Am. J. Trop. Med. Hyg., 75(2), 2006, pp. 251-255
Copyright © 2006 by The American Society of Tropical Medicine and Hygiene
RAPID ASSAY TO IDENTIFY THE TWO GENETIC FORMS OF CULEX (CULEX) PIPIENS L. (DIPTERA: CULICIDAE) AND HYBRID POPULATIONS
CAROLYN M. BAHNCK AND
DINA M. FONSECA*
Genetics Program, Smithsonian Institution, Washington, DC; Academy of Natural Sciences, Philadelphia, Pennsylvania
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ABSTRACT
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A previously developed method to identify members of the Culex pipiens complex exploiting polymorphisms in a nuclear intron (acetylcholinesterase [ACE] based-assay) cannot differentiate the two forms of Cx. pipiens: form pipiens and form molestus. Notably, the two forms seem to differ extensively in behavior and physiology and likely have very different epidemiologic importance. Because they are morphologically indistinguishable, molecular methods are critical for the evaluation of their relative importance. Although the two forms of Cx. pipiens have been distinguished using a panel of microsatellite loci, such a protocol is laborious and expensive. We developed a rapid assay based on polymorphisms in the flanking region of a microsatellite locus. Used in conjunction with the ACE-assay, this new assay allows the identification of pure and hybrid populations of the two Cx. pipiens forms as well as those including Cx. quinquefasciatus. We discuss the usefulness of the method as well as limitations to its application.
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INTRODUCTION
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Understanding vector population dynamics is fundamental to the development of disease control strategies. Complexes of sibling species present unique challenges because of the often-large differences in vectorial capacity between taxa that are morphologically indistinguishable.1 DNA-based rapid assays have emerged as tools to overcome the challenges of sibling species identification,2,3 but the Culex pipiens complex, which includes important disease vectors,4,5 has proven particularly difficult. This difficulty likely stems from the very recent divergence of behaviorally and physiologically distinct sub-groups, possibly associated with domestication. The nominal species of the complex, Culex (Culex) pipiens L., a temperate species, has two distinct forms: form pipiens and form molestus.6 Whereas Cx. pipiens f. pipiens diapauses, requires a blood meal to lay eggs (anautogeny), and is unable to mate in confined spaces, Cx. pipiens f. molestus does not diapause, is able to lay its first batch of eggs without a blood meal (autogeny), and mates in confined spaces (stenogamy).7 The combination of stenogamy and autogeny seems to allow Cx. pipiens f. molestus to occur in underground areas in urban settings.8 Although conclusive evidence is still lacking, the two forms are thought to have different blood host preferences (pipiens biting mainly birds and molestus mainly mammals, especially humans) and therefore very different vectorial capacities.9 The two forms have been shown to be genetically isolated in Northern Europe; but there is clear evidence of hybridization in North America.10 Further complicating the epidemiologic landscape in North America, Culex (Culex) quinquefasciatus Say, a tropical and sub-tropical species, hybridizes extensively with the temperate forms (D. M. Fonseca and others, unpublished data).10,11 Cx. quinquefasciatus is non-diapausing, anautogenous, and stenogamous. Also, as a species, it has no distinct preference for birds or mammals as blood sources and can be a vicious human biter.12,13 Although the shape of the male phallosome (genitalia) is diagnostic for Cx. pipiens and Cx. quinquefasciatus, the male phallosome is indistinguishable between the two forms of Cx. pipiens.6
With the objective of studying the phylogenetic relationships between members of the Cx. pipiens complex, we sequenced multiple alleles of several of the microsatellite loci currently available for the complex.14 During that process, we realized that locus CQ11 was diagnostic for the two forms of Cx. pipiens and decided to develop it as a rapid assay. The DNA-based rapid assay designed by Smith and Fonseca15 to identify most of the members of the Cx. pipiens complex, as well as other morphologically similar species, does not distinguish between the two forms of Cx. pipiens, indicating the need for an additional molecular assay. Although there has been a recent report that several transitions in the mitochondrial cytochrome oxidase subunit I allow the identification of the two forms,16 that assertion has since been refuted by the authors.17 Here, we describe a rapid assay that can be used to identify Cx. pipiens f. pipiens and Cx. pipiens f. molestus based on indels in the flanking region of a microsatellite locus. We also expand this assay to recognize the occurrence of populations that include hybrids of the two genetic forms as well as those that include Cx. quinquefasciatus.
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MATERIALS AND METHODS
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To prevent the need for cloning, we only identified specimens for sequencing that were homozygous at alleles representative of the range of allele sizes of CQ11 in Culex pipiens.14 We amplified using primer pairs CQ11F2/R3145 or CQ11F/R18 for Cx. pipiens and Cx. quinquefasciatus, respectively, using the conditions in Smith and others.14 To identify and discard Taq polymerase errors, two separate independent amplifications from each individual were sequenced. Cycle sequencing was performed using BigDye Terminator v.3.1 (Applied Biosystems [ABI], Foster City, CA). Sequences were analyzed using a capillary automated sequencer (ABI 3730) and aligned using Sequencher 4.2 (GeneCodes, Ann Arbor, MI). Based on observed diagnostic sequence differences between the two forms, we designed form-specific reverse primers using Primer3.19 We calculated the number of base pair differences between sequences using Arlequin 3.0.20 We excluded the actual microsatellite region contained in these sequences from this analysis and express the differences as average percent differences between taxa corrected for intra-taxa variation.20
We tested this assay in up to 14 specimens from four populations of Cx. pipiens f. pipiens, eight populations of Cx. pipiens f. molestus, and four populations of Cx. quinquefasciatus (Table 1
) that were carefully characterized as such by examination of behavioral and physiologic traits. All the specimens of form molestus were autogenous, bred easily in small cups, and were collected (or their parents were collected) in sheltered areas. Such sheltered areas range from flooded subterranean channels in a Menstrie factory and the London sewage (C. Malcolm, personal communication) to the warm water surrounding an electrical transformer in a clogged street drain in Philadelphia in December. The specimens of form pipiens were all collected outdoors, were anautogenous, and did not breed in laboratory cages. We included two locations (Menstrie in Scotland and Barking in London) where "molestus" and "pipiens" forms were collected within a few meters of each other. Populations of Cx. quinquefasciatus were identified both by genitalia analysis of male specimens as well as by the acetylcholinesterase (ACE) based rapid assay.15
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TABLE 1 Geographical location and sample size (n) of the pure populations of the two forms of C. pipiens as well as of Cx. quinquefasciatus used to test the validity of the rapid assay
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To allow the identification of the morphologically very similar Culex (Culex) torrentium Martini in Europe and Cx. quinquefasciatus in North America, we created a composite protocol using the assay we describe de novo here in conjunction with the ACE rapid assay.15 Briefly, the ACE reactions contain 1x polymerase chain reaction (PCR) buffer, 250 µmol/L of each dNTP, 2 mmol/L MgCl2, 0.15 mg/mL of BSA, 1 U of AmpliTaq (ABI), 0.1 µmol/L of both forward primers ACEtorr (or ACEquin) and ACEpip, 0.2 µmol/L of the reverse primer B1246s, and 1 µL of extracted genomic DNA (~6 ng). The PCR conditions used were as follows: 94°C for 5 minutes and then 35 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute, concluding with an extension of 72°C for 5 minutes. We tested the composite protocol in 26 populations of the Cx. pipiens complex and evaluated the ability of this rapid assay to identify hybrid populations.
We formally assessed the performance of the rapid assay using a subset of 18 representative populations (Table 2) by correlating the frequency of "pipiens" alleles in a group of specimens to their average probability of Cx. pipiens f. pipiens ancestry based on a full microsatellite analysis as in Smith and Fonseca.15 Probability of Cx pipiens f. pipiens ancestry was calculated using the program "Structure 2000"21 as in Fonseca and others.10 The least-squares regression was performed with JMP 3.1.6 (SAS Institute, Cary, NC). To provide a regression design not biased by an excessive number of pure populations, because in such populations the correlation between the two variables is 1, we removed eight pure populations of either Cx. pipiens forms or Cx. quinquefasciatus from the analysis. Their inclusion simply increases the correlation value.
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TABLE 2 Geographical location and sample sizes (n) of the specimens used to compare results of microsatellite analyses and rapid assays
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RESULTS
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We sequenced 10 specimens of Cx. pipiens and 7 of Cx. quinquefasciatus. We uncovered extensive variation in the flanking region of the CQ11 locus and found consistent sequence differences between the two Cx. pipiens forms and among all three taxa sequenced (Figure 1). On average, and after correcting for sequence variation within each group, we determined that Cx. pipiens form pipiens and Cx. pipiens form molestus differed by 14.7% (P < 0.001), a number that decreases to 2.6% (P < 0.001) when all indels are removed from the analysis. After testing multiple populations of Cx. pipiens f. molestus, we concluded CQ11 had only one allele size, and the microsatellite was composed of just four TG repeats. In contrast, Cx. pipiens f. pipiens had between 6 and 10 TG repeats (Figure 1).
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FIGURE 1. Polymorphisms occurring in the flanking regions and in the microsatellite locus CQ11 showing the relative position of the diagnostic primers. Each form and species is represented by the inclusive consensus of each allele size sequenced. The size of the alleles in base pairs is listed, and for comparative reasons, we calibrated all alleles to the sizes obtained using primer pair CQ11F2/R3. Position 1 corresponds to the first base pair of the original CQ11 clone, accession no. AF075420. A hyphen indicates a deletion, whereas a period indicates that the sequence is in agreement with Cx. pipiens f. pipiens allele 266. R, A/G; K, G/T; M, A/C; Y, C/T; W, A/T; S, C/G.
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Exploiting form-specific indels in the left flank of the microsatellite region, we developed several alternative diagnostic primers. Ultimately we optimized the protocol for pipCQ11R 5'-CATGTTGAGCTTCGGTGAA-3' and molCQ11R 5'-CCCTCCAGTAAGGTATCAAC-3'. The forward primer CQ11F2 5'-GATCCTAGCAAGCGAGAAC-3' is the same listed by Fonseca and others18 as CQ11F.14 We used a 20-µL reaction with a final concentration of 0.1 µmol/L of pipCQ11R and 0.15 µmol/L of molCQ11R and CQ11F2, 1x PCR buffer, 200 µmol/L of each dNTP, 2 mmol/L MgCl2, 0.15 mg/mL of bovine serum albumin (BSA), 1 U of AmpliTaq, and 1 µL of extracted genomic DNA. Conditions necessary for PCR amplification were as follows: 94°C for 5 minutes and then 40 cycles of 94°C for 30 seconds, 54°C for 30 seconds, and 72°C for 40 seconds, concluding with a final 5-minute extension at 72°C. Amplification products were run on a 2% agarose gel. Although the size of the DNA fragments varied in Cx. pipiens f. pipiens across the range of microsatellite alleles, they were always obviously smaller than the Cx. pipiens f. molestus DNA fragment. This is caused both by the larger flank of the molestus form (an extra 27 bp) and the fact that the pipCQ11R primer anneals 44 bp downstream from the molCQ11R primer (Figure 1
). The size difference allows us to easily distinguish the two forms with a single PCR run on a 2% agarose gel (Figure 2). It is important to point out, however, that the primers are also form specific, so in case of doubt, separate PCRs run with each set of primers will unambiguously identify each form.
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FIGURE 2. Fragments amplified using pipCQ11R, molCQ11R, and CQ11F2 primers and run on a 2% agarose gel. P, Cx. pipiens f. pipiens (from Nonnenweier, Germany); M, Cx. pipiens f. molestus (from Altenheim, Germany); MP, f. molestus and f. pipiens hybrid (from Greenville, SC); S, size standard (100-bp ladder; New England Biolabs, Beverly, MA).
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Although Cx. pipiens f. molestus and Cx. quinquefasciatus differ by 5.3% (1.5% without the indels; Figure 1), we were unable to design a compatible primer that would generate a band in Cx. quinquefasciatus with a diagnostic size. By combining our new assay with the ACE-assay, however, we were able to identify both the presence of Cx. quinquefasciatus (or Cx. torrentium in Europe) and American populations containing hybrids of Cx. quinquefasciatus and any of the two Cx. pipiens forms (Figure 3A and B). Specimens of the Cx. pipiens forms and of Cx. quinquefasciatus were identified unambiguously (Table 1). The probability of identifying hybrid populations was comparable when using the rapid assays or a full microsatellite analysis (Table 2; Figure 4). Pure populations of all three taxa are stacked on either extreme in Figure 4, with Cx. quinquefasciatus and Cx. pipiens f. molestus having a zero probability of Cx. pipiens f. pipiens ancestry and a zero frequency of "pipiens" bands. It is important to note that this analysis could be reproduced using the frequencies of the "molestus" or "quinquefasciatus" bands instead. We chose to use frequency of "pipiens" bands because identification of these Cx. pipiens form-specific PCR products in hybrid populations does not necessitate the use of the ACE-assay.
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FIGURE 3. Amplification products of (A) ACEpip, ACEquin, and B1246s primers run on a 1% agarose gel and (B) pipCQ11R, molCQ11R, and CQ11F2 primers run on a 2% agarose gel. P, Cx. pipiens f. pipiens; M, Cx. pipiens f. molestus; Q, Cx. quinquefasciatus;-c, negative control; S, size standard (100-bp ladder; New England Biolabs). Multiple letters within a lane denote hybrid ancestry. **ACE-assay and CQ11-assay do not agree for this specimen, indicating a recombination event. The sources of specimens used were (from left to right): Nonnenweier, Germany; Altenheim, Germany; Archer, FL; Greenville, SC; Walkertown, NC (the last three).
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FIGURE 4. Correlation between the frequency of PCR fragments diagnostic for Cx. pipiens f. pipiens and the average probability of ancestry from Cx. pipiens f. pipiens based on a panel of eight micro-satellite loci across 18 populations from North America and Europe. The numbers next to each data point (or group of data points) are the population identifiers (#) from Table 2.
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DISCUSSION
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We developed a new assay based on variation in the flanking regions of one of the microsatellite loci optimized for Cx. pipiens, CQ11. Although our current understanding of microsatellite evolution is wanting, it is widely accepted that, in general, microsatellites mutate at a much higher rate than other nuclear DNA and are usually in hyper-polymorphic regions.22 This observation may explain why the flanking region of locus CQ11 has already become different in the two Cx. pipiens forms that likely have a very recent shared history (D. M. Fonseca and others, unpublished data). It may also lead some to question the practicality of basing a molecular assay on a marker that we know is variable at the population level.14 However, our assay does not rely on microsatellite length variability; rather, it exploits several large (up to 18 bp) indels in the flanking region of the microsatellite that are specific to either Cx. pipiens f. pipiens or f. molestus. The generally accepted observation that smaller microsatellites experience lower mutation rates22 explains why Cx. pipiens f. molestus is fixed at the CQ11 locus, because replication slippage, the mechanism believed to generate the majority of microsatellite mutations, can no longer function once a threshold minimum number of repeats is reached.
We tested the rapid assay in well-characterized populations of the two forms of Cx. pipiens in northern Europe where they do not seem to hybridize and found that it performed well. The unique "pipiens" band was only recovered from specimens that matched the behavioral and physiologic characteristics of Cx. pipiens f. pipiens, and the converse was true for the unique "molestus" band. Furthermore, when we tested the assay in other populations, we obtained good concordance between a full microsatellite analysis and the rapid assays. However, because we sampled most extensively from North America and Europe, we are unable to confidently endorse its use in other areas of the world, particularly in Africa, without further sampling in these locations.
We also found that, in conjunction with the ACE-rapid assay, this new protocol provides a fast diagnostic of populations with hybrid specimens that involve one of the two Cx. pipiens forms. We emphasize that when hybrids are recognized, the full ancestry of the particular individual specimens requires a full microsatellite analysis. This is so for two reasons: first, the composite assay fails to separate between a hybrid of Cx. quinquefasciatus and Cx. pipiens f. pipiens from a hybrid that also has Cx. pipiens f. molestus ancestry (a three-way hybrid). This is because the pipCQ11R primer amplifies both in Cx. quinquefasciatus and in Cx. pipiens f. molestus (Figure 1); therefore, a full microsatellite analysis should be used if identifying such hybrids is paramount. Second, although the rapid assay will identify all first generation (F1) hybrids, backcrossing events result in recombination between loci leading to the independent assortment of the markers within hybrids. As a result, hybrid specimens may lose diagnostic bands. Because recombination occurs randomly across independent loci, the likelihood of loosing all traces of a hybrid ancestry decreases as the number of loci examined increases.
The decreased power of the rapid assay compared with a full microsatellite analysis, therefore, resides essentially in the lowered number of independent loci it samples. Take, for example, the specimen labeled with two asterisks in Figure 3B; although it generated the diagnostic band for Cx. quin-quefasciatus in the ACE locus, it does not have a diagnostic band for that species in the CQ11-assay, indicating recombination. More extensive recombination can lead to hybrid specimens actually lacking both ACE and CQ11 loci that are characteristic of one of the parental species or forms. Akin to increasing the number of molecular loci (as in a full microsatellite analysis), increasing the number of specimens tested per population decreases the probability of missing diagnostic markers and leads to correctly identifying a population as containing hybrids. We reiterate, however, that the results need to be interpreted at the population and not the individual level. A panel of multivariable microsatellite loci is the optimal method for determining the genetic ancestry of individual mosquitoes as well as of populations. The rapid assay we described, however, allows insight into the ancestry of Cx. pipiens complex populations without the added expense and time demands associated with a microsatellite analysis.
In conclusion, our rapid assay is fast and relatively inexpensive and has proven reliable. This approach will be useful in conjunction with ecological studies aiming at ascertaining the extent and epidemiologic significance of the multiple taxa in the Cx. pipiens complex, as well as offering insight into the consequences of their hybridization. The extensive sequence variation that forms the basis of this new assay also underscores the genetic uniqueness of the two forms of Cx. pipiens.
Received February 9, 2006.
Accepted for publication April 4, 2006.
Acknowledgments: We are deeply grateful to our collaborators for sending us samples. We also thank Kenli Okada and Andrea Widdel for extensive editorial comments and Tapan Ganguly and the DNA Sequencing Facility, University of Pennsylvania, for technical assistance.
Financial support: This study was supported by NIH Grant R01GM063258 and CDC/NIH Grant U50/CCU220532.
* Address correspondence to Dina M. Fonseca, Academy of Natural Sciences, 1900 Ben Franklin Parkway, Philadelphia, PA 19103. E-mail: fonseca{at}acnatsci.org 
Authors’ addresses: Carolyn Bahnck, Genetics Program, Smithsonian Institution, 3001 Connecticut Ave., NW, Washington, DC 20008-0551, E-mail: bahnck{at}acnatsci.org. Dina M. Fonseca, Academy of Natural Sciences, 1900 Ben Franklin Parkway, Philadelphia, PA 19103, E-mail: fonseca{at}acnatsci.org.
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ABSTRACT
INTRODUCTION
