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VECTOR COMPETENCE OF CULISETA INCIDENS ANDCULEX THRIAMBUS FOR WEST NILE VIRUSAuthor(s) WILLIAM K REISEN YING FANG VINCENT M MARTINEZSource Journal of the American Mosquito Control Association 22(4)662-6652006Published By The American Mosquito Control AssociationDOI httpdxdoiorg1029878756-971X(2006)22[662VCOCIA]20CO2URL httpwwwbiooneorgdoifull1029878756-971X28200629225B6623AVCOCIA5D20CO3B2

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VECTOR COMPETENCE OF CULISETA INCIDENS AND CULEXTHRIAMBUS FOR WEST NILE VIRUS1

WILLIAM K REISEN YING FANG AND VINCENT M MARTINEZ

Center for Vectorborne Diseases Department of Pathology Microbiology and Immunology School of VeterinaryMedicine University of California Old Davis Road Davis CA 95616

ABSTRACT The vector competence of Culiseta incidens (Thomson) and Culex thriambus Dyar for WestNile virus (WNV) were compared to Cx quinquefasciatus Say or Cx tarsalis Coquillett and Cxstigmatasoma Dyar collected concurrently in California Culiseta incidens were less susceptible to oralinfection than Cx quinquefasciatus but transmitted virus at a significantly higher rate thereby yieldingcomparable population transmission rates Culex thriambus was equally susceptible to oral infection andtransmitted virus at rates comparable to Cx tarsalis or Cx stigmatosoma A mammalian host selectionpattern most likely precluded detection of natural infection in Cs incidens a fairly abundant peridomesticspecies In contrast an avian host selection pattern and efficient vector competence resulted in repeateddetection of WNV in Cx thriambus however limited abundance and restrictive riparian larval habitatrequirements would seem to limit the involvement of Cx thriambus in WNV epidemiology

KEY WORDS Culiseta incidens (Thomson) Culex thriambus Dyar West Nile virus California vectorcompetence

The vector competence of 8 species of Califor-nia mosquitoes for West Nile virus (WNVFlaviviridae Flavivirus) was summarized previ-ously (Goddard et al 2002) Subsequent studieshave focused on several Culex species (Reisen etal 2005) because they have been infected mostfrequently during the ongoing epidemic (Hom etal 2004 2005) However other mosquito speciespossibly could be important focally because oftheir close proximity to humans in peridomestichabitats or because they repeatedly have beenfound infected during surveillance

Culiseta incidens (Thomson) is a commonperidomestic mosquito in coastal Californiaand utilizes larval habitats such as rain barrelsbirdbaths and surface pools (Bohart and Wa-shino 1978 Reisen and Reeves 1990) This speciesfeeds predominantly on mammals in Sacramento(Reisen and Reeves 1990) and Fresno (Miura etal 1976) counties In urban areas it frequently iscollected in association with Cx quinquefasciatusSay in gravid female traps Although repeatedlytested for arboviruses it has not been foundnaturally infected in California with westernequine encephalomyelitis virus (WEEV) StLouis encephalitis virus (SLEV) (Reeves andMilby 1990) or WNV (Table 1) During 2005a single WNV-positive pool was reported toArboNet from Nevada Previous experimentalinfection studies indicated that this species wascapable of transmitting both WEEV and SLEV(Hammon and Reeves 1943a 1943b) however

its vector competence for WNV has not beenreported

Culex thriambus Dyar is closely related andappears morphologically similar to Cx stigmato-soma Dyar and some field-collected females maybe misidentified (Bohart and Washino 1978Meyer and Durso 1993) However these speciesare very different biologically Culex thriambus isa relatively rare species that is collected in clean-water habitats along riparian corridors whereasCx stigmatosoma is abundant in periurbanhabitats where it exploits eutrophic situationssuch as dairy wastewater ponds Limited pre-cipitin testing indicated that in California Cxthriambus blood feeds predominantly on passer-ine birds (Tempelis and Reeves 1964) Thisspecies previously has not been found naturallyinfected with WEEV or SLEV (Reeves and Milby1990) however limited testing during expandedsurveillance associated with the ongoing WNVepidemic has revealed repeated infection withWNV (Table 1) Vector competence studies havenot been reported for this species

Herein we summarize preliminary data on thevector competence of Cs incidens and Cxthriambus for WNV and compare these resultsto those from concurrently collected and wellstudied Culex species

MATERIALS AND METHODS

Mosquitoes Culiseta incidens and Cx quin-quefasciatus were reared from larvae collected inBerkeley Alameda County on June 21 2004 andfrom egg rafts oviposited in gravid traps operatedin Los Angeles County at Whittier Narrows Damon June 10 2004 and in San Gabriel on May 3

1 Use of chickens as blood-meal hosts for field-caughtmosquitoes was approved under animal use and careprotocol No 11185 reviewed by the UC Davis In-stitutional Animal Care and Use Committee

Journal of the American Mosquito Control Association 22(4)662ndash665 2006Copyright E 2006 by The American Mosquito Control Association Inc

662

2005 Host-seeking females of Cx thriambus Cxtarsalis Coquillett Cx stigmatosoma and Csincidens were collected in dry-icendashbaited trapsoperated at Hansen Dam Los Angeles Countyon June 15 2005 and then blood fed on a non-infected chicken The F1 laboratory-reared prog-eny from these females were used for experimen-tation All larvae were reared in an insectary at22uC and 168 LD h photoperiod at a density of3ndash4 egg rafts per tray and fed a diet of finelyground alfalfa pellets and fish food (Mahmood1999) Adults were fed 10 sucrose but thenstarved for 24ndash48 h prior to infection attempts

Virus infection Methods of infection andviral assay were similar to those describedpreviously (Goddard et al 2002 Reisen et al2005) Fifty to 80 unfed female mosquitoes thatwere 3ndash8 days old and starved for 24ndash48 h wereoffered pledgets soaked with a 110 mixture ofWNV diluted in heparinized chicken blood and25 sucrose for 1ndash15 h In 2 experimentsadditional females were exposed to a 10-folddilution series of virus The WNV strain 35211AAF 92399 used in our experiments wasisolated from a flamingo during the 1999 NewYork outbreak and passaged twice in Vero cellcultures before use Engorged females were

maintained on 10 sucrose at 26uC and 168LD h photoperiod At 14 d females that fed onthe highest doses were anesthetized with triethyl-amine and then allowed to expectorate intoa capillary tube filled with a 11 mixture of fetalbovine serum and 10 sucrose for 15 min tomeasure their ability to transmit virus per os(Aitken 1977) Capillary tube contents wereexpelled into 300 ml of diluent (PBS 20 fetalbovine serum antibiotics) and then frozen at280uC for later assay

Assay Artificial blood meal expectorate andmosquito body samples were tested for virus byplaque assay on Vero cell culture in 6-well plates(Kramer et al 2002) Mosquito bodies weretriturated in 1 ml of diluent Plaque assays wereconducted by adding 100 ml of each sample toconfluent Vero cell monolayers and incubating at37uC for 15 h to allow for cell infection Followingincubation cells were rinsed and then covered witha 2 agarose overlay containing 0005 neutralred After 96 h and 120 h of incubation at 37uC ina 5 CO2 atmosphere plaques were counted andvirus concentrations were calculated as plaque-forming units (PFUs) per milliliter During 2005mosquito bodies were tested for infection bysingleplex RT-PCR using the primers and Taq-

Table 1 Natural infection of mosquitoes collected in California and tested for West Nile St Louis encephalitisand western equine encephalomyelitis viruses by a multiplex RT-PCR during 2004 and 2005 (Data from California

encephalitis virus surveillance program summaries)

Year Species Pools No testedWNV RNA

positiveMIR per

1000WEEV RNA

positiveMIR1 per

1000

2004 Total 14602 551095 1135 206 0Cx tarsalis 3536 138932 318 228 0Cs incidens 366 9180 0Cx thriambus 27 827 3 363 0

2005 Total 20795 746477 1236 166 51 007Cx tarsalis 7688 303832 458 151 51 017Cs incidens 431 11627 0 0Cx thriambus 98 3306 7 212 0

1 MIR 5 minimum infection rate

Table 2 Laboratory vector competence of Cs incidens and Cx thriambus for WNV compared to concurrentlycollected Culex mosquitoes

DateMosquito

source SpeciesPledget

virus titer1 nNo infec-

tionsNo trans-

mittingInfected

()2

Transmit-ting ()3

21-Jun-04 Berkeley Cs incidens 76 12 3 2 25 6710-Jun-04 Whittier Cs incidens 78 15 5 4 33 80

Cx quinquefaciatus 78 30 26 6 87 2303-May-05 San Gabriel Cs incidens 64 8 1 1 13 100

Cx quinquefaciatus 64 30 23 1 77 415-Jun-05 Hansen Dam Cx tarsalis 65 30 24 13 80 54

Cx stigmatosoma 65 22 20 9 91 45Cx thriambus 65 16 13 6 81 46Cs incidens 65 23 7 3 30 43

1 Titer of virus (log10 PFUml) on pledget used for mosquito infection2 (infectedtested) 3 1003 (transmittinginfected) 3 100

DECEMBER 2006 CS INCIDENS AND CX THRIAMBUS VECTOR COMPETENCE 663

Man platform after robotic RNA extractiondescribed previously (Shi et al 2001)

RESULTS AND DISCUSSION

Overall 16 (28) of 58 Cs incidens feeding onpledgets with an estimated 64ndash78 log10 PFU ofWNV per milliliter became infected This per-centage infection did not vary significantly (X2 5

13 df 5 3 P 010) among the 4 populationstested (Table 2) Similarly overall infection (82n 5 60) in concurrently collected Cx quinque-fasciatus did not vary (X2 5 10 df 5 1 P 010)between the 2 populations sampled but weresignificantly greater than percentage infection forCs incidens (X2 5 346 df 5 1 P 0001)However 10 (63) of 16 infected Cs incidensfemales expectorated virus and this was signifi-cantly greater (X2 5 141 df 5 1 P 0001) thanobserved for Cx quinquefasciatus (14 n 5 49)Therefore the percentage of total females of eachspecies that acquired and transmitted WNV athigh infectious doses was not significantly differ-ent (X2 5 07 df 5 1 P 010) However thepercentage of each species infected at progres-sively decreasing virus concentrations differedmarkedly in 1 experiment where sufficient femaleswere collected (Fig 1) Although Cs incidenswere common in peridomestic larval collectionsand relatively easy to rear they were difficult toblood feed and maintain Overall we offered 220females infectious blood meals on pledgets ofwhich 82 fed and of these only 58 survived the 2-week holding period at 26uC in our laboratoryBased on the absence of natural virus infectiona mammalian blood-meal host selection patternand moderate susceptibility to per os infection

this species should have limited involvement inencephalitis virus transmission in CaliforniaFailure to detect virus in this species during2004 and 2005 (Table 1) supports this conclusion

The laboratory infection and transmissionrates of Cx thriambus were not significantlydifferent (X2 5 12 and 04 respectively df 5 2 P 010) from those observed for concurrentlycollected Cx tarsalis and Cx stigmatosoma(Table 2) The percentage infection for all 3species decreased similarly as a function ofdecreasing viral titer in the infectious blood meal(Fig 1) In this single experiment Cx thriambusappeared to be equally or perhaps even moresusceptible than Cx tarsalis because 4 of 9females as compared to 0 of 12 females wereinfected at the 36 log10 PFUml viral doseBecause Cx thriambus has been found infectedrepeatedly in nature feeds predominantly onavian hosts and appears to be a highly competentexperimental host for WNV this species shouldbe considered important in virus maintenance oramplification within the avian-mosquito trans-mission cycle However the distribution andabundance of Cx thriambus seems limited inCalifornia to larval habitats associated with cleanwater along riparian systems Proposed reclama-tion of the original riparian systems in LosAngeles could expand larval habitats for thisspecies and possibly enhance its involvement inWNV activity

ACKNOWLEDGMENTS

We thank J R Rusmisel and staff AlamedaCounty MAD and M Madon J Wilson P

Fig 1 Percentage of females (n 5 7ndash30 females per data point) that were infected 2 weeks after feeding onpledgets with decreasing titers of West Nile virus

664 JOURNAL OF THE AMERICAN MOSQUITO CONTROL ASSOCIATION VOL 22 NO 4

OrsquoConnor and J Spoehl Greater Los AngelesCounty VCD for collecting the mosquitoes usedin these experiments Sandra Garcia and Sira-noosh Astari Center for Vectorborne Diseasesassisted with virus testing This research wasfunded by Research Grant No R01-A155607from the National Institutes of Allergy andInfectious Diseases NIH grants from the Uni-versity of California Mosquito Research Pro-gram and supplemental funds from the Centersfor Disease Control and Prevention and theGreater Los Angeles VCD Logistical supportwas provided by the Kern MVCD

REFERENCES CITED

Aitken THG 1977 An in vitro feeding technique forartificially demonstrating virus transmission by mos-quitoes Mosq News 37130ndash133

Bohart RM Washino RK 1978 Mosquitoes ofCalifornia Berkeley CA University of CaliforniaPress

Goddard LB Roth AE Reisen WK Scott TW 2002Vector competence of California mosquitoes forWest Nile virus Emerg Infect Dis 81385ndash1391

Hammon WM Reeves WC 1943a Laboratory trans-mission of St Louis encephalitis virus by three generaof mosquitoes J Exp Med 78241ndash253

Hammon WM Reeves WC 1943b Laboratory trans-mission of western equine encephalitis virus bymosquitoes of the genera Culex and Culiseta J ExpMed 78425ndash434

Hom A Houchin A McCaughey K Kramer VLChiles RE Reisen WK Tu E Glaser C Cossen CBaylis E Eldridge BF Sun B Padgett K Woods LMarcus L Hui LT Castro M Husted S 2004Surveillance for mosquito-borne encephalitis activityand human disease including West Nile virus inCalifornia 2003 Proc Mosq Vector Control AssocCalif 7248ndash54

Hom A Marcus L Kramer VL Cahoon B Glaser CCossen C Baylis E Jean C Tu E Eldridge BF

Carney R Padgett K Sun B Reisen WK Woods LHusted S 2005 Surveillance for mosquito-borneencephalitis virus activity and human disease in-cluding West Nile virus in California 2004 ProcMosq Vector Control Assoc Calif 7366ndash77

Kramer LD Wolfe TM Green EN Chiles RE FallahH Fang Y Reisen WK 2002 Detection ofencephalitis viruses in mosquitoes (Diptera Culici-dae) and avian tissues J Med Entomol 39312ndash323

Mahmood F 1999 Laboratory maintenance of mos-quitoes In Maramorosch K Mahmood F edsMaintenance of human animal and plant pathogenvectors Enfield NH Science Publishers p 3ndash31

Meyer RP Durso SL 1993 Identification of themosquitoes of California Sacramento CA CaliforniaMosquito Vector Control Association

Miura T Kliewer JW Tempelis CH 1976 Seasonaloccurrence of Culiseta incidens in the foothills ofFresno County California Mosq News 36343ndash349

Reeves WC Milby MM 1990 Natural infection inarthropod vectors In Reeves WC ed Epidemiologyand control of mosquito-borne arboviruses in Califor-nia 1943ndash1987 Sacramento CA California Mos-quito and Vector Control Association p 128ndash144

Reisen WK Fang Y Martinez VM 2005 Avian hostand mosquito (Diptera Culicidae) vector competencedetermine the efficiency of West Nile and St Louisencephalitis virus transmission J Med Entomol42367ndash375

Reisen WK Reeves WC 1990 Bionomics and ecologyof Culex tarsalis and other potential mosquito vectorspecies In Reeves WC ed Epidemiology and controlof mosquito-borne arboviruses in California 1943ndash1987 Sacramento CA California Mosquito andVector Control Association p 254ndash329

Shi PY Kauffman EB Ren P Felton A Tai JHDuPuis AP Jones SA Ngo KA Nicholas DCMaffei J Ebel GD Bernard KA Kramer LD 2001High-throughput detection of West Nile virus RNAJ Clin Microbiol 391264ndash1271

Tempelis CH Reeves WC 1964 Feeding habits of oneanopheline and three culicine mosquitoes by theprecipitin test J Med Entomol 1148ndash151

DECEMBER 2006 CS INCIDENS AND CX THRIAMBUS VECTOR COMPETENCE 665

2005 Host-seeking females of Cx thriambus Cxtarsalis Coquillett Cx stigmatosoma and Csincidens were collected in dry-icendashbaited trapsoperated at Hansen Dam Los Angeles Countyon June 15 2005 and then blood fed on a non-infected chicken The F1 laboratory-reared prog-eny from these females were used for experimen-tation All larvae were reared in an insectary at22uC and 168 LD h photoperiod at a density of3ndash4 egg rafts per tray and fed a diet of finelyground alfalfa pellets and fish food (Mahmood1999) Adults were fed 10 sucrose but thenstarved for 24ndash48 h prior to infection attempts

Virus infection Methods of infection andviral assay were similar to those describedpreviously (Goddard et al 2002 Reisen et al2005) Fifty to 80 unfed female mosquitoes thatwere 3ndash8 days old and starved for 24ndash48 h wereoffered pledgets soaked with a 110 mixture ofWNV diluted in heparinized chicken blood and25 sucrose for 1ndash15 h In 2 experimentsadditional females were exposed to a 10-folddilution series of virus The WNV strain 35211AAF 92399 used in our experiments wasisolated from a flamingo during the 1999 NewYork outbreak and passaged twice in Vero cellcultures before use Engorged females were

maintained on 10 sucrose at 26uC and 168LD h photoperiod At 14 d females that fed onthe highest doses were anesthetized with triethyl-amine and then allowed to expectorate intoa capillary tube filled with a 11 mixture of fetalbovine serum and 10 sucrose for 15 min tomeasure their ability to transmit virus per os(Aitken 1977) Capillary tube contents wereexpelled into 300 ml of diluent (PBS 20 fetalbovine serum antibiotics) and then frozen at280uC for later assay

Assay Artificial blood meal expectorate andmosquito body samples were tested for virus byplaque assay on Vero cell culture in 6-well plates(Kramer et al 2002) Mosquito bodies weretriturated in 1 ml of diluent Plaque assays wereconducted by adding 100 ml of each sample toconfluent Vero cell monolayers and incubating at37uC for 15 h to allow for cell infection Followingincubation cells were rinsed and then covered witha 2 agarose overlay containing 0005 neutralred After 96 h and 120 h of incubation at 37uC ina 5 CO2 atmosphere plaques were counted andvirus concentrations were calculated as plaque-forming units (PFUs) per milliliter During 2005mosquito bodies were tested for infection bysingleplex RT-PCR using the primers and Taq-

Table 1 Natural infection of mosquitoes collected in California and tested for West Nile St Louis encephalitisand western equine encephalomyelitis viruses by a multiplex RT-PCR during 2004 and 2005 (Data from California

encephalitis virus surveillance program summaries)

Year Species Pools No testedWNV RNA

positiveMIR per

1000WEEV RNA

positiveMIR1 per

1000

2004 Total 14602 551095 1135 206 0Cx tarsalis 3536 138932 318 228 0Cs incidens 366 9180 0Cx thriambus 27 827 3 363 0

2005 Total 20795 746477 1236 166 51 007Cx tarsalis 7688 303832 458 151 51 017Cs incidens 431 11627 0 0Cx thriambus 98 3306 7 212 0

1 MIR 5 minimum infection rate

Table 2 Laboratory vector competence of Cs incidens and Cx thriambus for WNV compared to concurrentlycollected Culex mosquitoes

DateMosquito

source SpeciesPledget

virus titer1 nNo infec-

tionsNo trans-

mittingInfected

()2

Transmit-ting ()3

21-Jun-04 Berkeley Cs incidens 76 12 3 2 25 6710-Jun-04 Whittier Cs incidens 78 15 5 4 33 80

Cx quinquefaciatus 78 30 26 6 87 2303-May-05 San Gabriel Cs incidens 64 8 1 1 13 100

Cx quinquefaciatus 64 30 23 1 77 415-Jun-05 Hansen Dam Cx tarsalis 65 30 24 13 80 54

Cx stigmatosoma 65 22 20 9 91 45Cx thriambus 65 16 13 6 81 46Cs incidens 65 23 7 3 30 43

1 Titer of virus (log10 PFUml) on pledget used for mosquito infection2 (infectedtested) 3 1003 (transmittinginfected) 3 100

DECEMBER 2006 CS INCIDENS AND CX THRIAMBUS VECTOR COMPETENCE 663

Man platform after robotic RNA extractiondescribed previously (Shi et al 2001)

RESULTS AND DISCUSSION

Overall 16 (28) of 58 Cs incidens feeding onpledgets with an estimated 64ndash78 log10 PFU ofWNV per milliliter became infected This per-centage infection did not vary significantly (X2 5

13 df 5 3 P 010) among the 4 populationstested (Table 2) Similarly overall infection (82n 5 60) in concurrently collected Cx quinque-fasciatus did not vary (X2 5 10 df 5 1 P 010)between the 2 populations sampled but weresignificantly greater than percentage infection forCs incidens (X2 5 346 df 5 1 P 0001)However 10 (63) of 16 infected Cs incidensfemales expectorated virus and this was signifi-cantly greater (X2 5 141 df 5 1 P 0001) thanobserved for Cx quinquefasciatus (14 n 5 49)Therefore the percentage of total females of eachspecies that acquired and transmitted WNV athigh infectious doses was not significantly differ-ent (X2 5 07 df 5 1 P 010) However thepercentage of each species infected at progres-sively decreasing virus concentrations differedmarkedly in 1 experiment where sufficient femaleswere collected (Fig 1) Although Cs incidenswere common in peridomestic larval collectionsand relatively easy to rear they were difficult toblood feed and maintain Overall we offered 220females infectious blood meals on pledgets ofwhich 82 fed and of these only 58 survived the 2-week holding period at 26uC in our laboratoryBased on the absence of natural virus infectiona mammalian blood-meal host selection patternand moderate susceptibility to per os infection

this species should have limited involvement inencephalitis virus transmission in CaliforniaFailure to detect virus in this species during2004 and 2005 (Table 1) supports this conclusion

The laboratory infection and transmissionrates of Cx thriambus were not significantlydifferent (X2 5 12 and 04 respectively df 5 2 P 010) from those observed for concurrentlycollected Cx tarsalis and Cx stigmatosoma(Table 2) The percentage infection for all 3species decreased similarly as a function ofdecreasing viral titer in the infectious blood meal(Fig 1) In this single experiment Cx thriambusappeared to be equally or perhaps even moresusceptible than Cx tarsalis because 4 of 9females as compared to 0 of 12 females wereinfected at the 36 log10 PFUml viral doseBecause Cx thriambus has been found infectedrepeatedly in nature feeds predominantly onavian hosts and appears to be a highly competentexperimental host for WNV this species shouldbe considered important in virus maintenance oramplification within the avian-mosquito trans-mission cycle However the distribution andabundance of Cx thriambus seems limited inCalifornia to larval habitats associated with cleanwater along riparian systems Proposed reclama-tion of the original riparian systems in LosAngeles could expand larval habitats for thisspecies and possibly enhance its involvement inWNV activity

ACKNOWLEDGMENTS

We thank J R Rusmisel and staff AlamedaCounty MAD and M Madon J Wilson P

Fig 1 Percentage of females (n 5 7ndash30 females per data point) that were infected 2 weeks after feeding onpledgets with decreasing titers of West Nile virus

664 JOURNAL OF THE AMERICAN MOSQUITO CONTROL ASSOCIATION VOL 22 NO 4

OrsquoConnor and J Spoehl Greater Los AngelesCounty VCD for collecting the mosquitoes usedin these experiments Sandra Garcia and Sira-noosh Astari Center for Vectorborne Diseasesassisted with virus testing This research wasfunded by Research Grant No R01-A155607from the National Institutes of Allergy andInfectious Diseases NIH grants from the Uni-versity of California Mosquito Research Pro-gram and supplemental funds from the Centersfor Disease Control and Prevention and theGreater Los Angeles VCD Logistical supportwas provided by the Kern MVCD

REFERENCES CITED

Aitken THG 1977 An in vitro feeding technique forartificially demonstrating virus transmission by mos-quitoes Mosq News 37130ndash133

Bohart RM Washino RK 1978 Mosquitoes ofCalifornia Berkeley CA University of CaliforniaPress

Goddard LB Roth AE Reisen WK Scott TW 2002Vector competence of California mosquitoes forWest Nile virus Emerg Infect Dis 81385ndash1391

Hammon WM Reeves WC 1943a Laboratory trans-mission of St Louis encephalitis virus by three generaof mosquitoes J Exp Med 78241ndash253

Hammon WM Reeves WC 1943b Laboratory trans-mission of western equine encephalitis virus bymosquitoes of the genera Culex and Culiseta J ExpMed 78425ndash434

Hom A Houchin A McCaughey K Kramer VLChiles RE Reisen WK Tu E Glaser C Cossen CBaylis E Eldridge BF Sun B Padgett K Woods LMarcus L Hui LT Castro M Husted S 2004Surveillance for mosquito-borne encephalitis activityand human disease including West Nile virus inCalifornia 2003 Proc Mosq Vector Control AssocCalif 7248ndash54

Hom A Marcus L Kramer VL Cahoon B Glaser CCossen C Baylis E Jean C Tu E Eldridge BF

Carney R Padgett K Sun B Reisen WK Woods LHusted S 2005 Surveillance for mosquito-borneencephalitis virus activity and human disease in-cluding West Nile virus in California 2004 ProcMosq Vector Control Assoc Calif 7366ndash77

Kramer LD Wolfe TM Green EN Chiles RE FallahH Fang Y Reisen WK 2002 Detection ofencephalitis viruses in mosquitoes (Diptera Culici-dae) and avian tissues J Med Entomol 39312ndash323

Mahmood F 1999 Laboratory maintenance of mos-quitoes In Maramorosch K Mahmood F edsMaintenance of human animal and plant pathogenvectors Enfield NH Science Publishers p 3ndash31

Meyer RP Durso SL 1993 Identification of themosquitoes of California Sacramento CA CaliforniaMosquito Vector Control Association

Miura T Kliewer JW Tempelis CH 1976 Seasonaloccurrence of Culiseta incidens in the foothills ofFresno County California Mosq News 36343ndash349

Reeves WC Milby MM 1990 Natural infection inarthropod vectors In Reeves WC ed Epidemiologyand control of mosquito-borne arboviruses in Califor-nia 1943ndash1987 Sacramento CA California Mos-quito and Vector Control Association p 128ndash144

Reisen WK Fang Y Martinez VM 2005 Avian hostand mosquito (Diptera Culicidae) vector competencedetermine the efficiency of West Nile and St Louisencephalitis virus transmission J Med Entomol42367ndash375

Reisen WK Reeves WC 1990 Bionomics and ecologyof Culex tarsalis and other potential mosquito vectorspecies In Reeves WC ed Epidemiology and controlof mosquito-borne arboviruses in California 1943ndash1987 Sacramento CA California Mosquito andVector Control Association p 254ndash329

Shi PY Kauffman EB Ren P Felton A Tai JHDuPuis AP Jones SA Ngo KA Nicholas DCMaffei J Ebel GD Bernard KA Kramer LD 2001High-throughput detection of West Nile virus RNAJ Clin Microbiol 391264ndash1271

Tempelis CH Reeves WC 1964 Feeding habits of oneanopheline and three culicine mosquitoes by theprecipitin test J Med Entomol 1148ndash151

DECEMBER 2006 CS INCIDENS AND CX THRIAMBUS VECTOR COMPETENCE 665

Man platform after robotic RNA extractiondescribed previously (Shi et al 2001)

RESULTS AND DISCUSSION

Overall 16 (28) of 58 Cs incidens feeding onpledgets with an estimated 64ndash78 log10 PFU ofWNV per milliliter became infected This per-centage infection did not vary significantly (X2 5

13 df 5 3 P 010) among the 4 populationstested (Table 2) Similarly overall infection (82n 5 60) in concurrently collected Cx quinque-fasciatus did not vary (X2 5 10 df 5 1 P 010)between the 2 populations sampled but weresignificantly greater than percentage infection forCs incidens (X2 5 346 df 5 1 P 0001)However 10 (63) of 16 infected Cs incidensfemales expectorated virus and this was signifi-cantly greater (X2 5 141 df 5 1 P 0001) thanobserved for Cx quinquefasciatus (14 n 5 49)Therefore the percentage of total females of eachspecies that acquired and transmitted WNV athigh infectious doses was not significantly differ-ent (X2 5 07 df 5 1 P 010) However thepercentage of each species infected at progres-sively decreasing virus concentrations differedmarkedly in 1 experiment where sufficient femaleswere collected (Fig 1) Although Cs incidenswere common in peridomestic larval collectionsand relatively easy to rear they were difficult toblood feed and maintain Overall we offered 220females infectious blood meals on pledgets ofwhich 82 fed and of these only 58 survived the 2-week holding period at 26uC in our laboratoryBased on the absence of natural virus infectiona mammalian blood-meal host selection patternand moderate susceptibility to per os infection

this species should have limited involvement inencephalitis virus transmission in CaliforniaFailure to detect virus in this species during2004 and 2005 (Table 1) supports this conclusion

The laboratory infection and transmissionrates of Cx thriambus were not significantlydifferent (X2 5 12 and 04 respectively df 5 2 P 010) from those observed for concurrentlycollected Cx tarsalis and Cx stigmatosoma(Table 2) The percentage infection for all 3species decreased similarly as a function ofdecreasing viral titer in the infectious blood meal(Fig 1) In this single experiment Cx thriambusappeared to be equally or perhaps even moresusceptible than Cx tarsalis because 4 of 9females as compared to 0 of 12 females wereinfected at the 36 log10 PFUml viral doseBecause Cx thriambus has been found infectedrepeatedly in nature feeds predominantly onavian hosts and appears to be a highly competentexperimental host for WNV this species shouldbe considered important in virus maintenance oramplification within the avian-mosquito trans-mission cycle However the distribution andabundance of Cx thriambus seems limited inCalifornia to larval habitats associated with cleanwater along riparian systems Proposed reclama-tion of the original riparian systems in LosAngeles could expand larval habitats for thisspecies and possibly enhance its involvement inWNV activity

ACKNOWLEDGMENTS

We thank J R Rusmisel and staff AlamedaCounty MAD and M Madon J Wilson P

Fig 1 Percentage of females (n 5 7ndash30 females per data point) that were infected 2 weeks after feeding onpledgets with decreasing titers of West Nile virus

664 JOURNAL OF THE AMERICAN MOSQUITO CONTROL ASSOCIATION VOL 22 NO 4

OrsquoConnor and J Spoehl Greater Los AngelesCounty VCD for collecting the mosquitoes usedin these experiments Sandra Garcia and Sira-noosh Astari Center for Vectorborne Diseasesassisted with virus testing This research wasfunded by Research Grant No R01-A155607from the National Institutes of Allergy andInfectious Diseases NIH grants from the Uni-versity of California Mosquito Research Pro-gram and supplemental funds from the Centersfor Disease Control and Prevention and theGreater Los Angeles VCD Logistical supportwas provided by the Kern MVCD

REFERENCES CITED

Aitken THG 1977 An in vitro feeding technique forartificially demonstrating virus transmission by mos-quitoes Mosq News 37130ndash133

Bohart RM Washino RK 1978 Mosquitoes ofCalifornia Berkeley CA University of CaliforniaPress

Goddard LB Roth AE Reisen WK Scott TW 2002Vector competence of California mosquitoes forWest Nile virus Emerg Infect Dis 81385ndash1391

Hammon WM Reeves WC 1943a Laboratory trans-mission of St Louis encephalitis virus by three generaof mosquitoes J Exp Med 78241ndash253

Hammon WM Reeves WC 1943b Laboratory trans-mission of western equine encephalitis virus bymosquitoes of the genera Culex and Culiseta J ExpMed 78425ndash434

Hom A Houchin A McCaughey K Kramer VLChiles RE Reisen WK Tu E Glaser C Cossen CBaylis E Eldridge BF Sun B Padgett K Woods LMarcus L Hui LT Castro M Husted S 2004Surveillance for mosquito-borne encephalitis activityand human disease including West Nile virus inCalifornia 2003 Proc Mosq Vector Control AssocCalif 7248ndash54

Hom A Marcus L Kramer VL Cahoon B Glaser CCossen C Baylis E Jean C Tu E Eldridge BF

Carney R Padgett K Sun B Reisen WK Woods LHusted S 2005 Surveillance for mosquito-borneencephalitis virus activity and human disease in-cluding West Nile virus in California 2004 ProcMosq Vector Control Assoc Calif 7366ndash77

Kramer LD Wolfe TM Green EN Chiles RE FallahH Fang Y Reisen WK 2002 Detection ofencephalitis viruses in mosquitoes (Diptera Culici-dae) and avian tissues J Med Entomol 39312ndash323

Mahmood F 1999 Laboratory maintenance of mos-quitoes In Maramorosch K Mahmood F edsMaintenance of human animal and plant pathogenvectors Enfield NH Science Publishers p 3ndash31

Meyer RP Durso SL 1993 Identification of themosquitoes of California Sacramento CA CaliforniaMosquito Vector Control Association

Miura T Kliewer JW Tempelis CH 1976 Seasonaloccurrence of Culiseta incidens in the foothills ofFresno County California Mosq News 36343ndash349

Reeves WC Milby MM 1990 Natural infection inarthropod vectors In Reeves WC ed Epidemiologyand control of mosquito-borne arboviruses in Califor-nia 1943ndash1987 Sacramento CA California Mos-quito and Vector Control Association p 128ndash144

Reisen WK Fang Y Martinez VM 2005 Avian hostand mosquito (Diptera Culicidae) vector competencedetermine the efficiency of West Nile and St Louisencephalitis virus transmission J Med Entomol42367ndash375

Reisen WK Reeves WC 1990 Bionomics and ecologyof Culex tarsalis and other potential mosquito vectorspecies In Reeves WC ed Epidemiology and controlof mosquito-borne arboviruses in California 1943ndash1987 Sacramento CA California Mosquito andVector Control Association p 254ndash329

Shi PY Kauffman EB Ren P Felton A Tai JHDuPuis AP Jones SA Ngo KA Nicholas DCMaffei J Ebel GD Bernard KA Kramer LD 2001High-throughput detection of West Nile virus RNAJ Clin Microbiol 391264ndash1271

Tempelis CH Reeves WC 1964 Feeding habits of oneanopheline and three culicine mosquitoes by theprecipitin test J Med Entomol 1148ndash151

DECEMBER 2006 CS INCIDENS AND CX THRIAMBUS VECTOR COMPETENCE 665

OrsquoConnor and J Spoehl Greater Los AngelesCounty VCD for collecting the mosquitoes usedin these experiments Sandra Garcia and Sira-noosh Astari Center for Vectorborne Diseasesassisted with virus testing This research wasfunded by Research Grant No R01-A155607from the National Institutes of Allergy andInfectious Diseases NIH grants from the Uni-versity of California Mosquito Research Pro-gram and supplemental funds from the Centersfor Disease Control and Prevention and theGreater Los Angeles VCD Logistical supportwas provided by the Kern MVCD

REFERENCES CITED

Aitken THG 1977 An in vitro feeding technique forartificially demonstrating virus transmission by mos-quitoes Mosq News 37130ndash133

Bohart RM Washino RK 1978 Mosquitoes ofCalifornia Berkeley CA University of CaliforniaPress

Goddard LB Roth AE Reisen WK Scott TW 2002Vector competence of California mosquitoes forWest Nile virus Emerg Infect Dis 81385ndash1391

Hammon WM Reeves WC 1943a Laboratory trans-mission of St Louis encephalitis virus by three generaof mosquitoes J Exp Med 78241ndash253

Hammon WM Reeves WC 1943b Laboratory trans-mission of western equine encephalitis virus bymosquitoes of the genera Culex and Culiseta J ExpMed 78425ndash434

Hom A Houchin A McCaughey K Kramer VLChiles RE Reisen WK Tu E Glaser C Cossen CBaylis E Eldridge BF Sun B Padgett K Woods LMarcus L Hui LT Castro M Husted S 2004Surveillance for mosquito-borne encephalitis activityand human disease including West Nile virus inCalifornia 2003 Proc Mosq Vector Control AssocCalif 7248ndash54

Hom A Marcus L Kramer VL Cahoon B Glaser CCossen C Baylis E Jean C Tu E Eldridge BF

Carney R Padgett K Sun B Reisen WK Woods LHusted S 2005 Surveillance for mosquito-borneencephalitis virus activity and human disease in-cluding West Nile virus in California 2004 ProcMosq Vector Control Assoc Calif 7366ndash77

Kramer LD Wolfe TM Green EN Chiles RE FallahH Fang Y Reisen WK 2002 Detection ofencephalitis viruses in mosquitoes (Diptera Culici-dae) and avian tissues J Med Entomol 39312ndash323

Mahmood F 1999 Laboratory maintenance of mos-quitoes In Maramorosch K Mahmood F edsMaintenance of human animal and plant pathogenvectors Enfield NH Science Publishers p 3ndash31

Meyer RP Durso SL 1993 Identification of themosquitoes of California Sacramento CA CaliforniaMosquito Vector Control Association

Miura T Kliewer JW Tempelis CH 1976 Seasonaloccurrence of Culiseta incidens in the foothills ofFresno County California Mosq News 36343ndash349

Reeves WC Milby MM 1990 Natural infection inarthropod vectors In Reeves WC ed Epidemiologyand control of mosquito-borne arboviruses in Califor-nia 1943ndash1987 Sacramento CA California Mos-quito and Vector Control Association p 128ndash144

Reisen WK Fang Y Martinez VM 2005 Avian hostand mosquito (Diptera Culicidae) vector competencedetermine the efficiency of West Nile and St Louisencephalitis virus transmission J Med Entomol42367ndash375

Reisen WK Reeves WC 1990 Bionomics and ecologyof Culex tarsalis and other potential mosquito vectorspecies In Reeves WC ed Epidemiology and controlof mosquito-borne arboviruses in California 1943ndash1987 Sacramento CA California Mosquito andVector Control Association p 254ndash329

Shi PY Kauffman EB Ren P Felton A Tai JHDuPuis AP Jones SA Ngo KA Nicholas DCMaffei J Ebel GD Bernard KA Kramer LD 2001High-throughput detection of West Nile virus RNAJ Clin Microbiol 391264ndash1271

Tempelis CH Reeves WC 1964 Feeding habits of oneanopheline and three culicine mosquitoes by theprecipitin test J Med Entomol 1148ndash151

DECEMBER 2006 CS INCIDENS AND CX THRIAMBUS VECTOR COMPETENCE 665