AJTMH Tropical Medicine and Hygiene News
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am. J. Trop. Med. Hyg., 73(4), 2005, pp. 744-748
Copyright © 2005 by The American Society of Tropical Medicine and Hygiene

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by WIDDEL, A. K.
Right arrow Articles by FONSECA, D. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by WIDDEL, A. K.
Right arrow Articles by FONSECA, D. M.
Related Collections
Right arrow Vector Biology
Right arrow Medical Entomology
Right arrow Mosquitoes

FINDING NEEDLES IN THE HAYSTACK: SINGLE COPY MICROSATELLITE LOCI FOR AEDES JAPONICUS (DIPTERA: CULICIDAE)

ANDREA K. WIDDEL, LINDA J. MCCUISTON, WAYNE J. CRANS, LAURA D. KRAMER, AND DINA M. FONSECA*
Molecular Ecology, PCER, Academy of Natural Sciences, Philadelphia, Pennsylvania; Department of Entomology, Rutgers University, New Brunswick, New Jersey; Wadsworth Center, New York State Department of Health, Slingerlands, New York


ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
First identified in three North American states in 1998, Aedes japonicus japonicus, the Asian bush mosquito, has since spread to 21 states, plus Ontario in Canada, northern France, and Belgium. Analyses of the introduction and expansion of this potentially deadly disease vector will be radically improved by including powerful genetic markers like microsatellites. Useful microsatellite loci have, however, been difficult to identify for mosquitoes in the genus Aedes because of the high amount of repetitive DNA in these species. We isolated single-copy DNA from Ae. j. japonicus and then used a standard enrichment method to identify regions containing microsatellites. Here we describe seven polymorphic microsatellite loci that were tested in American populations of Ae. j. japonicus. These loci were also found to be polymorphic in two other of the four Ae. japonicus subspecies and in Aedes koreicus.


INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In addition to the practical understanding required for successful control programs, population genetic studies of introduced disease vectors can provide excellent opportunities to examine evolutionary processes in complex systems. Genetic analyses enable us to locate putative origins of multiple introductions1,2 and evaluate differing vectorial capacity across populations.3,4 Changes in genetic makeup associated with introductions and expansions,5,6 as well as new associations with local or introduced hosts and pathogens, create dynamic systems that are amenable to examination and even experimentation.7 Change can be measured by comparison both to populations in the original range and, in some situations, to the earlier stages of the introduction. Though these systems are invaluable, thorough analyses are rare.8

The Asian bush mosquito, Aedes (Finlaya) japonicus japonicus Theobald (Diptera: Culicidae), was first collected outside its native range of northeast Asia9 in 1998. Although it is unclear when it was first introduced to the United States, in 1998 three collections of Ae. j. japonicus were made independently in three different states (CT, NY, and NJ).10,11 The fact that the extensive surveys associated with Aedes albopictus12 failed to uncover Ae. j. japonicus prior to 1998 argues that it must have been introduced no earlier than 1992.10 From three states in 1998, Ae. j. japonicus has since expanded in North America to a total of 19 U.S. eastern states (CT, DE, GA, IN, MA, ME, MD, NC, NH, NJ, NY, OH, PA, RI, SC, TN, VA, VT, and WV), Quebec, Canada, and the western state of Washington. Ae. j. japonicus is extremely common in many northeastern states,10,13 although its presence in southern and more western states is still very localized.14,15 Breeding populations were also found in France in 200016 and Belgium in 2002 (Schaffner F, personal communication). In July 2004, Ae. j. japonicus were collected on the Island of Hawaii (Burham Larish L, personal communication).

