3730xl DNA analyzer (Applied Biosystems) with Genescan 500-LIZ, using as a size standard for allele size scoring. Alleles were scored using the GeneMapper software version 4.0 (Applied Biosystems). Genetic Data Analysis. The number of alleles, allele frequencies, observed heterozygosities, expected heterozygosities, test of Hardy–Weinberg equilibrium for each locus, and linkage disequilibrium among loci were estimated using Arlequin ver. 3.5 Software (Excoffier and Lischer 2010). Null allele frequencies were calculated using MICRO-CHECKER 2.2.3 (Van Oosterhout et al. 2004). The statistical significance value for all multiple test was corrected by the sequential Bonferroni’s procedure (Rice 1989). Polymorphic Information Content (PIC) was calculated for each marker using following formula (Botstein et al. 1980).
PIC ¼ 1
k X i¼2
pi2
k1 X k X
2Pi2 Pj2
i¼1 j¼iþ1
Cross Cryptic Species Amplification. Microsatellite markers were also tested on other two members of Fluviatilis Complex, i.e., species S and U. Genotyping of all 20 microsatellite markers was done on five individuals of each of An. fluviatilis S collected from Sundergarh, Odisha (22.12 N, 84.03 E), and An. fluviatilis U collected from Haridwar, Uttarakhand (29.95 N, 78.17 E). Distribution of Microsatellite on Chromosome. To know the possible location of microsatellite markers on specific chromosome, DNA sequences of microsatellite markers were searched for homology with Anopheles gambiae Giles chromosome database available in VectorBase through BLAST. Results and Discussion A total of 142 clones from microsatellite-enriched DNA library were sequenced, out of which 83% were found positive for microsatellite repeats. Primer pairs
(CA)8
(GT)8
(CA)8CC(CA)12
(CA)10
(GA)8AA(GA)4
(GT)12
(CA)13
(CA)7
(AC)8
(CA)7
(CA)7
(CA)8
(CA)9
(CA)7
(GT)7
(GT)10,(CA)6
(TC)7(GT)6(GA)5TA(GA)7
(CA)7
(GAA)5
(CA)6
AF66
AF155
AF125
AF84
AF128
AF129
AF51
AF8
AF31
AF74
AF134
AF132
AF156
AF30
AF187
AF48
AF98
AF80
AF29
AF68
185–197 171–177 164–184 202–220 230–242 253–266 175–183 117–123 249–274
199 159 157 187 221 242 166 108 249
234–265 208–218 261 174
240 197 239 157
246–286
171–207
190
245
194–214
131–149
155 199
176–206
162–170
166 210
170–178
179
F: VIC-TGACCAAAGATGGTGTTCCA R: AGCAATCCACACTGGCTACC F: PET-TTCGAAGGAGCTTTCCAAGT R: GCACGTTATGTGCCCTCAAT F: FAM-GTGTGAGGTGGGAATTGTCC R: ACAATGAACGCTGGTACACG F: FAM-GCAGTCTGCATGGTAACGAA R: CTGCCGTAGCGAAATGGT F: PET-CGAAGCGAACCGTGTAGATT R: CTGGCGAACGATTGGACAT F: FAM-CGGAAGGTTCGATTTCCTTT R: GGCGAAGCAGTAAGAAGTGC F: NED-ACAAACGCACATACCGACAA R: ACTCATCACCGGTTTTACAGC F: M13-AACGGCGGCTAATGAGACT R: CATGCGTGTGTACATGAGTTTG F: M13-TCCCATCGACATAATCAGCA R: GATACGTTTGCATTCGTGGA F: M13-TACGCAGAGGTGCAGTGCTA R: CGTCCCTTCCCCCTAATAAA F: TCCTTTTCTCTGGTGGTTGG R: M13-CTCGGCAGTCTTTCTTGTCC F: M13-CCTCTCCGTGAGGTTTTGAA R: CAGTTTCCGGGTGTTGTTTT F: CGCATGTTTGATTCGTTTTG R: M13-CCAGATGCACATACGCAGTT F: CGAAGTAAGATGCACTCAACTCA R: M13-TCTGTGTTCCACGCTGATTTA F: GTTTGGCACACCGAGTTTTT R: M13-AGTCGTTCCGAAACATGCTC F: M13-AACCCCCAAAGGGAAAAATA R: CCCCACTAAGGTAGGGTGGT F: CCGTTCGGCTACATTTTCAT R: M13-TTCCCAACTCGCTTTCTCTC F: M13-AGAGTGTGTTAGCGGCAATG R: CGTCCCTCTCACTAGCAAGC F: CAGTCCAGCGCAATTTACCT R: M13-ATGCGCATTCCATCATCATA F: M13-TGTTGTGGGATTCATGCCTA R: TTCACACCAAGCCCTAAACC
Allele size range (bp)
Size of cloned allele (bp)
Primer sequence (50 -30 )
1
1
5
11
19
12
4
5
10
6
7
9
4
7
16
11
11
6
5
5
Na
–
–
0.714
0.352
0.708
0.677
0.514
0.484
0.666
0.382
0.457
0.857
0.371
0.606
0.687
0.852
0.606
0.600
0.555
0.638
HO
–
–
0.639
0.907
0.933
0.778
0.575
0.665
0.829
0.638
0.737
0.823
0.712
0.775
0.898
0.802
0.880
0.679
0.704
0.695
HE
–
–
0.373
0.000
0.015
0.586
0.118
0.164
0.169
0.003
0.001
0.505
0.000
0.010
0.003
0.880
0.000
0.273
0.013
0.404
PHWE
b
b
b
b
–
–
0.569
0.869
0.908
0.731
0.504
0.598
0.789
0.577
0.691
0.789
0.648
0.729
0.875
0.763
0.854
0.633
0.635
0.630
PIC
KJ482618
KJ482609
KJ482632
KJ482619
KJ482617
KJ482616
KJ482615
KJ482614
KJ482613
KJ482612
KJ482611
KJ482610
KJ482608
KJ482607
KJ482606
KJ482605
KJ482604
KJ482603
KJ482602
KJ482601
GenBank Accession Nos.
2R
2L
2R
2L
X
2R
2R
2L
3L
3L
3R
2R
Probable location on chromosomea
þ(M)
þ(M)
þ(M)
þ
þ
þ
þ(M)
þ
þ
þ
þ
þ
þ(M)
þ
þ
þ
þ(M)
þ
þ(M)
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
Species U
Species S
Cross-species amplification in
JOURNAL OF MEDICAL ENTOMOLOGY
Na, number of alleles; HO, observed heterozygosity; HE, expected heterozygosity; PHWE, P value for Hardy–Weinberg probability test; PIC, polymorphic information content; M, monomorphic; M13, TGTAAAACGACGGCCAGT. a Probable location on chromosome based on homology with An. gambiae database in VectorBase. b Indicated significant deviation from Hardy–Weinberg equilibrium after Bonferroni correction.
Repeat motif
Locus
Table 1. Characteristics of microsatellite markers developed for An. fluviatilis T
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LATHER ET AL: MICROSATELLITES FROM Anopheles fluviatilis T
for 35 microsatellite loci were tested for PCR amplification with An. fluviatilis T, of which 20 were selected for microsatellite genotyping that produced single discrete amplicon after PCR amplification. Eighteen microsatellite loci were found polymorphic and two were monomorphic based on DNA fragment analysis performed on 35 field-collected mosquitoes from Pauri Garhawal, Uttarakhand (Table 1). The number of alleles per locus varied from 4 to 19, with a mean of 8.5. The observed heterozygosities (HO) and expected heterozygosities (HE) ranged from 0.352 to 0.857 and from 0.575 to 0.933, respectively (Table 1). Polymorphic information content value ranged from 0.504 to 0.908. Four microsatellite loci (AF98, AF8, AF74, and AF84) showed significant deviations from Hardy–Weinberg equilibrium after Bonferroni correction (adjusted P-value
