AJTMH HINARI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am. J. Trop. Med. Hyg., 78(2), 2008, pp. 303-310
Copyright © 2008 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 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 Google Scholar
Google Scholar
Right arrow Articles by McAbee, R. D.
Right arrow Articles by Cornel, A. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by McAbee, R. D.
Right arrow Articles by Cornel, A. J.
Related Collections
Right arrow West Nile
Right arrow Medical Entomology
Right arrow Mosquitoes

Identification of Culex pipiens Complex Mosquitoes in a Hybrid Zone of West Nile Virus Transmission in Fresno County, California

Rory D. McAbee*, Emily N. Green, Jodie Holeman, Julie Christiansen, Niki Frye, Katherine Dealey, F. Steve Mulligan, III, Aaron C. Brault, AND Anthony J. Cornel
Department of Entomology, University of California at Davis, Mosquito Control Research Laboratory, Parlier, California; Center for Vector-Borne Diseases, University of California at Davis, California; Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California at Davis, California; Consolidated Mosquito Abatement District, Selma, California


ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Culex pipiens sensu lato mosquitoes were collected from 24 gravid traps (mid-June to mid-October, 2005) in Fresno County, CA. Captured gravid females were allowed to oviposit before sibling species identification by Ace.2 PCR and detection of West Nile virus (WNV) RNA by RT-PCR were performed on the mother and her offspring. Of the 442 Cx. pipiens s.l. female mosquitoes collected, 88 were positive for WNV viral RNA (peaked in August) with no significant differences among complex members or habitat. Vertical transmission was detected in 4 out of 20 families originating from WNV-positive mothers, however, in only a small number of offspring from each family. Out of 101 families that had PCR-based maternal and offspring identifications, the offspring from 15 families produced inexplicable amplicon patterns, suggesting ambiguities in the PCR assay identifications. Male genitalia (DV/D ratio) and Ace.2 PCR identifications revealed numerous discrepancies in our ability to accurately determine the identity of Cx. pipiens complex members in the hybrid zone of Fresno County.


INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In summer 2004, West Nile virus (family Flaviviridae, genus Flavivirus, WNV) activity was detected in most of California, including Fresno County. This activity was expected to continue and perhaps become more extensive in 2005. Given this scenario, we considered the summer of 2005 to be the optimal time to examine infection rates of WNV in a hybrid zone of Culex pipiens complex mosquitoes. Cx. pipiens s.l. mosquitoes have consistently been identified as major WNV vectors from numerous field isolations1–3 and from laboratory vector competence assays.4–7 In this paper no assumptions have been made about their taxonomic status, but herein after the terms Cx. pipiens and Cx. quinquefasciatus will be used to distinguish complex members.

Fresno County is located within the 36°N and 39°N latitude introgression zone in the Unites States where Cx. pipiens and Cx. quinquefasciatus and their hybrid populations occur in sympatry.8–10 Fresno County, therefore, represents a good locality to compare WNV infection rates among the two nominal taxa and hybrids of the Cx. pipiens complex temporally and spatially (urban versus rural versus peri-urban). Our original objectives where (1) to determine if specific members or populations of the Cx. pipiens complex mosquitoes were more commonly infected with WNV than others and (2) to determine if transovarial transmission occurs in the field by members of the Cx. pipiens complex. If spatial and temporal differences in infection rates were identified among the members of the complex, this information would be useful for mosquito abatement. For example, control and surveillance efforts could focus on sites that have a higher propensity to breed and maintain populations of Cx. quinquefasciatus versus Cx. pipiens and their hybrids in rural, peri-urban, or urban situations. Alternatively, if vertical WNV transmission is identified, control efforts could also be warranted in the winter to reduce over-wintering populations.

The justification for examining and comparing infection rates among Cx. pipiens s.l. stems from an ongoing debate about genetic differentiation and associated behavioral differences among the complex members,11,12 which was rekindled due to the invasion of WNV into the United States. Members of this complex display a variety of behavioral adaptations9,10,13–17 and show some morphologic differences8,18–22 and variation in WNV vector competency.4,23–25 It is not clear if the observed variation is associated with genetically discrete populations or taxa or if it represents a high degree of polymorphism within a single panmictic unit.

There are currently two methods to distinguish between Cx. pipiens and Cx. quinquefasciatus. One is based on relative overlap and measurements of the dorsal and ventral arms in male genitalia (DV/D ratio).26 Cx. pipiens have DV/D ratios of less than 0.2, Cx. quinquefasciatus has DV/D ratios greater than 0.4, and hybrids have intermediate ratios of 0.2–0.4. The other method that distinguishes the two nominal taxa target nucleotide differences in the Ace.2 gene that when amplified by polymerase chain reaction (PCR) produces a different size fragment for each of the two taxa.27,28 The Ace.2 gene is not associated with insecticide resistance.29,30

In this study we used a combination of male DV/D ratio measurements and the diagnostic PCR assay of Smith and Fonseca28 to identify wild females and their offspring. Identifications based on Smith and Fonseca28 were favored over the Aspen and Savage27 methods because of the simplicity of conducting one PCR procedure as opposed to two. Furthermore, Aspen and Savage27 PCR often did not amplify Cx. pipiens-specific product on specimens from California and elsewhere, such as in Africa, despite altering reaction conditions. After a wild female laid eggs, her DNA was extracted from a portion of the body and the rest of the carcass was used for WNV RNA detection. Wild female families were reared separately so that the DV/D ratios and PCR identifications of her male offspring could be matched to her identity to determine whether she mated with a conspecific or hybrid. Knowing the identity of the mother, in some instances PCR identification and DV/D ratios of the male offspring produced identifications that could not be possible genetically. This led us to consider evaluating the accuracies of both identification methods by setting up crosses and back-crosses between California Cx. pipiens north of 39°N and Cx. quinquefasciatus south of 36°N and comparing the hybrid and back-cross PCR and DV/D ratio identifications. These results indicate that both identification methods are unreliable, which has profound implications for systematic and ecological studies and resolving public health impacts of sibling species in the hybrid zone.


MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mosquito collection, rearing, and identification. Gravid female members of Cx. pipiens s.l. were captured in 8 rural, 8 urban (high-density housing, commercial areas), and 8 peri-urban (low-density housing on outskirts of cities) locations using modified gravid traps31 baited with 7-day-old Bermuda grass infusion.32 Previous evaluations found that gravid traps consistently collected more WNV positive Cx. pipiens complex mosquitoes than did CDC-style CO2 traps.32 We collected from a total of 24 trap sites, sampling 8 sites per week on a 3-wk rotation starting in June and ending in October.

Gravid females were placed into individual holding vials and provided with Bermuda grass-infused water to oviposit. After oviposition, the head and legs of the female were separated from the rest of the body for DNA extraction and the abdomen and thorax were stored in RNAlater (Ambion, Inc., Austin, TX) for RNA extraction and subsequent virus detection. In addition, all nongravid and nonovipositing mosquitoes were dissected and processed similarly.

Each egg raft (family) was reared separately in 2 L of Bermuda grass-infused water and larvae fed a diet of ground rodent diet 5001 (PMI Nutrition International LCC, Brent-wood, MO). Ten adult males (8 days old) from each family were separated into 3 parts. The head and legs were stored in 70% ethanol for DNA extraction, the thorax and abdomen were preserved in RNAlater for WNV RNA detection, and the genitalia were stored dry for DV/D ratio determination. PCR identification28 of the mother and her male offspring allowed for inferences to be made as to which complex member the mother mated with in the wild in the hybrid zone in Fresno County. Furthermore, congruence between PCR identification and DV/D ratio of the male offspring was evaluated. For the Smith and Fonseca28 PCR assay, the ACEpip, ACE-quin, and B1246s primers at the concentration recommended for the Americas were used. After PCR amplification, hybrids were scored on the basis of having 2 bands, each band corresponding to the fragment of diagnostic size for either Cx. pipiens or Cx. quinquefasciatus, regardless of the relative intensities of the bands. The remaining males and females from each family were pooled in groups of 25 and stored at –80°C for WNV RNA determination.

DNA was extracted from the head and legs of all dissected mosquitoes following the procedure described by Collins and others.33 Individual mosquitoes were identified by PCR,28 and male offspring were also identified by measurement of the DV/D ratio.26

WNV RNA detection from mosquitoes. Individual mosquitoes were ground in 0.5 mL of DMEM supplemented with 10% FBS, 10 U/mL penicillin/streptomycin, and 0.5 µg/mL Fungizone (Invitrogen, Carlsbad, CA) using a cordless motor and pellet pestles (Kontes, Vineland, NJ). RNA was extracted and eluted into 40 µL of nuclease-free water using the Viral RNA Mini-Kit (Qiagen, Valencia, CA) as per manufacturer specifications. Real-time PCR analysis used 10 µL of eluted RNA and the One-Step RT-PCR kit (Applied Biosystems, Foster City, CA) using an ABI 7500 Sequence Detection System. Primers spanning a portion of the envelope—WNENV-forward, WNENV-reverse, and WNENV-probe34—were used for the initial screening, whereas primers spanning a section of the NS1 region—3111V, 3239C, with probe 3136V35—were used for confirmatory purposes. Only samples that had a logarithmic increase in fluorescent signal above the threshold for both sets of primers and probes were considered positive.

Laboratory crosses. To evaluate the correlation of DV/D ratio to PCR identification we crossed males with females from laboratory colonies of Cx. quinquefasciatus (LA, originating in Los Angeles, CA) and Cx. pipiens (SH, originating from Redding in Shasta County, CA). The LA mosquitoes have been in colony since 2000 and have consistently produced Cx. quinquefasciatus-specific Ace.2 PCR products and DV/D ratios while in colony. The SH mosquitoes have been in colony since 2003 and have consistently produced Cx. pipiens-specific Ace.2 PCR products and DV/D ratios while in colony. Thirty individuals of each colony were first subjected to both DV/D ratio determination and PCR to ensure that the current generation still consistently produced identifications as either Cx. pipiens or Cx. quinquefasciatus. Virgin male and females were obtained by separating the sexes based on pupal morphology. All crosses were conducted in screened 28 cm x 28 cm x 28 cm cages in an insectary maintained at 26°C and 85–95% RH. The larvae were reared in 2 L of tap water and fed ground rodent diet 5001 (PMI Nutrition International LCC). Mice were used as a blood source for egg production 3 days after the virgin females and males were combined. The crossing schemes and the labels used are provided in Table 1Go. The crossing scheme was initiated by placing 20 virgin LA female with 20 SH male. The LA x SH hybrid egg rafts were reared, and the pupae were then sexed and reared to adults separately. Then, 20 hybrid male were inbred to 20 hybrid female, 20 hybrid male were back-crossed to 20 LA female, 20 hybrid male were back-crossed to 20 SH female, and the reciprocal back-cross of 20 hybrid female to males of each parental type was also done. The genitalia of 30 males not used in the inbreeding crosses or back-crosses were dissected for DV/D ratio measurements, and the corresponding remaining body was stored in 70% ethanol for DNA extraction and diagnostic PCR.28 This entire scheme was repeated except for initiating it with the reciprocal cross of 20 virgin LA female with 20 SH male . Each crossing series is designated by a letter A or B (Table 1Go). The A series consists of hybrids generated by a male SH and a female LA (AF1). These hybrids were then crossed with their siblings (AF2), and females were back-crossed to LA males (A2F2) and SH males (A3F2). Male AF1 mosquitoes were also back-crossed to LA females (A2F2) and SH females (A4F2). The B series of crosses is in the exact same pattern except the hybrids (BF1) are produced by mating between a male LA and a female SH.


View this table:
[in this window]
[in a new window]

 
TABLE 1
Designations for laboratory crosses between Cx. pipiens pipiens (Shasta) and Cx. pipiens quinquefasciatus (LA) colonies
 

RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
WNV infection in wild females and offspring. From mid-June until mid-October, 442 Cx. pipiens complex mosquitoes were collected from gravid traps in Fresno County and tested for presence of WNV RNA. The numbers of mosquitoes collected at each site varied considerably throughout this period. Overall we collected more mosquitoes in gravid traps in urban environments; however, there was much variation between collection sites. As an example, Figure 1Go depicts numbers collected in urban trap sites showing this variation for the duration of the study. Because of limited resources, we were unable to sample mosquitoes from all 24 traps every week; that would have enabled us to determine if trap counts showed significant differences between urban, rural, and peri-urban locations. The mosquitoes collected were identified by PCR28 of which 271 were Cx. quinquefasciatus, 73 Cx. pipiens, and 98 were hybrids. Of these 442 mosquitoes collected, 88 (19.9%) tested positive for WNV RNA based on the two RT-PCR primer-pair reactions. West Nile viral RNA-positive mosquitoes were collected in rural, urban, and peri-urban sites throughout Fresno County, with hot spots occurring in Navelencia and Selma (Figure 2Go).


Figure 1
View larger version (16K):
[in this window]
[in a new window]

  		    FIGURE 1. Numbers of Cx. pipiens s.l. collected in gravid traps from mid-June through to mid-October 2005 located in urban areas in Fresno County, California.

 

Figure 2
View larger version (45K):
[in this window]
[in a new window]

  		    FIGURE 2. Locations of gravid (triangles) and CO2-baited trap (squares) sites in Fresno County. The size of the triangle is proportional to the percentages of individual Cx. pipiens s.l. that were WNV RNA positive from the gravid traps. Black squares depict locations where WNV RNA-positive mosquito pools were reported. No WNV RNA-positive pools were reported for those sites with a white square.

 
Of the 88 mosquitoes that were positive for WNV RNA, 58 were Cx. quinquefasciatus, 10 were Cx. pipiens, and 20 were hybrids as determined by Ace.2 PCR. This overall infection rate of 19.9% was not significantly different among members of the Cx. pipiens complex at 95% confidence ({chi}2 = 0.85, Figure 3Go). There was also not a significant difference ({chi}2 = 0.93, 95% confidence) between the overall infection rate and mosquitoes collected in different local habitats (Figure 4Go). The vector index36 for each urban (0.73), peri-urban (0.50), and rural (0.61) site also indicated similar infection rates between habitats. There were significant differences ({chi}2 > 9.49) between the overall infection rate and the rates of infection determined for each month, with a peak of infection occurring in August (Figure 3Go).


Figure 3
View larger version (13K):
[in this window]
[in a new window]

  		    FIGURE 3. Percentage of WNV RNA positive Cx. pipiens s.l. as identified by Ace.2 PCR from mid-June through mid-October 2005.

 

Figure 4
View larger version (10K):
[in this window]
[in a new window]

  		    FIGURE 4. Percentage of WNV RNA positive Cx. pipiens s.l. collected from urban, peri-urban, and rural locations in Fresno County from mid-June through mid-October 2005.

 
Two hundred eight of the 442 females collected, oviposited, from which isofemale families were reared. Twenty of the females that oviposited were WNV RNA positive. From these 20 families, 5 individual offspring out of 47 tested (10.6%), representing 4 different families, were positive for WNV RNA, confirming that transovarial transmission occurred.

Identification of Cx. pipiens complex members in Fresno. Because we had offspring reared from a mother of known PCR Ace.2 Cx. pipiens complex identity for 208 families, we attempted, on the basis of offspring PCR identity, to predict which member of the complex the mother mated with in the wild. After the first round of PCR, 15 families produced combinations of offspring and mother identifications that could not be possible, such as producing offspring identifications homozygous for the Cx. pipiens-specific allele from mothers homozygous for the Cx. quinquefasciatus-specific allele. The opposite was also observed. A second round of PCR performed on the 15 un-interpretable families produced similar results.

DV/D measurements of the male offspring for which we had PCR identifications also revealed lack of congruence between the male genitalia morphology and the PCR identification (Figure 5Go). Both PCR and DV/D ratios were obtained for 475 male offspring. Ten males were selected from each of the 20 families originating from WNV RNA-positive mothers, and 10 males were chosen from 48 families originating from WNV RNA-negative mothers for male genitalia dissections. From each family selected, not all 10 of the males chosen were used to produce data as some of the genitalia could not be measured for DV/D ratios because of broken appendages. For males of DV/D ratios of less than 0.2 (diagnostic for Cx. pipiens), the PCR identified 26/55 as Cx. pipiens, 12/55 as hybrids, and 17/55 as Cx. quinquefasciatus. Males with DV/D ratios between 0.2 and less than 0.4 (hybrid category) produced PCR identifications of 42/293 as Cx. pipiens, 73/293 as hybrids, and 178/293 as Cx. quinquefasciatus. Males with DV/D ratios above 0.4 (Cx. quinquefasciatus category) produced PCR identifications 9/127 as Cx. pipiens, 21/127 as hybrids, and 98/127 as Cx. quinquefasciatus. Most lack of congruence occurred due to the PCR identifying many Cx. pipiens and hybrid DV/D-categorized specimens as Cx. quinquefasciatus. Although less frequent, identification of Cx. pipiens by PCR was found in DV/D hybrid and Cx. quinquefasciatus categories and PCR hybrid identifications were found in DV/D Cx. pipiens and Cx. quinquefasciatus categories. As we do not know the parents of these field-collected mosquitoes, it is impossible to tell which identification method is more flawed. These data indicate that either one or both methods of identification do not work for properly identifying Cx. pipiens complex mosquitoes of Fresno County.


Figure 5
View larger version (13K):
[in this window]
[in a new window]

  		    FIGURE 5. Proportions of male progeny identified by Ace.2 PCR and categorized according to DV/D ratio showing the lack of congruency between the 2 identification methods.

 
Inconsistent results were also found in hybrids originating from laboratory-generated crosses of mosquito colonies of known identity. Colony mosquitoes consistently produced congruent PCR and DV/D ratio identifications as either Cx. pipiens (SH colonies) or Cx. quinquefasciatus (LA colony) based on at least 100 individuals throughout their time in colony. This indicates that there is no polymorphism at the primer site of the Ace.2 locus, which would incorrectly identify them. Out of 30 hybrid males picked from the AF1 cross (LA female x SH male) the PCR identified 27 as hybrids (2 bands of equal intensity) and 3 as Cx. pipiens (Figure 6Go). In the reciprocal BF1 cross (LA male x SH female ), 16 males produced 2 bands, and in 14 only the Cx. quinquefasciatus diagnostic fragment was amplified (Figure 6Go). In all 16 cases, according to the naked eye, the Cx. quinquefasciatus fragment was more than twice the intensity of the Cx. pipiens diagnostic fragment.


Figure 6
View larger version (67K):
[in this window]
[in a new window]

  		    FIGURE 6. Example of 1.5% agarose gel showing the Ace.2 PCR bands characterizing Cx. pipiens s.l. hybrids produced from crosses of parent colonies from Northern (Shasta colony, Cx. pipiens) and Southern California (Los Angeles, Cx. quinquefasciatus). Lanes 1 and 18, 100-bp ladder; lanes 2–9, hybrids from LA female x SH male (AF1); lanes 10–17, hybrids from SH female x LA male (BF1). Three of the individuals produced the expected bands diagnostic for hybrids (lanes 5, 7, 9). Lanes 2 and 10–14 showed a weak Cx. pipiens-specific band (610 bp) and an intense Cx. quinquefasciatus-specific band (274 bp), while the rest produced only the Cx. quinquefasciatus-specific band.

 
A summary of DV/D ratios of 30 males each from the parents, hybrids, and back-crosses are given in Figures 7Go and 8Go. Figure 7Go represents the DV/D results from the cross originating from SH male x LA female and Figure 8Go from the reciprocal cross of SH female x LA male. DV/D ratios of hybrids from both the SH male x LA female and SH female x LA male were all smaller than 0.2, which is in fact within the Cx. pipiens range and not within the expected hybrid range between 0.2 and 0.4. Inbreeding the hybrids produced variable results where in the case of the SH male x LA female hybrids (AF2) the DV/D ratios were similar to their hybrids and in the case of the SH female x LA male hybrids (BF2) the DV/D values were mostly within the expected hybrid range of 0.2–0.4. Back-crossing to Cx. quinquefasciatus parents caused a general shift toward Cx. quinquefasciatus DV/D values but were mostly still within the hybrid range of 0.2–0.4. Because the SH female x LA male hybrids (BF2) had DV/D values typical for Cx. pipiens, back-crossing them to Cx. pipiens parents caused no change in values.


Figure 7
View larger version (10K):
[in this window]
[in a new window]

  		    FIGURE 7. DV/D ratios of male offspring (AF1) from crosses of known Cx. quinquefasciatus females (LA) and Cx. pipiens males (SH) and their back-crosses with their sibling hybrids (AF2) or LA (A1F2, A2F2) or SH (A3F2, A4F2). See Table 1Go for designations.

 

Figure 8
View larger version (9K):
[in this window]
[in a new window]

  		    FIGURE 8. DV/D ratios of male offspring (BF1) from crosses of known Cx. quinquefasciatus males (LA) and Cx. pipiens females (SH) and their back-crosses with their sibling hybrids (BF2) or LA (B1F2) or SH (B3F2, B4F2). See Table 1Go for designations.

 

DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
From mid-June to mid-October 2005, considerable WN virus activity was detected in Fresno County. While this study focused only on WNV infections in gravid Cx. pipiens s.l., at the same time, mosquito pool (from CDC-style carbon dioxide traps37) and sentinel chicken seroconversion surveillance for WNV was carried out by the Consolidated Mosquito Abatement District (CMAD) in collaboration with the California Vector-Borne Disease Surveillance System (http://www.calsurv.org/arbo.html). The CMAD submitted and received positive RT-PCR results from 22/87 Cx. tarsalis, 33/147 Cx. pipiens s.l., and 3/4 Cx. stigmatosoma for an overall minimum infection rate of 8/1000. Mosquito pool sizes ranged from 6 to 50, and pools that were WNV RNA positive were evenly spread throughout the CMAD (Figure 2Go). Cx. tarsalis and Cx. quinquefasciatus are competent bridge vectors that permit the virus to move from a strictly enzootic pattern to humans.38 This was confirmed by 43 human cases in people who live within the CMAD district boundaries. The CMAD set up 6 sentinel surveillance flocks of 10 chickens in each flock for a total of 60 chickens. The first chickens seroconverted in the 3rd week of July, about 5 weeks after the first Cx. pipiens member was positive. The next week (first week of Aug), a further 22 seroconverted, and by the middle of October 51/60 chickens had seroconverted, confirming once again intense WNV transmission in Fresno County.

The infection rate based on testing of individual gravid trap Cx. pipiens s.l. females conducted was double that of the MIR of CO2 Cx. pipiens s.l. pooled trap collections. The higher infection rates in gravid trap and individually sampled females can be explained. Firstly, MIRs were calculated assuming that each positive pool contained a single infected mosquito,39 which gives an underestimate of actual infection rates. Conversely, they can also give an inflated value if you have a positive in a pool with a small number of mosquitoes. Secondly, CO2-baited traps collect mostly young, unfed, nulliparous females (> 65%) of Cx. tarsalis and Cx. quinquefasciatus.40–42 Hence, the majority of the mosquitoes collected in CO2-baited traps have never come into contact with a viremic host, unlike that of gravid-female–based collections consisting of females who would have at least had one opportunity to come in contact with an infective host. From our experience, no members of the Cx. pipiens complex are autogenous in Fresno County (unpublished data). Many of the feral Cx. pipiens s.l. positive for WNV RNA will probably not transmit virus in nature. Several will succumb to predation, lethal insecticide exposure, or natural death before completion of the extrinsic incubation period. Furthermore, vector competence of different Cx. pipiens s.l. populations varies greatly depending on dose of infection and the genetic determinants of barriers to infection. For example, transmission rates can vary from 52% (Bakersfield Cx. quinquefasciatus) to as low as 6% (Coachella Valley Cx. quinquefasciatus) when infected with the same dose of WNV.4

WNV infection rates in Cx. quinquefasciatus rose steadily from early June, reaching a peak in August, which is the hottest time of the year, and then declining as the season progressed through to October (Figure 4Go). Total numbers of gravid Cx. pipiens s.l. collected stayed fairly constant throughout the season, peaking in July at 130 and declining to 100 in September (only 2 trapping rotations occurred in October, collecting 39 mosquitoes). This trend of peak WNV infection in mosquitoes coinciding with the warmest temperatures also occurred in 2004 in Coachella Valley, Los Angeles, and Kern County.38 According to Reisen and others,38 the invading NY99 WNV strain requires warm temperatures for efficient virus growth and transmission.

In this study, vertical transmission of WNV was confirmed among members of the Cx. pipiens complex by testing 10 male offspring from positive feral mothers. In all instances, only 1 or 2 out of the 10 male offspring tested were positive. This means that only a small proportion of the offspring become infected via transovarial transmission. If males become infected via transovarial transmission, then this raises the possibility of venereal transmission. Venereal transmission of WNV was demonstrated by force mating experiments; however, titers of virus in females infected via this route remained too low for this infection to be passed vertically or horizontally on to her progeny or vertebrate host, respectively.43

It is unfortunate that we cannot comment on differences in WNV infection rates among members of the Cx. pipiens complex in Fresno County because of inconsistency of the identification methods that distinguish complex members. Problems arose when assessments were made to ascertain, based on the mothers and her progeny PCR identifications, the degree of conspecific matings between Cx. pipiens and Cx. quinquefasciatus in the Fresno County hybrid zone. Identifications of offspring homozygous for the Cx. pipiens-specific Ace.2 allele were obtained from mothers homozygous for the Cx. quinquefasciatus-specific allele and vice versa. These offspring and mother identifications cannot be possible as no combinations of Cx. pipiens s.l. mating pair would give these results. This ambiguity of diagnostic PCR reaction results based on 15 families even raises concerns about the accuracy of the other 193 families with respect to with whom the mother mated and makes assessments of degree of conspecific mating, hybrid inbreeding, and back-crossing among complex members in Fresno County unjustified. There was also very poor congruence between DV/D ratio and PCR identifications of each of the males. No particular trend was evident in which direction the 2 diagnostic methods mismatched.

These incongruencies between DV/D ratio and PCR identifications required taking a step back to reconsider the validity of DV/D that is ubiquitously used as the a priori method to distinguish Cx. pipiens from Cx. quinquefasciatus and their hybrids. Crosses of northern California Cx. pipiens to southern California Cx. quinquefasciatus revealed several inconsistencies in the general understanding of the validity of DV/D. All the hybrids had ratios typical for Cx. pipiens, and only the back-crosses to the Cx. quinquefasciatus parents had intermediate ratios that, in fact, consisted of a continuous range of values between hybrids and the parental form. This means that, at the very least, among California Cx. pipiens s.l. categorizing DV/D into finite values corresponding to taxa and hybrids is a futile exercise. This suggests that the DV/D phenotype and expression occurs as a result of a complex interaction of underlying genetic and environmental (epigenetic) factors and cannot be considered a neutral genetic marker. The maintenance of the DV/D Cx. pipiens and Cx. quinquefasciatus phenotypes north of 39°N and south of 36°N latitudes, respectively, is most likely a result of environmental factors and perhaps, more specifically, temperature. The influence of temperature on DV/D ratio outcome has previously been shown by Wilton and Jakob.44 While differences in the clinal change in temperature can explain the DV/D ratio value clines in North America, it still remains to be explained why only the 2 discrete Cx. pipiens and Cx. quinquefasciatus DV/D ratio categories occur in sympatry in South Africa.10

Misidentification of Cx. pipiens/Cx. quinquefasciatus hybrids from known California parental origin by Ace.2 PCR identification28 warrants further discussion and investigation. The outcome and interpretation of the PCR are critically affected by the primer concentrations, as there is only one nucleotide that differentiates Cx. pipiens from the other complex members.28 In this study, we often obtained amplification of 1 band in hybrids, and when we did obtain 2 bands we observed a continuous range from that of equal intensities to faint bands (just visible to the eye) of fragment size diagnostic for one or other of the taxa. Separating hybrids from Cx. pipiens and Cx. quinquefasciatus remains an issue as there is still no appropriate diagnostic character.

An over-riding issue that still needs to be resolved in California is to assess gene flow among Cx. pipiens s.l. The widespread spatial distribution of similar insecticide resistance mechanisms and genes in Californian Cx. pipiens s.l. populations suggests that extensive gene flow is not just restricted to the hybrid zone in the Central Valley but extends further north and south. The organophosphate resistance-causing A2B2 esterase complex45 and the pyrethroid knock-down resistance (kdr-type) allele in the voltage-gated sodium channel gene46 occur in both northern Cx. pipiens (Shasta County) and the southern Cx. quinquefasciatus (San Diego County) populations (unpublished data). It is possible that these resistance mechanisms arose independently in the California Cx. pipiens and Cx. quinquefasciatus populations, but the spread of these mechanisms via gene flow between the 2 taxa seems a more plausible explanation. This extensive gene flow provides credence to Tabachnick and Powell’s8 conclusion that individuals classified as Cx. pipiens in northern California possess large amounts of the southern Cx. quinquefasciatus genotype and vice versa. This means that the current classification of Cx. pipiens and Cx. quinquefasciatus and hybrid populations/individuals used in systematics studies and comparative ecological, behavioral, vectorial capacity, and epidemiologic investigations may produce misleading results.

Received May 9, 2007. Accepted for publication November 25, 2007.

Acknowledgments: We thank members of the general public for permitting collection of mosquitoes from their properties in Fresno County.

Financial support: This project was funded by the University of California Mosquito Research Program (NIH R21-A155564-01) and Consolidated Mosquito Abatement District. Mosquito testing for viral RNA was partially funded by the Pacific Southwest Regional Center for Excellence (U54 AI-65359).

* Address correspondence to Rory D. McAbee, Mosquito Control Research Laboratory, 9240 S. Riverbend Avenue, Parlier, CA 93648. E-mail: rdmcabee{at}uckac.edu Back

Authors’ addresses: Rory D. McAbee, Julie Christiansen, Katherine Dealey, and Anthony J. Cornel, Department of Entomology, University of California at Davis, Mosquito Control Research Laboratory, Parlier, CA, and Center for Vector-Borne Diseases, University of California, Davis, CA, Telephone: +1 (559) 646-6581, Fax: +1 (559) 646-6593, E-mail: rdmcabee{at}uckac.edu. Emily N. Green and Aaron C. Brault, Center for Vector-Borne Diseases, University of California, Davis, CA, and Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California at Davis, CA. Jodie Holeman, Niki Frye, and F. Steve Mulligan III, Consolidated Mosquito Abatement District, PO Box 278, Selma, CA 93662.

Reprint requests: Rory D. McAbee, Mosquito Control Research Laboratory, 9240 S. Riverbend Avenue Parlier, CA 93648, Telephone: +1 (559) 646-6581, Fax: +1 (559) 646-6593, E-mail: rdmcabee{at}uckac.edu.


REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Lanciotti RS, Roehrig JT, Deubel V, Smith J, Parker M, Steele K, Crise B, Volpe KE, Crabtree MB, Scherret JH, Hall RA, MacKenzie JS, Cropp CB, Panigrahy B, Ostlund E, Schmitt B, Malkinson M, Banet C, Weissman J, Komar N, Savage HM, Stone W, McNamara T, Gubler DJ, 1999. Origin of the West Nile virus responsible for an outbreak of encephalitis in the northeastern United States. Science 286: 2333–2337.[Abstract/Free Full Text]
  2. Anderson JF, Andreadis TG, Vossbrink CR, Tirrell S, Wakem EM, French RA, Garmendia AE, van Kruiningen HJ, 2000. Isolation of West Nile from mosquitoes, crows, and a Coopers hawk in Connecticut. Science 286: 2331.[Web of Science]
  3. California Department of Health Services Vector-Borne Disease Section. 2004. Mosquito Pool Summary Report 12/03/04. Sacramento, CA: CDHS, 1–26.
  4. Goddard LB, Roth AE, Reisen WK, Scott TW, 2002. Vector competence of California mosquitoes for West Nile virus. Emerg Infect Dis 8: 1385–1391.[Web of Science][Medline]
  5. Goddard LB, Roth AE, Reisen WK, Scott TW, 2003. Vertical transmission of West Nile virus by three California Culex (Diptera: Culicidae) species. J Med Entomol 40: 743–746.[Web of Science][Medline]
  6. Turell MJ, O’Guinn M, Oliver J, 2000. Potential for New York mosquitoes to transmit West Nile virus. Am J Trop Med Hyg 62: 413–414.[Abstract]
  7. Dohm DJ, O’Guinn ML, Turell MJ, 2002. Effect of environmental temperature on the ability of Culex pipiens (Diptera: Culicidae) to transmit West Nile virus. J Med Entomol 39: 221–225.[Web of Science][Medline]
  8. Tabachnick WJ, Powell JR, 1983. Genetic analysis of Culex pipiens populations in the central valley of California. Ann Entomol Soc Am 76: 715–720.[Web of Science]
  9. Urbanelli S, Silvestrini F, Reisen WK, De Vito E, Bullini L, 1997. Californian hybrid zone between Culex pipiens pipiens and Cx. p. quinquefasciatus revisited (Diptera: Culicidae). J Med Entomol 34: 116–127.[Web of Science][Medline]
  10. Cornel AJ, McAbee RD, Rasgon J, Stanich M, Scott TW, Coetzee M, 2003. Differences in extent of genetic introgression between sympatric Culex pipiens and Culex quinquefasciatus (Diptera: Culicidae) in California and South Africa. J Med Entomol 40: 36–51.[Web of Science][Medline]
  11. Fonseca DM, Keyghobadi N, Malcolm CA, Mehmet C, Schaffner F, Motoyoshi M, Fleischer RC, Wilkerson RC, 2004. Emerging vectors in the Culex pipiens complex. Science 303: 1535–1538.[Abstract/Free Full Text]
  12. Spielman A, Andreadis TG, Apperson CS, Cornell AJ, Day JF, Edman JD, Fish D, Harrington LC, Kiszewski AE, Lampman R, Lanzaro GC, Matuschka FR, Munstermann LE, Nasci RS, Norris DE, Novak RJ, Pollack RJ, Reisen WK, Reiter P, Savage HM, Tabachnick WJ, Wesson DM, 2004. Outbreak of virus in North America. Science 306: 1473 (letter to the editor).
  13. Barr AR, 1982. The Culex pipiens complex. Steiner WWM, Tabachnik WJ, Rai WS, Narang S, eds. Recent Developments in the Genetics of Insect Disease Vectors. Urbana, IL: Stipes, 551–572.
  14. Urbanelli S, Bullini L, Villani F, 1985. Electrophoretic studies on Culex quinquefasciatus Say from Africa: genetic variability and divergence from Culex pipiens L. (Diptera: Culicidae). Bull Entomol Res 75: 291–304.[Web of Science]
  15. Byrne K, Nichols RA, 1998. Culex pipiens in London Underground tunnels: differentiation between surface and subterranean populations. Heredity 82: 7–15.[Web of Science]
  16. Chevillon C, Rivet Y, Raymond M, Rousset F, Smouse PE, Pasteur N, 1998. Migration/selection balance and ecotypic differentiation in the mosquito Culex pipiens. Mol Ecol 7: 197–208.
  17. Spielman A, 2001. Structure and seasonality of Nearctic Culex pipiens populations. Ann NY Acad Sci 951: 220–234.[Web of Science][Medline]
  18. Dobrotworsky NV, 1967. The problem of the Culex pipiens complex in the south Pacific (including Australia). Bull WHO 37: 251–255.[Web of Science][Medline]
  19. Cheng ML, Hacker CS, 1976. Inheritance of 6-phosphogluconate dehydrogenase variance in Culex pipiens quinquefasciatus Say. J Hered 67: 215–219.[Free Full Text]
  20. Cheng ML, Hacker CS, 1979. The genetics of hexokinase in a mosquito, Culex pipiens. Genetics 92: 903–913.[Abstract/Free Full Text]
  21. Jupp PG, 1978. Culex (Culex) pipiens pipiens Linnaeus and Culex (Culex) pipiens quinquefasciatus Say in South Africa: morphological and reproductive evidence in favour of their status as two species. Mosq Systematics 10: 461–473.
  22. Miles SJ, Paterson HE, 1979. Protein variation and systematics in the Culex pipiens group of species. Mosq Systematics 11: 187–202.
  23. Varma MGR, 1960. Preliminary studies on the infection of culicine mosquitoes with the Tamiland strain of West Nile virus. Indian J Med Res 48: 537–548.
  24. Jupp PG, McIntosh BM, 1970. Quantitative experiments on the vector capability of Culex (Culex) pipiens fatigans Weidemann with West Nile and Sindbis viruses. J Med Entomol 7: 353–356.[Web of Science][Medline]
  25. Hubalek Z, Halouzka J, 1999. West Nile fever: a reemerging mosquito borne viral disease in Europe. Emerg Infect Dis 5: 643–650.[Web of Science][Medline]
  26. Sundararaman S, 1949. Biometrical studies on intergradation in the genitalia of certain populations of Culex pipiens and Culex quinquefasciatus in the United States. Am J Hyg 50: 307–314.[Web of Science][Medline]
  27. Aspen and Savage, 2003. Polymerase chain reaction assay identifies North American members of the Culex pipiens complex based on nucleotide sequence differences in the acetylcholinesterase gene Ace.2. J Am Mosq Cont Assoc 19: 323–328.[Web of Science][Medline]
  28. Smith JL, Fonseca DM, 2004. Rapid assays for identification of members of the Culex (Culex) pipiens complex, their hybrids, and other sibling species (Diptera: Culicidae). Am J Trop Med Hyg 70: 339–345.[Abstract/Free Full Text]
  29. Bourguet D, Raymond M, Fournier D, Malcolm CA, Toutant JP, Arpagaus M, 1996. Existence of two acetylcholinesterases in the mosquito Culex pipiens (Diptera: Culicidae). J Neurochem 67: 2115–2123.[Web of Science][Medline]
  30. Malcolm CA, Bourguet D, Ascolillo A, Rooker SJ, Garvey CF, Hall LM, Pasteur N, Raymond M, 1998. A sex-linked Ace gene, not linked to insensitive acetylcholinesterase-mediated insecticide resistance in Culex pipiens. Insect Mol Biol 7: 107–120.[Web of Science][Medline]
  31. Reiter P, 1983. A portable, battery-powered trap for collecting gravid Culex mosquitoes. Mosquito News 43: 496–499.[Web of Science]
  32. Christiansen JA, Smith C, Madon MB, Albright J, Hazeleur W, Hazelrigg J, Kluh S, McAbee RD, Mulligan FS, Leal W, Cornel AJ, 2005. Use of gravid traps for collection of California West Nile vectors. Proc Papers Calif Mosq Control Assoc Ann Conf 73: 91–95.
  33. Collins FH, Mendez MA, Rasmussen MO, Mehaffey PC, Besansky NJ, Finnerty V, 1987. A ribosomal RNA gene probe differentiates member species of the Anopheles gambiae complex. Am J Trop Med Hyg 37: 37–41.[Abstract/Free Full Text]
  34. Lanciotti RS, Kerst AJ, Nasci RS, Godsey MS, Mitchell CJ, Savage HM, Komar N, Panella NA, Allen BC, Volpe KE, Davis BS, Roehrig JT, 2000. Rapid detection of West Nile virus from human clinical specimens, field-collected mosquitoes, and avian samples by a TaqMan reverse transcriptase-PCR assay. J Clin Microbiol 38: 4066–4071.[Abstract/Free Full Text]
  35. Shi P, Kauffman EB, Ren P, Felton A, Tai JH, Dupuis AP II, Jones SA, Ngo KA, Nichols DC, Maffei J, Ebel GD, Bernard KA, Kramer LD, 2001. High-throughput detection of West Nile virus RNA. J Clin Microbiol 39: 1264–1271.[Abstract/Free Full Text]
  36. Gujral IB, Zielinski-Gutierrez EC, LeBailly A, Nasci R, 2007. Behavioral risks for West Nile virus disease, Northern Colorado, 2003. Emerg Infect Dis 13: 419–425.[Web of Science][Medline]
  37. Pfuntner WC, 1979. A modified CO2-baited miniature surveillance trap. Bull Soc Vector Ecol 4: 31–35.
  38. Reisen WK, Barker CM, Carney R, Lothrop HD, Wheeler SS, Wilson JL, Madon MB, Takahashi R, Caroll B, Garcia S, 2006a. Role of corvids in epidemiology of West Nile virus in southern California. J Med Entomol 43: 356–367.[Web of Science][Medline]
  39. Nasci RS, Mitchell CJ, 1996. Arbovirus titer variation in field-collected mosquitoes. J Am Mosq Cont Assoc 12: 167–171.[Web of Science][Medline]
  40. Barr AR, Morrison AC, Guptavanji P, Bangs MJ, Cope SE, 1986. Parity rates of mosquitoes collected in San Joaquin marsh. Proc Calif Mosq Vector Control Assoc 54: 117–118.
  41. Reisen WK, Pfuntner AR, 1987. Effectiveness of five methods for sampling adult Culex mosquitoes in rural and urban habitats in San Bernardino County, California. J Am Mosq Control Assoc 3: 601–606.[Web of Science][Medline]
  42. Reisen WK, Lothrop HD, Hardy JL, 1995. Bionomics of Culex tarsalis (Diptera: Culicidae) in relation to arbovirus transmission in southeastern California. J Med Entomol 32: 316–327.[Web of Science][Medline]
  43. Reisen WK, Fang Y, Lothrop HD, Martinez VM, Wilson J, O’Conner P, Carney R, Cahoon-Young B, Shafii M, Brault AC, 2006b. Overwintering of West Nile virus in Southern California. J Med Entomol 43: 344–355.[Web of Science][Medline]
  44. Wilton JH, Jakob WL, 1985. Temperature-induced morphological change in Culex pipiens. J Am Mos Control Assoc 1: 174–177.
  45. Raymond M, Callaghan A, Fort P, Pasteur N, 1991. Worldwide migration of amplified insecticide resistance genes in mosquitoes. Nature 350: 151–153.[Medline]
  46. Martinez-Torres D, Chevillon C, Brun-Barale A, Berge JB, Pasteur N, Pauron D, 1999. Voltage-dependent Na+ channels in pyrethroid-resistant Culex pipiens L. mosquitoes. Pest Science 55: 1012–1020.




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 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 Google Scholar
Google Scholar
Right arrow Articles by McAbee, R. D.
Right arrow Articles by Cornel, A. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by McAbee, R. D.
Right arrow Articles by Cornel, A. J.
Related Collections
Right arrow West Nile
Right arrow Medical Entomology
Right arrow Mosquitoes

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS