Bangkok, Thailand; Department of Pathology, The University of Texas Medical ... nary data on inversion karyotypes of races B and E suggest ..... gene pools,.
Am. I. Trop. Med. Hyg., 55(6). 1996, pp. 589—594 Copyright C 1996 by The American society of Tropical Medicine and Hygiene
MICROSATELLITE
POLYMORPHISM IN ANOPHELES MACULATUS, A MALARIA VECTOR IN THAILAND
PORNPIMOL RONGNOPARUT, SARAPEE YAICHAROEN, NUNTAREE SIRICHOTPAKORN, Department
RAMPA RKVFANARITHIKUL, GREGORY C. LANZARO, 1@r KENNETH J. LINTHICUM of Entomology, United States Army Medical Component—Armed Forces Research Institute of Medical
Sciences
(USAMC-AFRJMS), Bangkok@Thailand; Department of Biochemistry, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand; Department of Pathology, The University of Texas Medical Branch, Galveston, Texas Abstract. Dinucleotide microsatellites were characterized from Anopheles maculatus, a species of mosquito that transmits malaria. A partial genomic library of An. maculatus, consisting of 3,960 kilobases (kb), was screened with either (GT),2 or (CT),2 probes. Approximately 1.5% of the recombinants contained sequences that hybridized to either (GT),2 or (CT),2 dinucleotide probes, suggesting that microsatellites are abundant in the genome of An. maculatus. Estimation of abundance of the two dinucleotide repeats revealed that (GT)@ or (CA)@ microsatellites occur on average every 68 kb and (CT), or (GA),, repeats every 495 kb. Among 23 microsatellite loci sequenced, four loci were selected to synthesize primers to perform polymerase chain reaction scoring for genetic polymorphism in a population of An. maculatus. A high level of polymorphism was observed with all four microsatellite loci analyzed. The number of alleles detected at each locus ranged from eight to 12 and the heterozygosities ranged from 0.25 to 0.54. A total of 42 alleles were found among four microsatellite loci. The large number of alleles and polymorphic nature resolved from microsatellite loci make these markers valuable for the study of population genetic structure and gene flow. Knowledge of gene flow is required to develop vector control strategies using genetic manipulations of malaria vector populations. The rapid spread of multidrug-resistant strains of human malaria parasites and insecticide resistance of mosquito vec tors highlights the need for the development of more effi cient malaria control methods.@4 One possible strategy would be to replace a wild vector population with a popu lation of the same species incapable of malaria transmission using genetic manipulation of mosquito vectors.5 An as sumption important to the execution of this strategy is that there is extensive gene flow among populations of the target
used for genetic analyses in many eukaryotic species, in cluding insects.'@9 Microsatellites are DNA sequences with short repeat motifs, such as (GT)5 or (CT)5)7 It has been observed that microsatellites show a high frequency of van ation in the number of repeats in different individuals,20 probably due to slippage during DNA replication.2' Further more, microsatellites are inherited in a Mendelian fashion and represent distinguishable loci with codominant alleles.― Compared with restriction fragment length polymorphisms, microsatellites typically have more alleles and the scoring procedures do not require Southern blotting.'2 Finally, be cause the microsatellite methods used the polymerase chain reaction (PCR), they requires minuscule amounts of tissue to be analyzed. Microsatellites have recently been developed and used for population genetic studies'8 and for developing a genetic map of An. gambiae;'9 however, there are no sequence data available for microsatellites in the An. maculatus group. The aims of this study were to determine the frequency and na
species. However, there is suggestive evidence that popula tions of many Anopheles species are subdivided by barriers to reproduction and that migration among geographic pop ulations is limited.6 At present, there is a general lack of knowledge concerning population genetic structure and corn plexity in mosquito species. Anopheles maculatus is a species complex that includes the major malaria vector of Malaysia and important second ary vectors in southern and western Thailand.7 Two geo graphic races of An. maculatus sensu stricto, races B and E, were found in southern, peninsular Thailand and have dis tinct patterns of polytene chromosome inversions.8 Their geographic distribution is nonrandom. Race B is found to extend northward from 13°Nlatitude, while race E extends southward from 12°Nlatitude9 (Rattanarithikul R, unpub lished data). Race E is the malaria vector in malaysia'° and southern Thailand;7 however, there is no evidence incrimi nating race B in malaria transmission in Thailand. Prelimi nary data on inversion karyotypes of races B and E suggest that there is little indication of hybridization (Rattanarithikul R, unpublished data). Classic allozyme analyses revealed lit lie or no polymorphisms
among
populations
of both
ture of dinucleotide microsatellites in An. maculatus and the level
of genetic
variability
at microsatellite
loci
in An. ma
culatus. MATERIALS AND METHODS
Specimen collection. Female specimens of An. maculatus were collected from bovine and human baits along penin sular Thailand. Specimens used in microsatellite assays were obtained from Uthaithani Province, Thailand, at approxi mately 14°Nlatitude. Mosquitoes were transported alive to the laboratory to produce F, progeny or to be kept frozen at —70°C.The F, specimens were derived from adult females that had been collected at human bait, blood fed on hamsters, allowed to lay eggs, and reared to adults. In conducting the research described in this report, the investigators adhered to the Guide for the Care and Use of Laboratory Animals
races
(Green CA, unpublished data). Recent methods of analyzing genetic variation in natural populations focus directly on the use of DNA markers. Among the many possible types of molecular markers, mi crosatellites may be especially useful for studies of popula tion 12 They have been found ubiquitously and
as promulgated
589
by the Committee
on Care
and Use of Lab
590
RONGNOPARUT
oratory Animals of the Institute of Laboratory Animal Re sources, National Research Council. Construction of mosquito partial genomic DNA II brary. Total genomic DNA from 10 F, progeny of a single An. maculatus family was extracted using previously pub lished methods.22 Genomic DNA was digested to completion with Sau 3A1 (Gibco-BRL, Gaithersburg, MD), ligated into the BamHI site of the plasmid pBluescriptll SK@ vector (Stratagene, La Jolla, CA), and subsequently transformed into competent XL1 blue cells (Stratagene) that allow blue/
white color selection. Transformants containing plasmid vec tors without inserts with be blue while those with plasmids containing DNA inserts will remain white. Screening of the library and sequencing. The library contained approximately 4,400 white transformants with in sert DNA sizes ranging from 300 to 1,200 basepairs (bp). To facilitate the identification of positive clones, each of the
4,400 colonies was double transferred onto Luria-Bertani (LB) medium plates, supplemented with 100 j.tg/ml of am picillin, over a squared matrix consisting of a grid drawn on a piece of paper. White transformants were individually transferred onto replica plates at the identical position. Each replica was lifted with nitrocellulose filters and treated with lysis solution containing 10% sodium dodecyl sulfate (SDS) to lyse bacteria. The filters were screened with 32P-labeled oligonucleotide probes by Southern blot hybridization. Two synthetic oligonucleotides, (GT),2 and (CT),2, were used as probes. About 1.8 p@gof each oligonucleotide sequence was 5' end-labeled
with
40
pCi
of 32P-dATP
using
T4 polynu
cleotide kinase (Gibco-BRL). The labeled oligonucleotides were purified on an STE SELECT-D G-25 spin column (5 Prime -9 3 Prime, Inc., Boulder, CO) and added to a hy bridization solution (6X SSC [0.9 M NaCl, 0.09 M sodium citrate], 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% bo vine serum albumin, 5 mM EDTA, pH 8.0, 0.5% SDS, 20 mM Tris, pH 7.6). Hybridization was carried out at 42°Cfor 12—16hr. The filters were washed twice in 2X SSC, 0.1% SDS for 20 mm each at room temperature, followed by washing twice in 0.2X SSC, 0.1% SDS for 20 mm at 50°C, and finally in 0.2X SSC at 50°Cfor 15 mm. The filters were then exposed to x-ray film (Kodak XAR-5; Eastman KOdak, Rochester, NY) overnight with an intensifying screen at —70°Cand putative positive clones were then identified. Plasmid DNAs were purified from putative positive clones using Quiagen tip-25 columns (Quiagen, Inc., Chatsworth, CA). Positive clones were confirmed by double digestion with Hind HI and Xba I and subjected to Southern blotting and/or
slot blotting,
followed
by hybridization
with
MD).
Prior
mal PCR conditions
to PCR
amplification
were determined
of specimens,
TABLE 1
Characteristics of polymerase chain reaction primers, annealing tern perature and expected product length for four microsatellite loci Ex Anneal ingpected
productLocusPrimer sequence (bp)GT18T7ACGATGArFGCGAGCAACG(5' -4 3')tempera
turelength
ACTCATCCTACCTAGTAC50°C225GT47RTGAGAAACTGAGTACCTAACC GGAAAACGrFACGCTCTAG56°C103GT68RTTG@GCTFAACCATCTAGC ACAGAACCGAATACCTAGC54°C143CT29RTTGATAATGACCAGTCGGTCG TCTFA1TFGAGCCGATGC50°C128
ers. The determination of conditions was carried out by test ing a series of annealing temperatures at 2°Cintervals below or above the melting temperatures of primers. In some cases, altering the amount of genomic DNA and/or the concentra tion of primers improved efficiency and specificity of PCR amplification.
The
PCR
products
were
analyzed
by
electro
phoresis through 4% agarose gels containing 1% Seakem and 3% Nusieve agarose (FMC BioProducts, Rockland, ME). After optimized conditions were established, standard PCR amplification was conducted using a primer labeled with ‘y32P-ATPby the following methods. Amplification of the DNA of each specimen was performed in a total volume of 20 pi using 2—5ng of genomic DNA, 0.4 mM dNTPs, 0.5 U of Taq polymerase (Perkin-Elmer Cetus, Norwalk, CT), 0.2 uM of each primer, and standard Perkin-Elmer Ce tus buffer. Each PCR included 0.04 p@Mof labeled primer. The forward primer was end-labeled using 0.4 p@Mof primer with 2 pCi of y32P-dATP and 0.5 U of polynucleotide kinase. After one denaturing step for 5 mm at 95°C, samples were processed through 29 cycles consisting of 20 sec at 94°C, 30 sec at the annealing temperature as specified in Table 1, and 30 sec at 72°C.The last elongation step was lengthened to 5 ruin at 75°C. Eight microliters of formamide stop so lution was mixed with labeled PCR products, denatured at 95°C for 5 mm, and electrophoresed on a standard DNA sequencing gel (6% polyacrylamide) for 1.5—2.5hr depend ing on the expected size of the amplified products from dif ferent loci. Gels were dried and exposed to x-ray film at —70°C with an intensifying screen for 2 hr. RESULTS
the end
labeled probes. Clones with strong positive signals were se quenced using 11 and RT primers using Sequenase 2 kit (United States Biochemicals, Cleveland, OH). Sequencing was performed following the supplier's instructions. Detection of length polymorphism in microsatellite loci. For detection of length polymorphisms, PCRs were per formed. Genomic DNA was extracted from adult female in dividuals using NP4O lysis solution as previously de scribed.23 The PCR primers corresponding to unique se quences flanking the repeats were synthesized and purified (Biopolymer Laboratory, University of Maryland, Balti more,
AND OTHERS
opti
for each pair of prim
Development of microsatellite markers. Two types of microsatellite sequences were examined, (GT)@ or (CA)@ and (CT),, or (GA),. These were obtained after screening a partial genomic library of An. maculatus for (GT)@ and (CT)@ se quences. Following Southern blot or slot blot hybridization to confirm positive clones, 58 clones were identified with a (GT),2 probe and eight with a (CT),2 probe. The average size of these cloned microsatellite inserts was 900 bp and a total of 4,400 clones were analyzed. A total of about 3,960 kb of genomic DNA was analyzed. With the genome size of 2— 2.6 X 108 bp per haploid for Anopheles species,24 our partial genomic library is estimated to represent approximately 1.9% of the An. maculatus genome. The average distance
591
MICROSATELLITEVARIATIONIN AN.MACUL4TUS
T@au@ 2
primer pairs for PCR amplification
Repeat sequences identified in the positive clones sequencePerfectGT34RT Repeat typeClone
flanking
Microsatelilte
numberRepeat
GT66T7 GT96RT GT52T7
GT41T7 GT67RT G67RT GT42RT GT6RT CT29RT GT18RT(CA)7 (CA),2ImperfectGT6T7
DNA survey.
We designed
primers
to de
repeats. Primers were tested in PCRs with genomic DNA from individual An. maculatus mosquitoes. The sequences and optimum annealing temperatures for primer pairs used in PCR amplification are shown in Table 1. Figure 1 shows an example of amplified DNA for the GT68RT (CA),, and CT29RT (GA)n loci in a sample of An. maculatus individ uals. Individual DNA bands were considered alleles and des ignated according to the mobility of a sequencing ladder of
(CA)8 (GT)8 (CA)9 (GT)9 (GT)9 (GT)9 (CA),,, (CA),, (GA),,
known
GT68RT GT22T7
(CA)6GA(CA)2 (GT)6CT(GT)3
GT9T7
(GT)7C(GT)2
GT4ORT (GT)4AT(GT)9(G)8 GT47RT(GT),0(T),, (A)8T(CA),7
between neighboring microsatellites was calculated by di viding the total length of DNA examined (3,960 kb) by the number of microsatellite sequences found within these se quences. It is estimated that (GT)5 or (CA)@ microsatellites occur on average every 68 kb and (CT)@ or (GA)@ repeats every 495 kb. The average distance between any of these two dinucleotide blocks is approximately 60 kb. The data indicated that in the genome of An. maculatus (GT)@ or (CA),, repeats are 7.3-fold more common than (CT)n or (GA)@ microsatellites. The abundance of (GT)@ or (CA)@ and (CT)@ or (GA)n sequences in the An. maculatus genome was confirmed by Southern blot hybridization using synthetic oh gonucleotide (GT),2 and (CT),2 probes. Hybridization of these probes onto genomic DNA of An. maculatus showed that (GT),2 probe hybridized much stronger than the (CT),2 probe. We sequenced 25 of 66 (GT),2 positive clones and eight (CT),2 positive clones. Sequencing allowed us to determine the number and nature of repetitive sequences, and the se quences that flank microsatellites. Poly (GT/CA) targets were found in 20 clones and poly (CT/GA) targets were found in three clones. Ten of the 33 clones were rejected. In one instance, duplicate targets were recognized by sequence comparison. In other instances, sequencing failed to find a
target in five clones, and four others had the target sequence too close to the vector cloning site to select suitable primers. Three types of repeats were found among these clones (Table 2) and they have been categorized according to Weber.@ Among the 23 described microsatellites, 1 1 were of the per fect type, with no interruptions of the dinucleotide repeat. Nine were of the imperfect type, with up to a 4-bp interrup tion within the repeat region. Three were of the compound type, with a run of (G)@,(A)9, or (T)@mononucleotide repeats adjacent to a run of dinucleotide repeat. The sequences microsatelhite
to
tect four microsatehhite loci that contained (GT or CA)@,7
GT3ORT (CA),AG(CA)9 GT87RT (CA)2AA(CA)7CG(CA), GT57T7 (GT)2AA(GT),0 CT48RT (GA)2GC(GA),9 CCT21RT(CA)2CC(AC)5GC(AC),CC(AC), (TC)7GC(TC)20TATF(TC),ØCompoundGT18T7
flanking
were made according
sequences.
regions
were
determined
and
the
sequence
as a size
standard
in denaturing
urea
gel.
Amplified DNA fragments shown in Figure 1 were in the expected size range for the GT68RT (CA)6GA(CA)2 and CT29RT (GA),, alleles amplified from the GT68RT and CT29RT plasmid controls. Each PCR band differed by mul tiples of 2 bp in length, suggesting that they vary in the number of repeat units in the microsatellite region. At locus GT18T7, a few individuals contained PCR bands that dif fered by 3 bp in length. The 3-bp differences are due to the nature of the compound repeat at this locus that contained a mononucleotide (T),3 repeat next to the (GT),0 repeat (Table 2). Although the GT47RT locus contained a run of mono nucleotides (Table 2), no 3-bp differences were found in the population tested. In some mosquito specimens, PCR am phification failed to produce products (see examples in Fig ure 1A, lane 17 and lB. lane 14). Repeated amplification on these samples with lower annealing temperature yielded PCR products with the expected size in the specimens that failed to amplify in the first attempt. Approximately 70% of
specimens that did not produce products during the first PCR run did yield products on the second attempt at PCR ampli fication with a lower annealing temperature. Pedigree studies have shown that microsatelhites are in herited in Mendehian codominant 20 and the band ing patterns are one or two bands, permitting unambiguous scoring of bands as alleles. For the purpose of scoring mi crosatellite loci, we refer to the product of each primer pair as a locus and each PCR variant as an allele. In theory, each allele of a target microsatelhite sequence should appear as a single band on a denaturing sequencing gel after PCR. Thus, the PCR product of a homozygote should appear as single band and a heterozygote as double bands. All four micro satellite loci examined were polymorphic. The number of alleles detected at each locus ranged from eight to 12 with a mean of 10.3 and the heterozygosities ranged from 0.25 to 0.54 (Table 3). The differences in PCR product size ranged from 2 to 22 bp. A total of 42 alleles were found among four microsatellite loci. Allele frequencies (size van ant) are shown for each locus in Figure 2. Except for locus GT47RT, all loci contained two or three common alleles with the frequencies of occurrence ranging from 0. 17 to 0.33. The GT47RT locus contained a more even distribution of several alleles that occurred at a moderate frequency. The GT47RT locus had a lower level of observed heterozygosity than oth er loci (Table 3). DISCUSSION This study used microsatellites to investigate versity in a small population of An. maculatus
genetic di specimens.
@
@.
592
RONGNOPARUT
AND
OTHERS
A GATC
i
2
34
567
8
9
101112131415161718192021222324
V
152...,. 150_. 148.....
I 46@ 144@ 142— 140— 138—
@
d@1,
.\
B 12 13 14 15 16 17 18 19 20 21 G A I
I 2 3 4 5 6 7 8 9 10 11 b@
.
@143 —
I 39
Autoradiograms
showing
microsatellite
patterns
of two loci, GT68RT
bp. —143
—139
!@@@!lJt :@ FIGURE 1.
C
-
_1 29 —127 —125 —123 @: —121
(A) and CT29RT
(B). Each
lane corresponds
to a different
Anopheles maculatus mosquito. Lanes GATC are sequencing standards. Lanes 17 in A and 14 in B show individual mosquitoes whose DNA failed to amplify
upon polymerase
chain reaction
amplification.
bp
We found that 1.5% (66 of 4,400) of all clones in a partial library contained sequences that hybridize to dinucleotide repeats. This suggests that microsatellite sequences are high ly abundant in the genome of An. maculatus. We chose di nucleotide microsatellites because they were more abundant than trinucleotide repeats, i.e., (CCT)@. Our initial attempt to screen an An. maculatus genomic DNA library with (CCT)7 resulted in only one clone. Southern blot hybridizations on An. maculatus genomic DNA with a (CCT)7 probe produced a much weaker signal than that produced with (GT),2 and (CT),2 probes. Since our partial genomic library represents a small part of An. maculatus genome, it is not surprising that we missed finding clones containing (CCT)@ repeats. Es timation of the abundance of (GT)@ or (CA),, and (CT),, or (GA),, dinucleotide repeats are one in every 68 kb and 495 kb, respectively. Microsatellite frequencies obtained for An. maculatus are comparable with those reported for other in vertebrate 617 In An. maculatus (GT)9 or (CA)@ di nucleotide repeats are more frequent than (CT)@ or (GA)@ repeats, similar to those in mammalian pec2° However, TABLE 3
Numbers
of alleles
detected
and observed
and expected
heterozy
gosities for four microsatellite loci examined in population of race B individuals from Uthaithani Province, Thailand heterozygosityGT18T7 LocusSample
sizeNumber
GT47RT GT68RT 0.87a CT29RT14
24 24 2711
Unbiased
estimate
of Nd.2'
of allelesObserved heterozygosityExpected
12 8 110.5
0.25 0.54 0.420.86
0.92 0.83
basepairs.
the opposite has been found in other insect species such as honey bee and bumble bee'6 where (CT), and (GT), repeats occur every 40 kb and 500 kb, respectively. Most of the sequenced An. maculatus microsatellites consisted of 7—21 repeats. Comparable lengths were reported for other insect 617
although
the
(GT)@
blocks
in
An.
gambiae
were
found to be relatively longer.'9 Microsatelhite loci often show extra bands. These bands are usually one or two repeat lengths different from the main product and probably anise from slipped-strand mispairing during PCR amplification.2' These extra bands could cause difficulty in distinguishing homozygotes from heterozygotes with alleles differing by one repeat unit. We conservatively scored these ambiguous cases as homozygotes. In a few cases, PCR bands of unexpected size additional to the cx pected polymorphic products were amplified; however, these additional bands were not polymorphic among the samples analyzed. We assumed that these nonpolymorphic PCR prod ucts were derived from sites not homologous to the micro satellite sites and thus did not include these bands in scoring. In the study of eukaryotic microsatellites, it has been ob served that dinucleotides with 10 or fewer repeats are not an'4 Each of the four loci we tested were all highly polymorphic with the total of 42 observed alleles. As shown in Table 3, observed heterozygosities were lower than cx pected for all four loci.26 This was especially true for locus GT47RT, where the observed heterozygosity was less than 50% of the expected value (Table 3). Possible explanations for such heterozygote deficiency could be due to the Wah lund Effect, inbreeding, or sampling effect. The Wahiund Effect is created when a mixture of more genetically differ
MICROSATELLITE
VARIATION
0.35
0.35
GT18RT
0.3
0.3 0.25
0.25 C 0
C 0
0.2
0.15
0.1
0.1
0.05
0.05
nfl 221
223
225
227
Fragment
229
H 231
233
235
237
0 77 79 81 83 85 87 89 91 93 95 97 99101103
239
Fragment aize (bp)
aize (bp)
0.35
035
GT68RT
0.3
0.3
0.25
0@
0I-
0.2
0.15
0
C 0
593
IN AN. MACULATUS
0.25 C 0
0.2
0.2
I@.
0.15
0.15
0.1
0.1
0.05
0.05
U-
0
136 138 140 142
0
44 146 148 150 152 154 156 158
121 123125127129131
Fragment size (bp) FIGURE 2.
Distribution
of allele
frequencies
133135137139141
143145
Fragment size (bp) for four
ent individuals are presumed to represent a single population in genetic analysis, resulting in a reduction in observed het erozygosity. Sampling effects may be introduced by the large numbers of alleles found and small number of An. ma culatus individuals (14—specimens) examined so that not all genotypes were observed in our sample. It is unlikely that the Wahlund Effect could be occurring since our chromo some inversion karyotyping and random-amplified polymor phic DNA PCR did not detect any more than one population in our sampling area (Rattanarithikul R, Sirichotpakorn N, unpublished data). Other indications could be associated with problems in differentiating between homozygotes and heterozygotes in the ambiguous cases, and/or the presence of null alleles.27 Any mutation or deletion that occurred at the PCR primer binding site of one homolog could result in the absence of one of the PCR product in heterozygous in dividuals. We speculate that mutations at priming sequences may have contributed to the lack of heterozygosity since our second attempt on PCR amplification with lower annealing temperature yielded PCR products with expected sizes. If null alleles were present in the population, heterozygous in dividuals could be mistyped as homozygotes, resulting in heterozygote deficiencies observed in the population. One of the four loci tested, GT68RT, contained
microsatellite
loci of Anopheles
maculatus.
bp
basepai.rs.
(CA)6GA(CA)2 repeat sequences that had fewer than 10 re peats (Table 2). Despite being short, the GT68RT locus was polymorphic, but was the least polymorphic. Our prehimi nary data suggest that the majority of individuals within each population differed with respect to multiple microsatellite locus genotype; fewer than 10% of the individuals within the sample possessed the same four microsatellite allelic pat tern. The allele numbers and observed heterozygosity shown on Table 3 indicate that there is a high level of genetic di versity within the An. maculatus population sampled. These findings are contrary to previous observation using classic allozyme analyses that revealed little or no polymorphisms, depending on the loci tested, within and among populations of races B and E of An. maculatus (Green JC, unpublished data). Results from this study suggest that microsatellite poly morphisms could provide a sensitive and efficient measure of genetic diversity. Primers developed for An. maculatus could be used in related species. The successful use of PCR primers for microsatellites in related species has been re ported in whale,28 cattle, and sheep.@ The abundance and highly polymorphic nature of these microsatellite loci, the ability to detect heterozygosity, and ease of genotype assay of large numbers of samples should permit studies of the
594
RONGNOPARUT AND OTHERS
genetic structure of natural populations of An. maculatus. Our future goals are to isolate and analyze more microsa tellite clusters in An. maculatus and to define the patterns of these markers in the different chromosomal forms within the An. maculatus group, especially in an effort to establish whether races B and E represent independent gene pools, and to estimate the extent of gene flow. Analyses of gene flow would provide information on the distance, direction, and rate of disperal of genes in An. maculatus populations. Knowledge on gene dispersal patterns is important in the development of malaria vector control strategies that use gene replacement in natural populations.
9. Green CA, Baimai V, Harrison BA, Andre RG, 1985. Cytoge netic evidence for a complex of species within the taxon Anopheles maculatus (Diptera:Culicidae). Biol J Linnean Soc 24: 321—328.
10. Yong HS, Chiang GL, Loong KP, Ooi CS, 1988. Genetic van ation
in the malaria
mosquito
vector
Anopheles
maculatus
from peninsular Malaysia. Southeast Asian J Trop Med Pub lic Health
19: 68 1—687.
11. Bruford MW, Wayne RK, 1993. Microsatellites and their ap plication to population 3: 939—943.
genetic studies.
Curr Opin Genet Dev
12. Queller DC, Strassmann JE, Hughes CR, 1993. Microsatellites and kinship. Trends Ecol Evol 8: 285—288. 13. Tautz D, Renz M, 1984.
Simple
sequences
are ubiquitous
re
petitive components of eukaryotic genomes. Nucleic Acids Res 12: 4127—4138.
Acknowledgments:
We thank
many
staff
of the Ministry
of Public
Health (Bangkok, Thailand) for cooperation during the course of this study. At AFRIMS, Somporn Chanaimongkol, Pradith Mahapibul, Chumnong Noigamol, Suda Ratanawong, and Prasertsri Rohitara tuna provided expert technical support. Financial
support:
This work was supported
in part by the UNDP/
World Bank/WHO Special Programme for Research and Training in Tropical Diseases. Disclaimer: The views of the authors do not necessarily reflect the position of the Department of the Army or the Department of Dc fense. Authors'
addresses:
Pornpimol
Rongnoparut,
Nuntaree
Sirichotpa
korn, Rampa Rattanarithikul, and Kenneth Linthicum, Department of Entomology, USAMC-AFRIMS, Bangkok 10400, Thailand. Sar apee Yaicharoen, Department of Biochemistry, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand. Gregory Lanzaro, Department of Pathology, The University of Texas Medical Branch, Galveston, TX 77555. Reprint requests: Pornpimol Rongnoparut, Department of Entomol ogy, USAMC, AFRIMS, 315/6 Rajvithi Road, Bangkok 10400, Thailand (from the United States, send to Kenneth J. Linthicum, USAMC-AFRIMS, APO 96546).
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