In its native range in northern Japan, Korea, and Eastern Russia, Ae. j. japonicus is mostly a forest and low-density mosquito that is not considered an important disease vector, although it is known to be a laboratory vector of Japanese encephalitis.17 In contrast, American Ae. j. japonicus have been shown to be effective vectors of West Nile virus,18,19 Eastern equine encephalitis,20 La Crosse virus,21 and St. Louis encephalitis.22 The expansion of Ae. j. japonicus in the United States has rivaled that of Aedes albopictus in its speed and current abundance.23 This expansion was unexpected considering the restricted and relatively unchanged ranges of two other introduced Aedes species: Aedes togoi was introduced in the 1960s to the state of Washington24 and Aedes bahamensis to Florida in 1986,25 but neither has spread beyond a few contiguous counties.26,27 In contrast, Ae. j. japonicus spread from three U.S. states to 21 in only 6 years.

After introduction, the average genetic makeup of Ae. j. japonicus may change as a result of bottlenecks and possibly new selection pressures.6,28 This might lead to behavioral and/or physiologic changes that could reveal the potentially critical role of this species as a disease vector. Indeed its current abundance in states like Pennsylvania, New Jersey, and New York, where it has become one of the most abundant species in both rural and urban environments10,13 seems to indicate change. Investigating the genetic makeup associated with the introduction and range expansion of Ae. j. japonicus in the United States will allow us to test hypotheses of evolutionary change as well as examine the role of multiple introductions of phenotypically divergent populations. To do so we require highly polymorphic genetic markers such as microsatellites. Microsatellites are our marker of choice because of their relative ease of use, hypervariablity, and low cost compared with sequencing.29 Although processing large numbers of samples is fairly inexpensive, the investment associated with microsatellites comes during the development process that is both time and equipment intensive.

Isolating microsatellites for mosquitoes in the genus Aedes (or Ochlerotatus30) has proven problematic in the past.31 Although microsatellite regions are present in Aedes mosquitoes,31 they are commonly duplicated throughout the genome so that a single set of primers will amplify several different loci. These "locus families" render the primers useless for standard population analysis that require single-locus markers with Mendelian inheritance.32 To avoid this problem, we used a protocol designed to remove highly repetitive DNA and survey only single copy DNA for the presence of microsatellite regions.33


MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
We obtained 88 frozen larvae of Ae. j. japonicus from the colony at Rutgers University and extracted their DNA using a DNeasy Tissue Kit from Qiagen (Valencia, CA) including the optional RNAse step. The resulting 34.5 µg of DNA were sonicated to generate fragments ranging from 300 to 1,000 base pairs (bp). To eliminate interference from secondary structure, the sample was heated to 95°C for 10 minutes then promptly chilled on ice for 10 minutes before sonication.

Following Elsik and Williams,33 approximately 25 µg of DNA (average size of approximately 700 bp) were cleaned by ethanol precipitation and resuspended in 25 µL (1 µg/µL) of 0.4 M phosphate buffer (PB), denatured at 100°C for 10 minutes and allowed to reanneal slowly at 60°C for 20 hours (derived from the C0T 1000 for Ae. albopictus).34,35 During this period the repetitive regions reassociate and, by interrupting this process before the DNA has completely rean-nealed, we were able to recover single-stranded low copy DNA. This DNA is separated from the double stranded DNA with the use of a hydroxyapatite (HAP) column, which was prepared in 0.03 M PB with a pH of 7. The DNA was applied to the column and washed with 3–5 mL of 0.03 M PB. Single-stranded DNA was recovered with a 1 mL wash of 0.12 M PB. Double stranded DNA was collected with a 1 mL wash of 0.4 M PB. The recovered DNA was cleaned using a QIAquick PCR Purification column (Qiagen). This recovered low copy DNA is biotinylated and used to fish out the matching low copy sequences from a separate linker-ligated pool of DNA.

Approximately 1 µg of sonicated DNA isolated from 3 Ae. j. japonicus specimens was digested with mung-bean exonuclease (New England Biolabs [NEB], Beverly, MA) to remove single-stranded overhangs, dephosphorylated using calf intestinal phosphatase (NEB), and ligated to SNX linkers following the guidelines in Hamilton and others.36,37 Both this pool of DNA and the biotinylated low copy DNA were denatured (95°C for 10 minutes) and hybridized (ratio of 1:2) overnight at 65°C in 6X SSC and 1% SDS.38 Streptavidin coated beads (Dynabeads, Oslo, Norway) were used to isolate the SNX-linked low copy DNA.33

The SNX-linked low copy DNA was denatured and hybridized with biotin end-labeled oligonucleotides (GT15), (GA15), (CAC10), (GCT10), (GGT10), (GTC10) at 55°C and hybridized with (AAC10) and (CAT10) repeats at 45°C. As in Key-ghobadi and others,36 streptavidin-coated magnetic beads (Dynabeads) were used to isolate the DNA fragments that hybridized to the biotynelated repeats. Those fragments were digested with NheI (NEB) and ligated to XbaI-cut, dephos-poralated pbluescript (SK+ plasmid; Stratagene, La Jolla, CA), transformed into Escherichia coli XL1-Blue MRF’ cells (Stratagene), and plated onto selective agar medium. According to the guidelines in Keyghobadi and others,36 positive colonies were identified and sequenced.

Initially we screened all microsatellite loci using a panel of 14 specimens from locations across the United States and Japan collected for a previous study.39 Once a panel of informative loci was established, we examined their polymorphism in eleven sets of eight individuals obtained from the Vector Control offices in twelve eastern Pennsylvania counties (Susquehanna, Wayne, Wyoming, Pike, Monroe, Northampton, Lehigh, Berks, Bucks, Montgomery, Chester). We also examined loci OJ5 and OJ338 in 70 specimens from Japan (Sapporo, Obihiro, Tokyo, Hiroshima, Nagasaki, Saga).39 To examine the broader usefulness of the loci, we went further and tested the panel of seven microsatellites on two other field collected subspecies of Ae. japonicus and related species obtained from collaborators in Japan, Korea, and the United States: Aedes japonicus yayamensis, Aedes japonicus shintienensis, Aedes koreicus, Aedes albopictus, and Aedes aegypti. Furthermore, to confirm the microsatellite loci were inherited following Mendelian assortment,36,40 we examined the parents and progeny of two family groups with 20 and 17 offspring respectively, We created the families by setting aside male and female pupae and subsequently force mating the females to individual males.


RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
An estimated 875 ng of single-stranded low copy DNA was recovered from the HAP column and used to develop a suite of microsatellites (Table 1Go). Our enrichment was successful, with an estimated 15,000 positive colonies from a total of approximately 70,000 (21%). A total of 233 positive clones were sequenced but there was a substantial amount of repetition in the sequences recovered. The most common locus was found 42 times (~18% of clones sequenced), and only three microsatellite sequences were completely unique. Twenty eight clones did not have a well defined microsatellite region, 6 sequences had little or no flank to design primers in, and 13 had microsatellite repeats and were used to design primers using Primer3 software.41 A total of 18 primer pairs were designed for those 13 loci; 7 loci proved to be polymorphic and useful in population analysis. Of the rejected loci, one locus was not single-copy (a GT repeat), four often did not amplify suggesting a large number of null alleles, and one locus was monomorphic.


View this table:
[in this window]
[in a new window]
 
TABLE 1
Characterization of seven microsatellite loci in Aedes japonicus japonicus
 
The PCR conditions for all seven microsatellite loci are the same and were optimized as two different multiplexes (Table 1Go) that were run separately in an automated sequencer. Final concentrations of the PCR reagents in a 20 µL volume are as follows: 1x PCR buffer (GeneAmp, Applied Biosystems Inc., ABI), 2.5 mM MgCl2, 0.2 µM of each primer, 200 µM of each dNTP, 0.2 mg/mL BSA, and 1 unit of AmpliTaq DNA polymerase (ABI). The PCR amplification was performed in MJ Research PTC100 and 200 thermocyclers and was preceded by a 5-minute denaturation at 96°C, followed by 30 cycles of 30 seconds at 94°C, 30 seconds at 54°C, and 30 seconds at 72°C. The amplification ended with an elongation step of 5 minutes at 72°C. The PCR products were sized in an ABI3100 in GENESCAN mode, binned and sized with GeneMapper 3.5 (ABI).

We examined the polymorphism of the seven informative markers across Ae. j. japonicus populations in eastern Pennsylvania (Table 1Go). We observed 3 to 9 alleles per locus and expected heterozygosities ranged from 0.32 to 0.80 (Table 1Go). Statistical tests for Hardy-Weinberg equilibrium and linkage were conducted in GENEPOP.42 There was no significant deviation from Hardy-Weinberg equilibrium, and all pairwise tests of linkage disequilibrium between loci were non-significant especially after sequential Bonferroni correction. A significant linkage between OJ5 and OJ338 in the specimens from eastern Pennsylvania was not significant in tests using the specimens from Japan. Tests of inheritance of the seven loci in two family groups showed no significant departure from expected Mendelian patterns ({chi}2 test).

We found that several of the microsatellite loci were also polymorphic in Ae. j. shintienensis and Ae. j. yayamensis (Table 2Go) and, interestingly, were overall as variable or more variable in Ae. koreicus. When applied to Ae. albopictus and Ae. aegypti, the primers did not amplify.


View this table:
[in this window]
[in a new window]
 
TABLE 2
Variability of the panel of microsatellite loci developed for Aedes japonicus japonicus across related species (the allelic size range (in bp) is listed, followed by the number of alleles in parenthesis)
 

DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We successfully isolated seven microsatellite markers useful for fine scale genetic analysis. We found these markers to have a Mendelian inheritance and a high degree of polymorphism in Pennsylvania, as well as being informative in related species and subspecies. It seems that adding a few extra steps for the isolation of single-copy DNA paid off.

Previous attempts to isolate usable microsatellite loci from Aedes species have been generally frustrating, often unsuccessful, or marginally so31,43 (D. Fonseca, unpublished). Huber and others43 failed to develop microsatellites for Ae. aegypti without enrichment. Using an enrichment protocol, Fagerberg and others31 were unable to isolate any useful microsatellites, but instead found only multi locus families.31 With an enrichment of GT repeats, Fonseca (unpublished) similarly found only locus familes in Ae. j. japonicus. Although Huber and others43 claimed they found three useful microsatellite loci for Ae. aegypti after using an enrichment protocol, most of the loci currently in use44–47 were developed from sequences obtained from the Ae. aegypti Genome Project.48 The drawback of this approach is that microsatellites are often in coding regions that are under selection and often have low polymorphism.45 On the other hand, Behbahani and others49 developed microsatellites for Aedes polynesiensis using an enrichment protocol apparently without any overt problem.

The success of our isolation of low copy microsatellite loci from Ae. j. japonicus was not without tribulation. We had a large amount of repetition in the clones, which may be partially explained by the proposition of Fagerberg and others31 that some DNA fragments are more likely to be cloned than others (clone specific redundancy). Also, the use of PCR to create complements for the single-stranded DNA recovered from the streptavidin beads certainly generates redundant copies. Our efforts to isolate single-locus DNA reduced the risk of finding loci in locus families, but did not safeguard us from the repetition seen in the clones. To eliminate this problem we propose probing the colonies with the most repetitious flanking regions to avoid wasting resources resequencing the same locus.

The seven markers we describe have also proven to be informative for other subspecies in the Ae. j. japonicus complex as well as Ae. koreicus. Unlike mitochondrial DNA for which quasiuniversal primers are available,50 microsatellites tend to be very species specific. Mutations accumulate in the flanking regions resulting in the amplification of a suite of primers in another species being inversely related to the evolutionary distance between the two species.51 The fact that there was more polymorphism in Ae. koreicus, compared with the tested subspecies, agrees with the findings using sequence data (ND4, COII, and D2), that show Ae. j. japonicus is more closely related to Ae. koreicus than to Ae. j. shintienensis and Ae. j. yayamensis (D. Fonseca and others, unpublished data).

The microsatellite markers we report were developed to illuminate genetic differences in recently introduced populations. The newness of Ae. j. japonicus in the United States allows a unique opportunity to uncover some of the dynamics of introductions that are masked with time or obscured by less sensitive indicators. These primers will enable us to take an in depth look at patterns of expansion of this species in the United States and possibly make inferences about putative source populations.

Received January 27, 2005. Accepted for publication May 19, 2005.

Acknowledgments: We are indebted to Nusha Keyghobadi for proposing the low-copy DNA protocol to develop microsatellites for Aedes japonicus; Sven Erik Spilchiger and Michael Hutchinson (PA-DEP) for providing us with specimens of Aedes japonicus japonicus from eastern Pennsylvania; Drs. Ichiro Miyagi and Takako Toma for specimens of related subspecies and species in Japan; Heung-Chul Kim for Aedes koreicus from Korea; Peter Armbruster for specimens of Aedes albopictus and Ae. aegypti from Florida; Julie Smith, Kenli Okada, Carolyn Bahnck, Erika Butler, and an anonymous reviewer for comments on an earlier version of the manuscript; Tapan Ganguly and the DNA Sequencing Facility, University of Pennsylvania, for technical assistance.

Financial support: This project has been funded in whole with federal funds from the National Institute of Allergy and Infectious Disease, National Institutes of Health, under contract no. N01-AI-25490.

* Address correspondence to Dina M. Fonseca, Academy of Natural Sciences, 1900 Ben Franklin Parkway, Philadelphia, PA 19103. E-mail: fonseca{at}acnatsci.org Back

Author’s addresses: Andrea K. Widdel, Academy of Natural Sciences, 1900 Ben Franklin Parkway, Philadelphia, PA 19103, E-mail: widdel{at}acnatsci.org. Linda J. McCuiston, Entomology Department, Rutgers University, 180 Jones Avenue, New Brunswick, NJ 08901. Wayne J. Crans, Entomology Department, Rutgers University, 180 Jones Avenue, New Brunswick, NJ 08901. Laura D. Kramer, Wad-sworth Center, New York State Department of Health, 5668 State Farm Rd., Slingerlands, NY 12159. Dina M. Fonseca, Academy of Natural Sciences, 1900 Ben Franklin Parkway, Philadelphia, PA 19103, Telephone: 215-299-1177, Fax: 215-299-1182, E-mail: fonseca{at}acnatsci.org.

Reprint requests: Dina M. Fonseca, Academy of Natural Sciences, 1900 Ben Franklin Parkway, Philadelphia, PA 19103, Telephone: 215-299-1177, Fax: 215-299-1182, E-mail: fonseca{at}acnatsci.org.


REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Meixner MD, McPheron BA, Silva JG, Gasparich GE, Sheppard WS, 2002. The Mediterranean fruit fly in California: evidence for multiple introductions and persistent populations based on microsatellite and mitochondrial DNA variability. Mol Ecol 11: 891–899.[Medline]
  2. Silva JG, Meixner MD, McPheron BA, Steck GJ, Sheppard WS, 2003. Recent Mediterranean fruit fly (Diptera: Tephritidae) infestations in Florida–a genetic perspective. J Econ Entomol 96: 1711–1718.[ISI][Medline]
  3. Paupy C, Girod R, Salvan M, Rodhain F, Failloux AB, 2001. Population structure of Aedes albopictus from La Reunion Island (Indian Ocean) with respect to susceptibility to a dengue virus. Heredity 87: 273–283.[ISI][Medline]
  4. Knox TB, Kay BH, Hall RA, Ryan PA, 2003. Enhanced vector competence of Aedes aegypti (Diptera: Culicidae) from the Torres Strait compared with mainland Australia for dengue 2 and 4 viruses. J Med Entomol 40: 950–956.[ISI][Medline]
  5. Ross KG, Trager JC, 1990. Evolution. Systematics and population genetics of fire ants (Solenopsis saevissima complex) from Argentina. Evolution 44: 2113–2134.
  6. Tsutsui ND, Suarez AV, Holway DA, Case TJ, 2000. Reduced genetic variation and the success of an invasive species. Proc Natl Acad Sci U S A 97: 5948–5953.[Abstract/Free Full Text]
  7. Fonseca DM, Keyghobadi N, Malcolm CA, Mehmet C, Schaffner F, Mogi M, Fleischer RC, Wilkerson RC, 2004. Emerging vectors in the Culex pipiens complex. Science 303: 1535–1538.[Abstract/Free Full Text]
  8. Kolbe JJ, Glor RE, Rodriguez Schettino L, Lara AC, Larson A, Losos JB, 2004. Genetic variation increases during biological invasion by a Cuban lizard. Nature 431: 177–181.[Medline]
  9. Tanaka K, Mizusawa K, Saugstad ES, 1979. A revision of the adult and larval mosquitoes of Japan (including the Ryukyu Archipelago and the Ogasawara islands) and Korea (Diptera: Culicidae). Contributions of the American Entomological Institute 16: 1–987.
  10. Andreadis TG, Anderson JF, Munstermann LE, Wolfe RJ, Florin DA, 2001. Discovery, distribution, and abundance of the newly introduced mosquito Ochlerotatus japonicus (Diptera: Culicidae) in Connecticut, USA. J Med Entomol 38: 774–779.[ISI][Medline]
  11. Peyton EL, Campbell SR, Candeletti TM, Romanowski M, Crans WJ, 1999. Aedes (Finlaya) japonicus japonicus (Theobald), a new introduction into the United States. J Am Mosq Control Assoc 15: 238–241.[ISI][Medline]
  12. Moore CG, Mitchell CJ, 1997. Aedes albopictus in the United States: ten-year presence and public health implications. Emerging Infectious Diseases 3: 329–334.[ISI][Medline]
  13. Falco RC, Daniels TJ, Slameck MC, 2002. Prevalence and distribution of Ochlerotatus japonicus (Diptera: Culicidae) in two counties in southern New York State. J Med Entomol 39: 920–925.[ISI][Medline]
  14. Roppo MR, Lilja JL, Maloney FA, Sames WJ, 2004. First occurrence of Ochlerotatus japonicus in the state of Washington. J Am Mosq Control Assoc 20: 83–84.[ISI][Medline]
  15. Joy JE, 2004. Larval mosquitoes in abandoned tire pile sites from West Virginia. J Am Mosq Control Assoc 20: 12–17.[ISI][Medline]
  16. Schaffner F, Chouin S, Guilloteau J, 2003. First record of Ochlerotatus (Finlaya) japonicus japonicus (Theobald, 1901) in metropolitan France. J Am Mosq Control Assoc 19: 1–5.[ISI][Medline]
  17. Takashima I, Rosen L, 1989. Horizontal and vertical transmission of Japanese encephalitis virus by Aedes japonicus (Diptera: Culicidae). J Med Entomol 26: 454–458.[ISI][Medline]
  18. Turell MJ, O’Guinn ML, Dohm DJ, Jones JW, 2001. Vector competence of North American mosquitoes (Diptera: Culicidae) for West Nile virus. J Med Entomol 38: 130–134.[ISI][Medline]
  19. Sardelis MR, Turell MJ, Andre RG, 2001. Ochlerotatus j. japonicus in Frederick County, Maryland: discovery, distribution, and vector competence for West Nile virus. J Am Mosq Control Assoc. 2: 137–141.
  20. Sardelis MR, Dohm DJ, Pagac B, Andre RG, Turell MJ, 2002. Experimental transmission of eastern equine encephalitis virus by Ochlerotatus j. japonicus (Diptera: Culicidae). J Med Entomol 39: 480–484.[ISI][Medline]
  21. Sardelis MR, Turell MJ, Andre RG, 2002. Laboratory transmission of La Crosse virus by Ochlerotatus j. japonicus (Diptera: Culicidae). J Med Entomol 39: 635–639.[ISI][Medline]
  22. Sardelis MR, Turell MJ, Andre RG, 2003. Experimental transmission of St. Louis encephalitis virus by Ochlerotatus j. japonicus. J Am Mosq Control Assoc Jun 19: 159–162.
  23. Kutz FW, Wade TG, Pagac BB, 2003. A geospatial study of the potential of two exotic species of mosquitoes to impact the epidemiology of West Nile virus in Maryland. J Am Mosq Control Assoc 19: 190–198.[ISI][Medline]
  24. Belton P, Belton OC, 1990. Aedes togoi comes aboard. J Am Mosq Control Assoc 6: 328–329.[ISI][Medline]
  25. Pafume BA, Campos EG, Francy DB, Peyton EL, Davis AN, Nelms M, 1988. Discovery of Aedes (Howardina) bahamensis in the United States. J Am Mosq Control Assoc 4: 380.[ISI][Medline]
  26. Lounibos LP, 2002. Invasions by insect vectors of human disease. Annu Rev Entomol 47: 233–266.[ISI][Medline]
  27. Sames WJ, Herman WE, Florin DA, Maloney FA, 2004. Distribution of Ochlerotatus togoi along the Pacific coast of Washington. J Am Mosq Control Assoc 20: 105–109.[ISI][Medline]
  28. Tsutsui ND, Suarez AV, Holway DA, Case TJ, 2001. Relationships among native and introduced populations of the Argentine ant (Linepithema humile) and the source of introduced populations. Mol Ecol 10: 2151–2161.[Medline]
  29. Zhang DX, Hewitt GM, 2003. Nuclear DNA analyses in genetic studies of populations: practice, problems and prospects. Mol Ecol 12: 563–584.[Medline]
  30. Reinert JF, 2000. New classification for the composite genus Aedes (Diptera: Culicidae: Aedini), elevation of subgenus Ochlerotatus to generic rank, reclassification of the other subgenera, and notes on certain subgenera and species. J Am Mosq Control Assoc 16: 175–188.[ISI][Medline]
  31. Fagerberg AJ, Fulton RE, Black WC, 2001. Microsatellite loci are not abundant in all arthropod genomes: analyses in the hard tick, Ixodes scapularis and the yellow fever mosquito, Aedes aegypti. Insect Mol Biol 10: 225–236.[ISI][Medline]
  32. Sunnucks P, 2000. Efficient genetic markers for population biology. Trends in Ecology and Evolution 15: 199–203.
  33. Elsik CG, Williams CG, 2001. Low-copy microsatellite recovery from a conifer genome. Theor Appl Genet 103: 1189–1195.
  34. McLain DK, Rai KS, Fraser MJ, 1987. Intraspecific and interspecific variation in the sequence and abundance of highly repeated DNA among mosquitoes of the Aedes albopictus subgroup. Heredity 58: 373–381.
  35. Rao PN, Rai K, 1987. Inter and intraspecific variation in nuclear DNA content in Aedes mosquitoes. Heredity 59: 253–258.
  36. Keyghobadi N, Matrone MA, Ebel GD, Kramer LD, Fonseca DM, 2004. Microsatellite loci from the northern house mosquito (Culex pipiens), a principal vector of West Nile virus in North America. Molecular Ecology Notes. 4: 20–22.
  37. Hamilton MB, Pincus EL, Di Fiore A, Fleischer RC, 1999. Universal linker and ligation procedures for construction of genomic DNA libraries enriched for microsatellites. Biotechniques 27: 500-2–504-7.
  38. Sambrook J, Fritsch EF, Maniatis T, 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
  39. Fonseca DM, Campbell S, Crans WJ, Mogi M, Miyagi I, Toma T, Bullians M, Andreadis TG, Berry RL, Pagac B, Sardelis MR, Wilkerson RC, 2001. Aedes (Finlaya) japonicus (Diptera: Culicidae), a newly recognized mosquito in the United States: analyses of genetic variation in the United States and putative source populations. J Med Entomol 38: 135–146.[ISI][Medline]
  40. Fonseca DM, Atkinson CT, Fleischer RC, 1998. Microsatellite primers for Culex pipiens quinquefasciatus, the vector of avian malaria in Hawaii. Mol Ecol 7: 1617–1619.[Medline]
  41. Rozen S, Skaletsky HJ, 2000. Primer 3 on the WWW for general users and for biologist programmers. Krawetz S. MS, ed. Bioinformatics Methods and Protocols: Methods in Molecular Biology. Totowa, NJ: Humana Press, 365–386.
  42. Raymond M, Rousset F, 1995. Genepop (version1.2): population-genetics software for exact tests and ecumenicism. J Heredity 86: 248–249.[Free Full Text]
  43. Huber K, Mousson L, Rodhain F, Failloux AB, 1999. Short report: microsatellite sequences as markers for population genetic studies of the mosquito Aedes aegypti, the vector of dengue viruses. Am J Trop Med Hyg 61: 1001–1003.[Abstract]
  44. Barbazan P, Dardaine J, Gonzalez JP, Phuangkoson N, Cuny G, 1999. Characterization of three microsatellite loci for Aedes aegypti (Diptera: Culicidae) and their use for population genetic study. Southeast Asian J Trop Med Public Health 30: 482–483.[Medline]
  45. Ravel S, Herve JP, Diarrassouba S, Kone A, Cuny G, 2002. Microsatellite markers for population genetic studies in Aedes aegypti (Diptera: Culicidae) from Cote d’Ivoire: evidence for a microgeographic genetic differentiation of mosquitoes from Bouake. Acta Trop 82: 39–49.[ISI][Medline]
  46. Huber K, Loan LL, Hoang TH, Tien TK, Rodhain F, Failloux AB, 2002. Temporal genetic variation in Aedes aegypti populations in Ho Chi Minh City (Vietnam). Heredity 89: 7–14.[ISI][Medline]
  47. Paupy C, Chantha N, Huber K, Lecoz N, Reynes JM, Rodhain F, Failloux AB, 2004. Influence of breeding sites features on genetic differentiation of Aedes aegypti populations analyzed on a local scale in Phnom Penh Municipality of Cambodia. Am J Trop Med Hyg 71: 73–81.[Abstract/Free Full Text]
  48. Severson DW, Knudson DL, Soares MB, Loftus BJ, 2004. Aedes aegypti genomics. Insect Biochem Mol Biol 34: 715–721.[ISI][Medline]
  49. Behbahani A, Dutton TJ, Raju AK, Townson H, Sinkins SP, 2004. Polymorphic microsatellite loci in the mosquito Aedes polynesiensis. Molecular Ecology Notes 4: 59–61.
  50. Simon C, Frati F, Beckenbach A, Crespi B, Liu H, Flook P, 1994. Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers. Ann Entomol Soc Am 87: 651–701.
  51. Primmer CR, Moller AP, Ellegren H, 1996. A wide-range survey of cross-species microsatellite amplification in birds. Mol Ecol 5: 365–378.[Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by WIDDEL, A. K.
Right arrow Articles by FONSECA, D. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by WIDDEL, A. K.
Right arrow Articles by FONSECA, D. M.
Related Collections
Right arrow Vector Biology
Right arrow Medical Entomology
Right arrow Mosquitoes

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS