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Population genetic structure of the Culex pipiens (Diptera: Culicidae) complex, vectors of West Nile… Joyce, Andrea L; Melese, Etienne; Ha, Phuong-Thao; Inman, Allan Jan 4, 2018

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RESEARCH Open AccessPopulation genetic structure of the Culexpipiens (Diptera: Culicidae) complex, vectorsof West Nile virus, in five habitatsAndrea L. Joyce1*, Etienne Melese2, Phuong-Thao Ha3 and Allan Inman4AbstractBackground: The Culex pipiens complex consists of several morphologically similar, closely related species. In the UnitedStates, Cx. pipiens L. is distributed North of 39° latitude, while Cx. quinquefasciatus Say occurs South of 36° latitude; a hybridzone occurs between these two latitudes including in the Central Valley of California. Members of the Cx. pipiens complexand their hybrids are vectors for West Nile virus (WNv). Hybrid offspring of Cx. pipiens and Cx. quinquefasciatus have beenfound to have enhanced transmission rates of WNv over those of pure populations of each species. We investigated whetherhybrids of Cx. pipiens and Cx. quinquefasciatus occurred more frequently in any of five habitats which were dairies, rural,suburban, and urban areas, and wetlands. In addition, the proportion of alleles unique to Cx. quinquefasciatus and Cx.pipiens found in each habitat-associated population were determined.Methods: Amplified fragment length polymorphism (AFLP) markers were used to compare the population structure ofthe Cx. pipiens complex from each habitat to geographically distant populations considered pure Cx. pipiens and Cx.quinquefasciatus. Structure analyses were used to assign individuals to either Cx. pipiens, Cx. quinquefasciatus, or hybridsof the Cx. pipiens complex. The ancestry of hybrids (F1, F2, or backcrossed) in relation to the two parent populationswas estimated for each Central Valley population. Loci unique to the pure Cx. pipiens population and the pure Cx.quinquefasciatus population were determined. The proportion of loci unique to Cx. pipiens and Cx. quinquefasciatuspopulations were subsequently determined for each population from the five Merced habitats and from the OrovilleCalifornia population. The unique loci found in Merced populations and not in Cx. pipiens or Cx. quinquefasciatus werealso determined. A principal components analysis was run, as was an analysis to determine loci under putative selection.Results: The Structure Harvester analysis found K = 3, and the Culex pipiens complex mosquitoes formed a genetic clusterdistinct from Cx. quinquefasciatus and Cx. pipiens. Individuals collected from each habitat were nearly all hybrids. However,Cx. pipiens complex collected near dairies had more individuals categorized as Cx. pipiens than collections from the otherhabitats. None of the mosquitoes collected in Merced or Oroville were considered pure Cx. quinquefasciatus. Significantgenetic divergence was detected among the Cx. pipiens complex from the five habitats in Merced; Cx. pipiens complexmosquitoes from dairies were divergent from the urban and suburban populations. New Hybrids analysis found thatindividuals from all five Merced habitat-associated populations and the population from Oroville were primarily categorizedas hybrids backcrossed to the Cx. pipiens population. Finally, all five habitat-associated populations shared more alleles withCx. pipiens than with Cx. quinquefasciatus, even though the pure Cx. quinquefasciatus population was more geographicallyproximate to Merced. Results from the principal component analysis, and the occurrence of several unique loci in Mercedpopulations, suggest that Cx. pipiens molestus may also occur in the habitats sampled.(Continued on next page)* Correspondence: ajoyce2@ucmerced.edu1Public Health, University of California Merced, 5200 North Lake Road,Merced, CA 95343, USAFull list of author information is available at the end of the article© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Joyce et al. Parasites & Vectors  (2018) 11:10 DOI 10.1186/s13071-017-2594-6(Continued from previous page)Conclusions: Nearly all mosquitoes in the five habitats in Merced in the Central Valley of California area were hybrids of Cx.pipiens and Cx. quinquefasciatus, consisting of hybrids backcrossed to Cx. pipiens. Habitat-associated mosquitoes collectednear dairies had more individuals consisting of pure Cx. pipiens, and no mosquitoes from Merced or Oroville CA classified aspure Cx. quinquefasciatus. The genetic distances among Cx. pipiens and Cx. quinquefasciatus, and hybrid populations agreewith previous studies using other molecular markers. Cx. pipiens hybrids in Merced shared more alleles with Cx. pipiens thanCx. quinquefasciatus which was unexpected, since Merced is geographically closer to the northern limit of Cx. quinquefasciatusdistribution. Culex pipiens molestus may occur in more habitats in the Central Valley than previously suspected, which warrantsfurther investigation. Future studies could investigate the vector competence of hybrids backcrossed to either Cx. pipiens or Cx.quinquefasciatus parent for their ability to transmit West Nile virus.Keywords: AFLPs, Culex pipiens, Culex quinquefasciatus, Culex pipiens molestus, HybridsBackgroundThe Culex pipiens species complex consists of severalmorphologically similar closely related mosquito speciesinvolved in the transmission of West Nile virus [1]. WestNile virus (WNv), originally described from Uganda andintroduced on the East Coast of the United States in 1999,spread rapidly across North America and reached theWest Coast in several years. West Nile virus is enzootic,primarily contained in a bird-mosquito transmission cyclewith humans being incidental hosts, although preventionof human infection with West Nile remains a publichealth concern where the virus has been introduced [2].In the United States, Culex pipiens L. and Cx. quinquefas-ciatus Say are introduced species, with Cx. pipiens generallyfound North of 39° latitude, and Cx. quinquefasciatus Southof 36° latitude; the region between these latitudes whichincludes the Central Valley of California is a contact zonewhere hybrids of Cx. pipiens and Cx. quinquefasciatus occur[3–6] (Fig. 1). One ecological factor in California which con-tributes to hybrid formation and maintenance of the hybridzone is that Cx. pipiens inhabits cooler northern latitudesand undergoes a winter reproductive diapause, while Cx.quinquefasciatus resides in more southern latitudes and canoverwinter without diapausing. Both Cx. pipiens and Cx.quinquefasciatus are efficient vectors of WNv [7], but thetwo species differ in their host preferences. Cx. pipiens hostfeeding preference is primarily ornithopihlic [4, 8], and Cx.quinquefasciatus feeds on both birds and mammals, whichcan bridge the transmission of WNv between avian andmammalian hosts [9–11]. In addition, another very closelyrelated member of the complex, Culex pipiens molestus, isreported in some areas of the United States, and typically in-habits primarily underground areas such as sewers andbasements [12, 13], while in Europe Cx. pipiens molestushybrids have been found underground as well as above-ground in animal shelters [8, 14].The morphological identification and separation of mem-bers of the Culex pipiens species complex is difficult. Priorto genetic studies, the primary morphological method usedto separate these two species was the DV/D ratio of malegenitalia [15]. Molecular studies have since been developedsuch as rapid genetic assays which can be used to distin-guish species within the complex [16–18], and the ace-2gene can be useful to distinguish some populations morethan others [16]. Culex pipiens and Cx. quinquefasciatusare known to hybridize. Hybrids of the two mosquito spe-cies might have biological traits of both parent species,which could broaden their host preference and increase thetransmission and infection rates of West Nile virus [19, 20].Hybrids have been challenging to identify as well. For ex-ample, neither the DV/D ratio nor the ace-2 gene couldFig. 1 Map of the Pacific Coast of the United States, showing the39° latitude southern boundary of the distribution of Cx. pipiens andthe 36° latitude northern boundary of Cx. quinquefasciatus. The zonebetween the two latitudes (between 39° and 36°) is the hybrid zonefor the two Culex species. Collections from this study are Cx. pipiensfrom Lewis and Clark Reservoir Washington, Cx. pipiens complexfrom Oroville California and Merced County California, and Cx.quinquefasciatus from Coachella California. Merced collections arefurther detailed in Table 1 and Fig. 2Joyce et al. Parasites & Vectors  (2018) 11:10 Page 2 of 15distinguish hybrid populations of the Cx. pipiens complexnear Fresno, California [5].Hybrids of Cx. pipiens and Cx. quinquefasciatus havebeen demonstrated to have higher transmission rates ofWNv than those of each parent species [21]. For example,crosses of male × female Cx. pipiens had offspring with a6% WNv transmission rate after a 13–14 d extrinsic incu-bation period (EIP), while progeny of male × female Cx.quinquefasciatus crosses had a 63.4% transmission rate[21]; hybrid offspring of female Cx. quinquefasciatus × maleCx. pipiens had even higher transmission rates (80.3%),while hybrids of female Cx. pipiens and male Cx. quinque-fasciatus had a 62% transmission rate [21]. Similarly, hybridoffspring from Cx. quinquefasciatus or Cx. pipiens crossedwith Cx. pipiens molestus had higher transmission ratesthan those from offspring of pure parental crosses. Hybridsof the Cx. pipiens complex are potentially a greater threatto public health if they have higher transmission rates ofWest Nile virus than offspring from pure parental crosses.The proportion of hybrids and the WNv transmission ratehave been investigated in the hybrid zone of Cx. pipiens andCx. quinquefasciatus in the Central Valley of California.Using the DV/D ratio measurement of genitalia, mosquitoesidentified as the Cx. pipiens complex from dairy lagoons inMerced County consisted of 11% Cx. pipiens complexhybrids and 89% Cx. pipiens [22]. Another study using theDV/D ratio to examine individuals from Merced found that32% of the Cx. pipiens complex were classified as hybrids,62% as Cx. quinquefasciatus and 6% were Cx. pipiens [3].Variation in the percentage of Cx. pipiens to Cx. quinquefas-ciatus was found among multiple sites in Stockton, CA, sug-gesting population structure can occur on a relatively smallscale [3]. Using field-collected mosquitoes from FresnoCounty and the ace-2 gene PCR assay for identification, 22%(98/442) of the Cx. pipiens complex collections were identi-fied as hybrids; these field-collected hybrids had a 20% WNvinfection rate (20/98 collected) compared with 21% (58/271)of those identified as Cx. quinquefasciatus or 14% (10/73)identified as Cx. pipiens [5]. However, the same study byMcAbee et al. [5] used the DV/D ratio and found 66% (293/442) of individuals in Fresno classified as hybrids. In Cx.pipiens populations fromMerced to Bakersfield, WNv trans-mission rates varied from 12.5 to 40% and 50 to 69% afteran EIP of 7 and 14 d, while populations of ‘pure’ Cx. pipiensand Cx. quinquefasciastus from northern and southernCalifornia did not transmit West Nile virus [22]. Goddardet al. [1] found a Bakersfield population of Cx. quinquefas-ciatus had a higher transmission rate of WNv (52%) after14 d EIP than did those from Riverside or Orange California(19 and 36%, respectively), while Cx. pipiens from Shastahad a 71% transmission rate after 14 d extrinsic incubationperiod [1]. The transmission rate of WNv for the Cx. pipienscomplex varies among these studies. However, populationsfrom the hybrid zone have typically demonstrated higherWNv transmission rates than pure parental populations,and transmission rates of WNv for Cx. quinquefasciatus aregenerally greater than for Cx. pipiens and Cx. pipiens moles-tus [1, 5, 21, 22].Habitat can influence the species composition, the abun-dance of hybrids, and the WNv infection rate of the Cx.pipiens complex [23]. In other insect systems, adjoininghabitats can contribute to genetically divergent populations,which has been termed ecological speciation [24, 25]. Simi-larly, mosquito populations with distinct host preferencescan have genetic differences as well [26]. Culex pipiens andCx. pipiens molestus can occur in different habitats in closeproximity [13], demonstrating that habitat can be associ-ated with the abundance of Cx. pipiens complex hybrids.Culex pipiens molestus is commonly associated with under-ground areas such as basements and sewers, yet recentlyhybrids of Cx. pipiens and Cx. pipiens molestus have beenfound indoors in animal shelters [8]. In California, previousstudies have focused on hybridization of Cx. quinquefascia-tus and Cx. pipiens; several studies have found evidence ofCx. pipiens molestus [12, 13]. The role of habitat in hybridformation for Cx. pipiens and Cx. quinquefasciatus hasrarely been investigated [27]. Although the proportion ofhybrids in the Cx. pipiens complex was similar in urbanand rural areas (34 and 36%, respectively), the abundanceof the Cx. pipiens complex and the WNv infection rate washigher in urban low income areas than in urban middleincome or in rural areas [23]. The general pattern of humanWNv cases in North America has been that most casesoccur in urban and agricultural (rural) habitats [28]. TheCentral Valley of California is largely rural, but habitatswithin Merced County vary greatly. Eastern Merced Countyhas an urban corridor along a major highway and numerousdairies and potential hosts for Cx. quinquefasciatus, whilewestern Merced County is rural with smaller communities,agricultural areas, and extensive wetland habitat for migrat-ing birds, the preferred hosts of Cx. pipiens. Previous studieswhich included Cx. pipiens complex mosquitoes from Mer-ced included few samples and varied greatly in the propor-tion of hybrids found. Given that laboratory studies havedemonstrated that pure Cx. quinquefasciatus and its hybridswith Cx. pipiens have higher West Nile virus transmissionrates, a more thorough investigation of the Cx. pipiens com-plex in the region would be beneficial.The objective of this study was to determine the popu-lation genetic structure of Cx. pipiens complex mosqui-toes in five habitats in Merced County, which is locatedin the Central Valley of California. This study investi-gated whether a particular habitat had a higher fre-quency of hybrids relative to abundance of pure Cx.quinquefasciatus or pure Cx. pipiens. Similarly, we wereinterested in whether any habitat had more pure Cx.quinquefasciatus due to its higher transmission rate ofWNv than that of Cx. pipiens. Areas or habitats foundJoyce et al. Parasites & Vectors  (2018) 11:10 Page 3 of 15to have more hybrids or more Cx. quinquefasciatus couldbe prioritized as targets for vector control.MethodsCollecting samplesMosquitoes from five habitats were collected in the hybridzone of Cx. pipiens and Cx. quinquefasciatus in MercedCounty in the Central Valley of California (Figs. 1 and 2).Traps were set in four quadrants of the county; Northwestnear Gustine, Southwest near Los Banos, Northeast nearHilmar and Livingston, and Southeast near Merced(Fig. 2). The distance across the sampled area in MercedCounty was 30 miles from North to South, and 30 milesfrom East to West. Mosquito samples were collected dur-ing 2012–2014. Each year, traps were set in the five habi-tats in each quadrant which included near dairies, rural,suburban, urban areas, and in wetlands. All traps were setin open outdoor areas with the intent to trap populationsof Cx. quinquefasciatus, Cx. pipiens, and their hybrids; notraps were placed underground in sewers or basements, orin animal shelters, where Cx. pipiens molestus was pre-sumed to occur. Dairies were typically in rural areas andhad nearby dairy lagoons with runoff water polluted withmanure. Rural areas were farmland or countryside withless than 2500 people in the area [29]. Suburban areaswere residential areas located on the outskirts of a city ortown [30], while urban areas were characterized bydensely developed locations with at least 2500 residents[29]. Finally, wetlands were defined as land where watershallowly covers the soil at the surface and includes areassuch as swamps and marshes [31]. Many collection site loca-tions are sites regularly surveyed by the Merced CountyMosquito Abatement District (MCMAD) surveillance pro-gram and are included in the CaliforniaVectorborne DiseaseSurveillance (CalSurv) database used for West Nile surveil-lance in California [32].Adult female mosquitoes were trapped using CDC stylelight traps baited with CO2 set in the afternoon andretrieved the following morning during the months ofJune–October. Adult mosquitoes from traps were frozenand subsequently identified using light microscopy [33].Culex pipiens and Culex quinquefasciatus are morpho-logically identical and thus classified as the Culex pipienscomplex. The difficulty in distinguishing these two speciesmorphologically, and the inconsistency of results usingthe DV/D ratio to separate hybrids of the two species,have led to a number of investigations using molecularmarkers to distinguish the two species. In Merced County,there are no other Culex species which resemble theCulex pipiens complex. Females identified as belonging tothe Cx. pipiens complex were frozen or saved in ethanolfor subsequent DNA extraction. Samples were collectedfrom 31 unique sites in Merced County (Table 1), whichwere classified into five habitats of interest. The followingnumber of collection sites within Merced county were usedfor each of the five habitats: dairy (n = 5), rural (n = 9),Fig. 2 Collections of the Cx. pipiens complex in Merced County, California from five habitats. The habitats include dairies, rural, suburban, urbanareas and wetlandsJoyce et al. Parasites & Vectors  (2018) 11:10 Page 4 of 15suburban (n = 5), urban (n = 7) and wetlands (n = 5) (Table1, Fig. 2). Geographical coordinates of trap locations wererecorded at each sampling site (Table 1, Fig. 2).A population of Cx. pipiens from Washington StateNorth of 39°, and one of Cx. quinquefasciatus fromsouthern California South of 36° were included in thestudy for comparison with samples from our five Mercedhabitats. The pure Cx. pipiens population was collectedfrom Lewis and Clark Reservoir, Washington (46°21′32″N,-119°25′29″E) and the Cx. quinquefasciatus population wascollected from Mecca, Coachella, California (33°34′47″N,-116°4′37″E) (Fig. 1). We obtained a population fromTable 1 Location of sample collections in Merced County by habitatHabitat Site name or site code CalSurv Map code (Fig. 2) GPS coordinates Individuals usedDairy 000402 D1 37.431944, -120.864167 2001225 D2 37.44205, -120.625528 42Baker Dairy D3 37.105948, -120.781767 9F&A Brooks Dairy D4 37.294767, -120.58755 11Stevenson/Hilmar D5 37.0985, -120.7701933 8Total 72Rural 001219 R1 37.426017, -120.745781 1000407 R2 37.255278, -120.992222 3000102 R3 37.261667, -120.518889 10001103 R4 37.389842, -120.661219 11Netherton R5 37.2819444, -121.0138888 9000413 R6 37.045, -120.713056 2Old Romero School R7 37.130233, -121.0147166 9Stevenson Ranch R8 37.290767, -120.96598 8Gustine Airport R9 37.260866, -120.96598 1Total 54Suburban909 S1 37.3375, -120.456111 6000506 S2 37.393333, -120.719444 16000507 S3 37.378611, -120.719444 4000403 S4 37.408056, -120.85 18KM’s House S5 37.319008, -120.4422833 3Total 47Urban 000916 U1 37.302222, -120.481389 8001205 U2 37.312183, -120.492539 11001227 U3 37.310103, -120.496792 4001304 U4 37.247411, -121.010942 1001301 U5 37.307947, -120.452553 6Sonora Pool U6 37.324713, -120.465416 1Sycamore U7 37.252366, -120.004333 4Total 35Wetlands000417 W1 37.338333, -120.526667 3001302 W2 37.099117, -120.816314 2000005 W3 37.364444, -120.578056 5Gustine Water Trtmnt W4 37.252817, -120.973617 2Gustine Duck Club W5 37.1750166, -120.947466 4Total 16Joyce et al. Parasites & Vectors  (2018) 11:10 Page 5 of 15Oroville in northern California which we originally plannedto use as the ‘pure’ Cx. pipiens population. However, the hy-brid zone of Cx. pipiens and Cx. quinquefasciatus is nowconsidered to extend North of its original 39° latitudeboundary (and North of Oroville California), so we insteadchose to use the Washington State population as our pureCx. pipiens population (Fig. 1). All mosquito samples col-lected and used for DNA in this study were female.DNA extraction, amplified fragmented lengthpolymorphisms (AFLPs)DNA was extracted from the entire mosquito body (head,thorax, and abdomen) for all individuals using the QiagenDNeasy Blood and Tissue kit (Venlo, Netherlands) followingthe protocols for animal tissue with an overnight incubationtime of ~ 24 h at 65 °C [34]. Final products were eluted in100 μl of AE buffer. The DNA quantity was measured usingthe Qubit® dsDNA HS Assay kit (Life Technologies-ThermoFisher Scientific, Waltham, MA, USA). The quantity ofDNA in samples averaged 5–10 ng/μl. Only female adultswere used for molecular work.Amplified fragment length polymorphisms were pro-duced as described by Vos et al. [35] and modified byJoyce et al. [36]. Three primer combinations were used,(i) M-CAT and E-ACG, (ii) M-CAC and E-ACT and (iii)M-CAC and E-ACA (Table 2). Individuals from the fivehabitats (dairy, rural, suburban, urban and wetlands) inMerced, California and from three other locations [Mecca,Coachella, California (COA); Oroville California (ORO);and Lewis and Clark Reservoir, Washington (WA)] wereall randomized on five 96-well plates for AFLP reactions.Eleven individuals were run in duplicate in order to testthe error rate of the AFLP markers.Each restriction/ligation reaction (well) consisted of thefollowing: 0.05 μl each of EcoRI and MseI, 1.1 μl of T4DNA ligase buffer, 1.1 μl of 0.5 M NaCl, 0.55 μl of dilutedBSA (bovine serum albumin), 0.03 μl of T4 DNA ligase,1.0 μl each of EcoRI and MseI adaptor pairs (LifeTechnologies-Thermo Fisher Scientific, Waltham, MA,USA), and 0.61 μl of sterile distilled water. The plate withrestriction⁄ ligation reactions was held at room temperatureovernight (12 h at 25 °C) to ensure complete digestion [37].The amplified product was diluted 20-fold using 15 mMTris-HCl buffer (pH 8.0) containing 0.1 mM EDTA. Pre-selective PCR amplification was performed on a Thermo-Fisher Arktik thermal cycler. Each reaction contained 15 μlof AFLP preselective mix (all Life Technologies/Thermo-Fisher), 1 μl of each amplification primer (Life Technolo-gies), along with 4 μl of the diluted restriction⁄ ligation mix-ture. The PCR program for pre-selective amplificationconsisted of an initial warm-up of 95 °C for 1 min followedby 20 cycles at 95 °C for 20 s, 56 °C for 30 s, and 72 °C for90 s with a final hold at 75 °C for 5 min. The amplifiedproduct was diluted 20-fold using 15 mM Tris-HCl buffer(pH 8.0) containing 0.1 mM EDTA. Selective amplificationwas conducted using two primer combinations. For eachselective amplification, a reaction consisted of 15 μl ofAFLP platinum supreme mix, 1.0 μl of EcoRI + 3 selectiveprimers, and 1.0 μl of MseI + 3 selective primers (all LifeTechnologies-Thermo Fisher Scientific Waltham, MA,USA)(Table 2). The PCR program for selective amplifica-tion consisted of an initial warm-up of 95 °C for 1 min,12 cycles of 95 °C for 20 s, 65 °C for 40 s with a lowering of0.7 °C per cycle, 72 °C for 90 s, followed by 35 cycles of 95 °C for 20 s, 56 °C for 40 s, 72 °C for 90 s, and finally a holdof 72 °C for 7 min before storing the samples at 4 °C. Priorto capillary electrophoresis, 0.4 μl of the Genescan LIZ 500size standard and 0.9 μl of HiDi formamide (all Life Tech-nologies) were added to 1 μl of the final product of eachsample. The LIZ 500 size standard allows for detection offragments between 50 and 500 bp. Sample fragments wereseparated using automated capillary electrophoresis by theABI 3730 XL automated capillary DNA sequencer (AppliedBiosystems-Thermo Fisher, Waltham, MA, USA).GeneMapper version 5.0 (Life Technologies, Thermo-Fisher) was used to determine presence or absence of frag-ments. The peak detection threshold was set for eachprimer combination, and was typically 150 luminescent units.Each AFLP marker was considered a locus and assumed tohave two possible alleles (0 = absent, 1 = present). Bands notpresent in more than one individual were eliminated (i.e. pri-vate alleles) prior to further analyses, as they were not consid-ered informative. For samples which were run in duplicate,each marker was examined to determine whether markerswere scored identically at each locus by GeneMapper, anddata were used to calculate the mismatch error rate [38].Structure 2.3.4 software [39] was used to group individualswith similar genotypes within each species. Structure uses aBayesian algorithm to cluster individuals into K, which isdefined as the number of genetically distinct populations in adata set. Parameters used for the analyses include the follow-ing: no a priori assignment of individuals to a known popula-tion, analysis for diploid insects, a burn-in of 100,000 and200,000 subsequent iterations, an admixture model, and inde-pendent loci.For runs in Structure software, the number of poten-tial populations for K was estimated as the number ofTable 2 Primer combinations used for selective polymerasechain reaction of amplified fragment length polymorphisms(AFLPs), number of markers produced by each primercombination, number < 125 bp, and the percent mismatch errorratePrimercombinationEcoR1- Mse1- No. of markers Markers <125 bp Percentmismatch1 ACG CAT 120 38/120 2.02 ACT CAC 110 29/110 2.03 ACA CAC 120 25/120 1.6Joyce et al. Parasites & Vectors  (2018) 11:10 Page 6 of 15geographical sampling locations plus 4 as suggested byPritchard et al. [40]. At the completion of Structure runs,K was calculated for each species using Structure Har-vester using the Evanno method [41, 42], to determine themost likely number of population clusters (K) for the pop-ulations sampled. A Structure analysis was first run for allpopulations, and K was estimated as the number of geo-graphic sampling locations plus 4 [5 Merced habitats +Oroville + pure Cx. quinquefasciatus + pure Cx. pipienspopulations (8 pops +4, K = 12)] for the overall analysis assuggested by Pritchard et al. [40], and each iteration wasrun 20 times. A Structure analysis was also run for mos-quitoes collected in each Merced habitat and compared tothe Cx. pipiens and Cx. quinquefasciatus populations, aswas the Oroville population. For each habitat analysis inStructure, the number of potential populations for K wasestimated as the number of geographic sampling locationsplus 4 (a single habitat + pure Cx. quinquefasciatus + pureCx. pipiens populations = 3 populations +4, K = 7 for eachhabitat analysis) as suggested by Pritchard et al. [40]. Simi-larly, mosquitoes collected in all five Merced habitats werecompared in an additional Structure analysis (K estimated as5 habitats +4, K = 9), and examined with Structure Harvesteras well. A q value of >0.80 from Structure was used to assignindividuals to clusters while individuals with a q value <0.8were considered admixed [13]. Structure results were used inClumpak software to run Distruct to permutate runs to bestvisualize results.New Hybrids v.1.1 software was used to examine theprobability of each Cx. pipiens complex mosquito’s assign-ment of membership into a number of groups, includingpure Cx. quinquefasciatus, pure Cx. pipiens, F1 hybrids ofthe two Culex species, F2 hybrids, or backcrosses to eitherparent [43]. Individuals were assigned to a pure species ifq > 0.9, F1 if q = ~0.5, F2 if q < 0.5 for both parent species,and considered a hybrid backcross to a parent species if0.5 ≤ q < 0.9. Individuals were not assigned a priori to a par-ticular population, and runs were conducted with Jefferylike priors. One hundred thousand iterations were run andthe posterior probability of each individual’s assignment tothe above genetic classes was determined.An analysis of molecular variation (AMOVA) was run tocompare the molecular variation of individuals of the Cx.pipiens complex from populations collected in four regions,including Cx. quinquefasciatus (COA), Cx. pipiens (WA),Oroville California (ORO) and all individuals from MercedCalifornia [44]. The AMOVA was run using 999 permuta-tions, and pairwise comparisons of the genetic divergence(FST) values between populations were made, using Bonfer-roni corrections for multiple comparisons. A secondAMOVA was run for individuals from the 5 Merced habitatpopulations also using 999 permutations. Similarly, the Fstvalues were compared for significance between pairs ofpopulations, with Bonferroni corrections for multiplecomparisons. Analyses were run using GenAlEx 6.5 soft-ware [45]. Output from the AMOVA Fst values were usedto run a principal component analysis among the eightpopulations using GenAlEx 6.5.Nei’s genetic distance was determined among all 8 pop-ulations using GenDist in Phylip 3.695 [46]. A Mantel testwas run to determine if genetic distance was correlated togeographical distance between populations. Since collec-tions from each Merced habitat came from 5 to 9 sites inthe county (Table 1), we chose a representative locationand genetic distance for Merced to run the Mantel testbetween Merced and the three other collection regions.We used the genetic distance from a downtown Mercedurban population (U6) for comparison with Oroville CA,Cx. pipiens (WA) and Cx. quinquefasciatus (COA).For Cx. pipiens complex mosquitoes from the five habitats,the proportion of unique alleles from Cx. quinquefasciatus(COA) and Cx. pipiens (WA) populations was determinedfor each Merced mosquito. Alleles unique to Cx. quinquefas-ciatus were defined as those which were present only in thepure Cx. quinquefasciatus population and not in in the pureCx. pipiens population; similarly, loci unique to Cx. pipienswere those only found in the pure Cx. pipiens population.Unique loci from the pure Cx. quinquefasciatus and the pureCx. pipiens population in each Merced mosquito were firstidentified. Next, the number of unique Cx. quinquefasciatusalleles and unique Cx. pipiens alleles were determined forindividuals in each Merced habitat-associated population,and used to produce the mean proportion of Cx. quinquefas-ciatus and Cx. pipiens alleles for each of the five habitats andthe Oroville population. For each habitat, we used a Chi-square goodness of fit test to compare whether the propor-tion of unique Cx. pipiens: Cx. quinquefasciatus alleles var-ied from a 1:1 equal distribution [47]. One-way ANOVAwas then used to determine whether one habitat had a sig-nificantly higher proportion of Cx. quinquefasciatus allelesthan other habitat-associated populations. Unique alleleswhich were found only in the Merced habitat populationsbut were not found in the Oroville, the pure Cx. quinque-fasciatus or the Cx. pipiens populations were also identifiedas well. Loci present in higher frequency and found innearly half the individuals (30 or more) of at least 2 or morehabitat-associated populations were determined, followedby unique alleles found in very low frequencies (typically in1–10 individuals) in the Merced populations.The software Mcheza was used to examine candidateloci that may be under selection in the habitat associatedpopulations [48]. Mcheza is available from popgen.net,and is a selection workbench developed for dominantmarkers such as AFLPs. The file with presence orabsence of AFLP loci was converted to the Genepop for-mat. MCheza was run to examine which loci were Fstoutliers. The following default settings were used; confi-dence interval 0.95, false discovery rate of 0.1, Theta 0.1,Joyce et al. Parasites & Vectors  (2018) 11:10 Page 7 of 15beta-a 0.25 and beta-b 0.25. The option of neutral meanFst was chosen and 500,000 simulations were run. Can-didate loci under positive selection, balancing selectionand neutral selection were determined.ResultsCollecting samplesFemale mosquitoes identified as the Culex pipiens complexwere collected from 5 dairies, 9 rural, 5 suburban, 7 urbanand 5 wetland sites in Merced County (Table 1, Fig. 2).Culex pipiens complex mosquitoes were generally moreabundant from collections near dairies and were relativelycommon in rural, suburban and urban samples (Table 1).Most wetlands collections in this study yielded few Cx.pipiens complex mosquitoes, as the wetlands sites weredominated by Culex tarsalis Coquillett.Population genetic structure: amplified fragment lengthpolymorphisms (AFLPs)Amplified fragment length polymorphisms (AFLPs) wereproduced using 3 primer combinations (Table 2). Therewere 350 alleles produced for comparison of the 278 indi-viduals from the 8 populations in the study, which includedCoachella California (Cx. quinquefasciatus), 5 habitat-associated populations of Cx. pipiens complex from MercedCA, Oroville CA (Cx. pipiens complex) and Cx. pipiensfrom Washington State. From Merced County, there were72 Cx. pipiens complex mosquitoes included from dairies,54 from rural areas, 52 from suburban areas, 35 from urbansites and 16 from wetland habitats. There were also 17 mos-quitoes of pure Cx. quinquefasciatus from Coachella, 17 Cx.pipiens complex from Oroville, and 15 pure Cx. pipiensfrom Washington. Fragments produced were viewed withGeneMapper 5.0 and scored as present or absent for eachindividual. Three primer combinations were used to pro-duce AFLPs, resulting in 120, 110, and 120 fragments (Table2). The number of fragments which were sized <125 bp foreach primer were 38/120 for primer combination 1, 29/110for primer 2, and 25/120 for primer 3 (Table 2). A test ofthe error mismatch rate for the AFLP markers for 8 individ-uals from Merced found that fragments had a 2, 2 and 1.6%mismatch error rate for each of the three primers, respect-ively (Table 2).The Structure analysis of all 8 populations and subse-quent Structure Harvester analysis of the data found thehighest Delta K Evanno value at K = 9, followed by K = 3(Figs. 3 and 4, Additional file 1: Figure S1). However, theresult of K = 9 populations was not biologically relevant,so the value of K = 3 was chosen indicating that there were3 genetically distinct groups (Figs. 3 and 4, Additional file1: Figure S1, Additional file 2: Figure S2). Culex quinque-fasciatus from Coachella California formed the first group,the Merced and Oroville California collections formed asecond central valley group, and the last cluster was Cx.pipiens from Washington (Fig. 3). The Structure analysisfound that all individuals of Cx. quinquefasciatus fromCoachella California had a q value of >0.8 and were con-sidered pure Cx. quinquefasciatus (Fig. 3), and most indi-viduals of Cx. pipiens from Washington had q values >0.8.From the Washington Cx. pipiens population, there were6 individuals considered admixed. Nearly all individualsfrom Merced classified their own cluster (97%, 233/237green) (Fig. 3). The Cx. pipiens complex mosquitoes fromthe Merced habitats had 3% (6/239) individuals that classi-fied as pure Cx. pipiens (blue bars); one was from dairy,one from rural, two from suburban and two from urbanareas (Fig. 3). None of the mosquitoes from Merced habi-tats classified as pure Cx. quinquefasciatus. Oroville CAmosquitoes all classified as admixed (Fig. 3).Subsequent Structure analyses compared individuals fromeach Merced habitat to those of the pure Cx. pipiens andCx. quinquefasciatus populations. The Structure Harvesteranalysis for each habitat found the number of geneticallydistinct populations was K = 2; individuals from eachMerced habitat shared alleles with both Cx. pipiens and Cx.quinquefasciatus, suggesting hybrid populations in eachMerced habitat as well as in Oroville CA (Additional file 3:Figure S3a-f). In all habitat analyses and the Oroville ana-lysis, the q values were >0.9 for all Cx. quinquefasciatusfrom Coachella California, and >0.9 for all Cx. pipiens fromWashington. In the Structure analysis of mosquitoes fromdairy habitat, most (69%, 50/72) were admixed, while 31%(22/72) had q values >0.9 for assignment to pure Cx. pipiens(Additional file 3: Figure S3a). For rural areas, most (88%,45/51) were admixed, and 12% (6/51) of mosquitoes had qvalues >0.9 for assignment to Cx. pipiens (Additional file 3:Figure S3b). For suburban mosquito collections, most 85%(40/47) again were admixed, and 15% (7/47) of mosquitoeswere assigned to Cx. pipiens (Additional file 3: Figure S3c).Urban mosquitoes followed a similar pattern; 89% of urbanmosquitoes (31/35) were admixed, and 11% (4/35) classifiedas Cx. pipiens (Additional file 3: Figure S3d). Finally, wet-lands had 100% of individuals admixed (16/16) (Additionalfile 3: Figure S3e). The collection from Oroville Californiahad 65% (11/17) of individuals admixed, and 35% (6/17)classified as Cx. pipiens (Additional file 3: Figure S3f). Anadditional Structure and Structure Harvester analysis of thefive Merced habitat-associated populations found the high-est Evanno Delta K value was at K = 8, followed by K= 6and K= 4; however, all Structure graphic files illustrating theprobability of assignment for individuals for all of theaforementioned K values indicated one genetic cluster,and it was concluded that K = 1.The software New Hybrids was used to classify theancestry of mosquitoes into groups, and determinewhether they were parental species, F1 or F2 hybrids,or backcrosses to pure parental species. The analysisfound the Coachella Ca mosquitoes were all classifiedJoyce et al. Parasites & Vectors  (2018) 11:10 Page 8 of 15as pure Cx. quinquefasiatus (100%), and the Washing-ton mosquitoes were all classified as parental Cx.pipiens, as expected (Table 3). Individuals from Mer-ced dairies had 3% (2/72) assigned to pure Cx.pipiens, 0% assigned to Cx. quinquefasciatus, 0% F1or F2 hybrids, and 97% (70/72) assigned to hybridbackcrosses to pure Cx. pipiens. Rural areas in Mer-ced had 2% (1/51) assigned to Cx. pipiens, 0% to Cx.quinquefasciatus, 0% F1 and F2, and 98% (50/51)were hybrids backcrossed to Cx. pipiens. Suburbanareas in Merced had 100% (47/47) classify as hybridsbackcrossed to Cx. pipiens, as did those from urbanareas (35/35) and wetlands mosquitoes (16/16). Fi-nally, Oroville had all but one (16/17, 94%) individualclassified as hybrids backcrossed to Cx. pipiens.Analysis of molecular variation (AMOVA)AMOVA of the Cx. pipiens complex populations fromfour geographic areas found that collection region had asignificant effect on genetic variation (Table 4), accountingfor 12% of variation. Pairwise Fst values of genetic diver-gence between the four groups were significant (P < 0.01).The genetic distance estimate between Cx. quinquefascia-tus from Coachella and Cx. pipiens from Washington was0.360, Cx. quinquefasciatus from Coachella to Merced andOroville were 0.164 and 0.232, respectively, and Cx.pipiens from Washington to Oroville and Merced was0.108 and 0.106, respectively (Table 5). Finally, Mercedand Oroville which are both considered to be in thehybrid zone were less genetically distant (0.046) from eachother than with the other populations.Fig. 3 A Structure 2.3.4 analysis of the 8 populations in the study was run using the following parameters: diploid individuals, 100,000 iterations,admixed data, and independent loci. Each vertical bar represents an individual mosquito. The y-axis shows the probability of an individual beingassigned to one of the three genetic clusters. Red bars (Cx. quin) represent the Cx. quinquefasciatus individuals from Coachella, California. Greenbars represent individuals from five habitat-associated populations from Merced; dairy, rural, suburban, urban and wetlands. Oroville (Oro) California is amixture of green and blue bars, and blue bars (Cx. pip) represent individuals of Cx. pipiens from Washington. Structure Harvester found that K = 3; therewere three genetically distinct populationsJoyce et al. Parasites & Vectors  (2018) 11:10 Page 9 of 15Molecular variation between the five Merced habitatcollections was significant as well (AMOVA, P = 0.008),and accounted for 1% of the variation (Table 4). Pairwisegenetic divergence tests found that individuals collectednear dairies were significantly different than those col-lected near suburban and urban areas (P < 0.01) (Table 6).Pairwise comparisons among genetic divergence of otherhabitats were not significantly different.The principal components analysis found that the firstaxis accounted for 61.82% of the variation, while the sec-ond and third axes explained 22.56% and 11.77% of thevariation, respectively (Fig. 5). Examining axis 1, fromleft to right, there is a clear separation of the Cx. pipiensand Cx. quinquefasciatus populations into the top twoquadrants (Fig. 5). The population from Oroville nearlyclustered into the quadrant with Cx. pipiens, and it wasintermediate between the Cx. pipiens population and theMerced habitat-associated populations. The second axis(y-axis) separates the Cx. pipiens population from theMerced habitat associated populations, which fall into athird quadrant, yet are positioned on axis 1 between Cx.pipiens and Cx. quinquefasciatus (Fig. 5).Genetic distance and mantel testNei’s genetic distance was determined between all 8 popu-lations in the study (Table 7). The largest genetic distancewas between the Cx. pipiens and Cx. quinquefasciatuspopulations from Coachella California and Washington(0.134, 13%). Culex quinquefasciatus from southern Californiawas 0.064–0.067 distant from the five habitats in Merced and0.087 distant from Oroville. Culex pipiens from Washingtonwas 0.05 distant from Oroville, California and 0.042–0.49 dis-tant from Merced populations. Finally, the Merced popula-tions were 0.021–0.032 distant from Oroville (Table 7). AMantel test found no significant relationship between thegenetic distance and geographic distance for the Cx. pipienspopulations (r= 0.686, P= 0.110).Proportion of unique Cx. quinquefasciatus and Cx. pipiensalleles by habitatThe proportion of alleles unique to the COA Cx. quinque-fasciatus and WA Cx. pipiens populations were deter-mined for each individual from the five Merced habitats(Table 8). The number of unique alleles in the pure Cx.quinquefasciatus population which were not found in pureCx. pipiens was 73, while 122 unique alleles were found inthe pure Cx. pipiens which were not present in the pureFig. 4 Results from a Structure Harvester analysis of all eightpopulations. Structure Harvester uses the results from Structure tocalculate the Delta K value, the change in likelihood, for the numberof potential clusters. Structure Harvester calculated the most likelynumber of clusters was 3 (K = 3)Table 3 Frequency of individuals in pure and hybrid classes from a New Hybrids analysisPure-bred A Cx. pipiens Hybrids Pure-bred B Cx.quinqueLineage Pure A F1 F2 Backcross A Backcross B Pure BCx. pipiens 15/15 0 0 0 0 0Dairy 2/72 0 0 70/72 0 0Rural 1/51 0 0 50/51 0 0Suburban 0 0 0 47/47 0 0Urban 0 0 0 35/35 0 0Wetland 0 0 0 16/16 0 0Oroville 1/17 0 0 16/17 0 0Cx. quinquefasciatus 0 0 0 0 0 17/17Table 4 Results of analysis of molecular variation (AMOVA) testsSource df Sum of squares Variation (%) PAmong regionsa 3 563.80 12 0.001Individuals within regions 274 10,694.66 88Among habitatsb 4 204.86 1 0.008Individuals within habitats 224 8904.05 99aCx. pipiens complex populations from four regions including Cx.quinquiefasciatus from Coachella CA, Cx. pipiens complex from Merced andOroville, and Cx. pipiens from WashingtonbPopulations of Cx. pipiens complex from five Merced habitatsAbbreviation: df degrees of freedomJoyce et al. Parasites & Vectors  (2018) 11:10 Page 10 of 15Cx. quinquefasciatus population. For each of the fivehabitat-associated populations in Merced, the averagenumber of Cx. quinquefasciatus alleles in all individuals ineach populations was 9, while the average number of Cx.pipiens alleles was 15; Oroville individuals had an averageof 10 unique alleles from Cx. quinquefasciatus and 20from Cx. pipiens. The proportion of unique Cx. quinque-fasciatus alleles in Merced populations ranged from anaverage of 38% in dairy and urban populations up to 42%in rural populations (Table 8), while the proportion ofunique Cx. pipiens alleles ranged from 0.58 in rural areasto 0.62 in dairies (Table 8). Within each habitat, a Chi-square test found that the proportion of Cx. pipiens: Cx.quinquefasciatus varied significantly from 1:1 (P < 0.05)(Table 8), with a lower proportion of alleles contributedfrom Cx. quinquefasciatus than from Cx. pipiens. A one-way ANOVA found there was no significant difference inthe proportion of unique Cx. quinquefasciatus allelesamong all 5 habitat-associated populations (F(4,265) = 0.65,P = 0.623). The Oroville population had a larger propor-tion of Cx. pipiens alleles than of Cx. quinquefasciatus (χ2= 38.72; df = 1; P < 0.001) (Cx. pipiens 72%, Cx. quinque-fasciatus 28%) (Table 8).Merced habitat-associated populations and the Oro-ville population were examined as well to determinetheir unique alleles. Merced habitats had three alleles atrelatively high frequency, which were found in nearlyhalf the individuals in 2 or 3 habitats, yet they were notpresent in the Oroville population, nor in the Cx. pipiensor Cx. quinquefasciatus populations. One of these lociwas present in 34, 30, 30, 15 and 9 individuals of dairy(D), rural (R), suburban (S), urban (U) and wetland (W)habitat collections, respectively, while a second and thirdunique loci were found in 31D, 21R, 3S, 4 U, 7 W and1D, 16R, 13S, 6 U and 0 W individuals of the same habi-tats. In additional, there were fifteen alleles found at avery low frequency (in 1–10 individuals) in several habi-tats in Merced, and which were also not found in theOroville, Cx. pipiens, or the Cx. quinquefasciatus popu-lations. The Mcheza analysis to examine loci under se-lection found 10 loci under putative positive selection,and 36 loci possibly under balancing selection (Fig. 6).DiscussionThe Cx. pipiens species complex consists of morpho-logically similar, but genetically, behaviorally and eco-logically distinct populations. Numerous studies haveconsidered whether Cx. pipiens and Cx. quinquefasciatusare distinct species or subspecies of Cx. pipiens, espe-cially with respect to variation in host feeding preferenceand reproductive diapause [49, 50]. Previous studiesused the DV/D ratio of the genitalia to separate thesetwo groups and their hybrids [15], but environmentalTable 5 Results of pairwise comparisons of genetic divergenceestimates (FST) between Cx. pipiens complex populationsPopulation 1 2 3 41 Cx. quinquefasciatus COA 02 Cx. pipiens complex Merced 0.164* 03 Cx. pipiens complex ORO 0.232* 0.046* 04 Cx. pipiens WA 0.360* 0.106* 0.108* 0*P < 0.01 indicates comparison between populations is significant. All valueswere significant at P < 0.01 after a Bonferroni correctionAbbreviations: COA Coachella CA, ORO Oroville CA, WA Lewis and ClarkeReservoir, WATable 6 Results of pairwise comparisons of genetic divergence estimates (FST) from five Merced habitatsPopulations in Merced habitat 1 2 3 4 51 Dairy 02 Rural 0.004 ns 03 Suburban 0.007* 0.001 ns 04 Urban 0.011* 0.008 ns 0.006 ns 05 Wetland 0.009 ns 0.011 ns 0.010 ns 0.008 ns 0*P < 0.01 indicates comparison between populations is significant. Results were corrected for multiple comparisons with a Bonferroni correctionAbbreviation: ns not significant at P < 0.01Fig. 5 Principal components analysis using genetic distance output ofAMOVA of the eight populations in the study. The eight populationsincluded pure Cx. pipiens from Washington, pure Cx. quinquefasciatus(Cx. quin) from Coachella, California, a population from Oroville (Oro)Ca, and five habitat-associated populations from MercedJoyce et al. Parasites & Vectors  (2018) 11:10 Page 11 of 15conditions and food availability could influence the sizeof individuals in a population, and subsequent workfound that DV/D ratios do not consistently correspondto genetic differences in populations [3, 5]. In the caseof a species complex, molecular markers are helpful andcould be more accurate to separate populations within aspecies group. Different insect orders vary in the level ofgenetic variation considered sufficient to warrant separ-ate species status [51, 52]. A study of Culex annulirostrisin Australia and Papua New Guinea found morphologic-ally similar but genetically divergent lineages just 3% di-vergent, one able to transmit Japanese encephalitis virus(JEV) while another lineage did not [53]. In this study,Cx. pipiens and Cx. quinquefasciatus had a genetic dis-tance of 0.134 (13%) indicating moderate genetic diver-gence, which supports that these two groups are distinctspecies [49].The Structure analysis of all eight populations consid-ered in this study found K = 3 (three distinct groups) withMerced and Oroville populations in the Central Valleymore similar to each other than to either the Cx. pipiensor Cx. quinquefasciatus populations. The AMOVA ana-lysis found 12% of molecular variation among the eightpopulations in this study, similar to the 11% and 10% vari-ation observed in two previous studies of Cx. pipiens pop-ulations in the Midwest and in California, USA [13, 20].The populations in this study from the Cx. pipiens complexhybrid zone had genetic distances between Cx. pipiens andCx. quinquefasciatus in the range of 0.04–0.05, close to 5%,suggesting the stable interbreeding hybrid population isundergoing significant divergence from Cx. pipiens and Cx.quinquefasciatus. This is also supported by the finding of anumber of loci putatively under positive selection (Fig. 6).Hybrid offspring of other species are often sterile, but theCx. pipiens complex hybrids in the Central Valley are fertileand can interbreed with both Cx. pipiens and Cx. quinque-fasciatus [13]. An interesting biological attribute of Cx.pipiens and Cx. quinquefasciatus is their ability to hybridizein areas where they have been introduced, yet not tohybridize in South Africa where they are thought to haveoriginated, and where they occur together sympatrically [4].A stable interbreeding population in the hybrid zone ofCalifornia has been observed previously [13]. A study of thepopulation genetic structure of the Cx. pipiens complexfrom southern to northern California and also includingWashington found four genetically distinct groups, with twoclusters in the Central Valley. One Central Valley Californiapopulation which occurred in collections from northernCalifornia near Shasta and South to Turlock was calledCluster X, which Merced Cx. pipiens hybrids may belong to.The FST pairwise genetic divergence estimates in this studyfor Cx. quinquefasciatus from Coachella and Cx. pipiensfrom Oroville were 0.236, while those of Kothera et al. [13]for Cx. quinquefasciatus from Coachella and Cx. pipiensfrom Shasta were 0.27. From the Coachella population tothat of Merced, the pairwise genetic divergence estimatewas 0.164, while in Kothera et al. [13] their measurementsfor two Coachella populations to Turlock ranged from 0.174to 0.187. The study by Kothera et al. [13] used microsatel-lites, while this study determined genetic structure usingAFLPs. The similarity of results between the two studiessupports the utility of both types of molecular markers forpopulation comparisons.We sampled different ecological habitats of Merced todetermine whether Cx. pipiens and Cx. quinquefasciatuscould occur in close proximity in different habitats, sincethey were found to occur together in previous studies ofthe Central Valley of California, as well as in South Africa[3–5, 22]. In Merced, the majority of individuals sampledwere hybrid mosquitoes; however, dairy populations hadmore pure Cx. pipiens individuals. Populations in all fivehabitats had more alleles specific to the Cx. pipiens popula-tion from Washington than alleles unique to Cx. quinque-fasciatus from Coachella, even though the geographicaldistance between Merced and Washington is almost twicethe distance as between Merced and Coachella, California(~700 miles vs ~400 miles). Another study of Cx. pipiensthrough the middle of the USA similarly found that inMemphis, near the center of the hybrid zone, nearly allindividuals sampled were hybrids [20].Table 8 The proportion of fixed unique alleles derived frompure populations of Cx. pipiens and Cx. quinquefasciatusHabitat No. ofindividualsProportion ofCx. pipiensallelesProportion ofCx. quinquefasciatusallelesχ2-valueP-valueDairy 72 0.620 0.380 11.49 0.0007*Rural 54 0.580 0.420 5.12 0.024*Suburban 47 0.600 0.400 8.00 0.005*Urban 35 0.620 0.380 11.49 0.0007*Wetlands 16 0.625 0.385 10.58 0.001**P < 0.05Table 7 Nei’s genetic distance among populations. Populationsfrom Coachella, California (COA), Merced County populations fromdairy, rural, suburban (Suburb.), urban and wetland (Wet.) areas,Oroville California, and Lewis and Clark Reservoir, Washington(L&C, WA). Locations of all populations in Table 1 and Fig. 2COA Dairy Rural Suburb. Urban Wet. Oroville L&C,WACOA. – 0.064 0.064 0.065 0.067 0.067 0.087 0.134Dairy – 0.006 0.007 0.009 0.012 0.021 0.042Rural – 0.006 0.009 0.014 0.027 0.048Suburb – 0.009 0.014 0.028 0.049Urban – 0.014 0.032 0.045Wetland – 0.030 0.048Oroville – 0.050L&C, WA –Joyce et al. Parasites & Vectors  (2018) 11:10 Page 12 of 15Habitats where Cx. pipiens complex mosquitoes werecollected in Merced had a small but significant influenceon the genetic composition of hybrids. AMOVA analysisrevealed 1% of genetic variation in Merced collections dueto habitats, with dairy collections being genetically diver-gent from rural and suburban habitats. The analysis whichexamined the number of unique Cx. pipiens or Cx. quin-quefasciatus alleles in the Merced populations found thaton average each habitat had a similar proportion of Cx.pipiens: Cx. quinquefasciatus alleles, with rural collectionshaving a trend toward a higher percentage of Cx. quinque-fasciatus alleles (42%); perhaps this small but statisticallyinsignificant difference is enough to influence wherehuman WNv cases occur.There were several alleles found in the Merced popula-tions at relatively high frequency, and fifteen alleles foundin the Merced populations at low frequency, none of whichwere found in the Cx. pipiens, Cx. quinquefasciatus or theOroville populations. The principal components analysisfound that Cx. pipiens and Cx. quinquefasciatus fell intoseparate quadrants. However, the Merced habitat associatedpopulations were in a third quadrant, but with respect tothe x-axis, they were in close proximity to being intermedi-ate between Cx. pipiens and Cx. quinquefasciatus. Thereare several explanations as to why the Merced hybrids arenot placed exactly in between the two upper quadrants withCx. pipiens and Cx. quinquefasciatus. The Merced popula-tions consist of many more individuals than included in theCx. pipiens or Cx. quinquefasciatus populations, whichmight have captured some alleles not present in the smallerparent populations. However, the Oroville population wassimilar sized to the Cx. pipiens and Cx. quinquefasciatuspopulations, and it lies between the Cx. pipiens quadrantand the third quadrant with the Merced populations. It isunlikely that the Merced mosquito populations are mis-identified; there are no known mosquito species in theMerced area which could be confused morphologically withthe Cx. pipiens complex. The placement of the Mercedpopulations in the third quadrant in the principal compo-nent analysis suggests a genetic contribution from a closelyrelated member of the Cx. pipiens complex, perhaps Cx.pipiens molestus.Previous studies of the Cx. pipiens complex in the Mercedarea did not consider the presence of Cx. pipiens molestus.We focused this study on whether Cx. quinquefasciatus orCx. pipiens complex hybrids might be more common in aparticular habitat; for that reason, mosquito collections werefocused outdoors in aboveground habitats where it was be-lieved that Cx. pipiens molestus would not commonly occur.However, several recent studies including have found Cx.pipiens molestus hybrids more widespread and in more hab-itats than previously expected [8, 13]. In this study, Mercedcollections did not include sewers or underground struc-tures such as basements which Cx. pipiens molestus wastraditionally considered to inhabit. Future studies in Mercedand the southern San Joaquin Valley should collect under-ground in sewers and basements along with other outdoorhabitats including in animal shelters, to determine whereCx. pipiens molestus may be present.The genetic composition of hybrids is just one factorthat could influence where WNv positive mosquitoes areabundant. Most hybrids were of similar genetic compos-ition, consisting primarily of hybrids backcrossed to Cx.pipiens. Thus, they may be likely to have similar vectorcapacities and transmission rates of West Nile virus. How-ever, this would need to be determined experimentally toascertain with certainly the ability of hybrids backcrossedto parental species to transmit West Nile virus.Temperature has been suggested as a limiting environ-mental factor which affects the distribution of Cx. pipiensand Cx. quinquefasciatus along with the extent of theirhybrid zone [5]. Warmer winters in southern latitudesallow Cx. quinquefasciatus to overwinter as reproductiveadults, which contributes to higher Cx. quinquefasciatusabundance in the spring, more generations per year, and alonger WNv disease transmission season. In the CentralValley of California, cooler winter temperatures may pre-vent Cx. quinquefasciatus from surviving. In northern lati-tudes where Cx. pipiens is abundant, adults undergoreproductive diapause and are less numerous in spring. InFig. 6 Loci under possible positive selection shown in red, neutral selection in the gray region, and those under balancing selection are shown inthe yellow region, using Mcheza softwareJoyce et al. Parasites & Vectors  (2018) 11:10 Page 13 of 15addition, Cx. pipiens have a low vertical transmission rateof West Nile virus, making the disease slower to increaseeach year in local mosquito populations [5]. Some havesuggested the southern range of the hybrid zone hasmoved North of 39° latitude [20]. The Oroville populationin this study which was collected near 39° latitude wasfound to be a hybrid population, not pure Cx. pipiens.Models of climate warming and increasing average tem-peratures predict the northern expansion of Cx. pipienscomplex hybrids, and an increasing number of humanWest Nile virus cases [53]. Studies have demonstratedvariation in vector capacity of Cx. pipiens complex popu-lations, with hybrids having higher transmission rates thannon-hybrids, and pure Cx. quinquefasciatus having amongthe highest transmission rates [5, 21].ConclusionsThis study investigated the hybrid composition of Cx.pipiens complex populations from five Merced habitats todetermine whether some habitats had more hybrids thanothers. Nearly all mosquitoes collected were hybrids back-crossed to Cx. pipiens; collections from dairies had more in-dividuals than the other habitats that classified as pure Cx.pipiens, while none of the habitats in Merced or Orovillehad mosquitoes that classified as pure Cx. quinquefasciatus.Mosquitoes collected in dairies were genetically divergentfrom those collected in suburban and urban areas. All hy-brids had a larger proportion of alleles shared with the pureCx. pipiens population than with the pure Cx. quinquefas-ciatus population. Results also suggest the presence of Cx.pipiens molestus in Merced, but this needs to be confirmed.The Cx. pipiens complex in the Merced shared more alleleswith the pure Cx. pipiens population, even though it wasmore geographically distant from the pure Cx. pipiens popu-lations than from the pure Cx. quinquefasciatus population.Cold winter temperatures may limit the northern introgres-sion of Cx. quinquefasciatus alleles into the hybrid zone.Future studies might examine the ability of hybrids back-crossed to Cx. pipiens and Cx. quinquefascitus to transmitWest Nile virus.Additional filesAdditional file 1: Figure S1. Results from a Structure Harvester analysisof all eight populations of the Cx. pipiens complex in this study. Each rowshows the probability of K populations and delta K. The most likely numberof populations was K = 3. (TIFF 2681 kb)Additional file 2: Figure S2. The results from Distruct using output fromStructure for K = 3 populations. (TIFF 2317 kb)Additional file 3: Figure S3. Structure analyses for each of five Mercedhabitat-associated populations and for the Oroville California populationindividually compared to Cx. quinquefasciatus from Coachella, California andto Cx. pipiens from Washington. Structure was run using the following parameters:diploid individuals, 100,000 iterations, admixed data, and independent loci. Eachvertical bar represents an individual mosquito. Structure Harvester found K= 2 clusters. The y-axis shows the probability of an individual being assignedto one of the two genetic clusters. Panels include a dairy (D) collections, brural (R) habitat collections, c suburban (S) collections, d urban (U) collections,e wetland (W) collections, and f Oroville California, each compared to pure Cx.quinquefasciatus and Cx. pipiens populations. (PDF 659 kb)AcknowledgmentsWe thank the staff at Merced County Mosquito Abatement District (MCMAD) forassisting in collecting samples and for assisting with identification of Culex pipienscomplex species, especially Jason Bakken and Arlilla Bueno. Ashley Valley andMaria Martinez of the University of California Merced helped with DNA extractionand AFLP work. Additional mosquito collections were obtained by Lindsay Robson,Kylie McMillan, and Austyn Smith. We thank Coachella Valley Mosquito and VectorControl District, Butte County Mosquito and Vector Control District and BentonCounty Mosquito Control District Washington for contributing insects.FundingThis project was supported by the Merced County Mosquito Abatement District(MCMAD) in Merced, California, United States. MCMAD supported the cost ofmosquito collections, genetic work, and UC Merced students who worked on theproject. The project was also supported by the University of California Merced,who contributed to the cost of research supplies and supporting undergraduatestudent assistants.Availability of data and materialsThe data supporting the conclusions of this article are included within the articleand its additional files. The datasets used and analyzed during the current studyavailable from the corresponding author upon reasonable request.Authors’ contributionsStudy design: ALJ and AI. Data collection: ALJ, EM, PTH and AI. Data analysis:ALJ, EM and PTH. Manuscript preparation: ALJ. All authors read and approvedthe final manuscript.Ethics approval and consent to participateNot applicable.Consent for publicationNot applicable.Competing interestsThe authors declare that they have no competing interests.Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations.Author details1Public Health, University of California Merced, 5200 North Lake Road,Merced, CA 95343, USA. 2Department of Microbiology and Immunology, LifeSciences Institute, University of British Columbia, Vancouver, BC V6T 1Z3,Canada. 3Santa Clara County Vector Control District, 1580 Berger Dr, SanJose, CA 95112, USA. 4Merced County Mosquito Abatement District, 3478Beachwood Dr, Merced, CA 95348, USA.Received: 22 February 2017 Accepted: 17 December 2017References1. Goddard LB, Roth AE, Reisen WK, Scott TW. Vector competence of Californiamosquitoes for West Nile virus. Emerg Infect Dis. 2002;8:1385–91.2. 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