• 1

    Mackenzie JS, Lindsay MD, Coelen RJ, Broom AK, Hall RA, Smith DW, 1994. Arboviruses causing human disease in the Australasian zoogeographic region. Arch Virol 136 :447–467.

    • Search Google Scholar
    • Export Citation
  • 2

    Russell RC, 2002. Ross River virus: ecology and distribution. Annu Rev Entomol 47 :1–31.

  • 3

    Hanna JN, Ritchie SA, Phillips DA, Serafin IL, Hills SL, van den Hurk AF, Pyke AT, McBride WJ, Amadio MG, Spark RL, 2001. An epidemic of dengue 3 in far north Queensland, 1997–1999. Med J Aust 174 :178–182.

    • Search Google Scholar
    • Export Citation
  • 4

    Ritchie SA, Long S, Smith G, Pyke A, Knox TB, 2004. Entomological investigations in a focus of dengue transmission in Cairns, Queensland, Australia, by using the sticky ovitraps. J Med Entomol 41 :1–4.

    • Search Google Scholar
    • Export Citation
  • 5

    Mackenzie JS, Johansen CA, Ritchie SA, van den Hurk AF, Hall RA, 2002. Japanese encephalitis as an emerging virus: the emergence and spread of Japanese encephalitis virus in Australasia. Curr Top Microbiol Immunol 267 :49–73.

    • Search Google Scholar
    • Export Citation
  • 6

    Jansen CC, Webb CE, Northill JA, Ritchie SA, Russell RC, van den Hurk AF, 2008. Vector competence of Australian mosquito species for a North American strain of West Nile virus. Vector Borne Zoonotic Dis 8 :805–811.

    • Search Google Scholar
    • Export Citation
  • 7

    Kay BH, Boreham PFL, Williams GM, 1979. Host preferences and feeding patterns of mosquitoes at Kowanyama, Cape York Peninsula, northern Queensland. Bull Entomol Res 69 :441–457.

    • Search Google Scholar
    • Export Citation
  • 8

    Kay BH, Boyd AM, Ryan PA, Hall RA, 2007. Mosquito feeding patterns and natural infection of vertebrates with Ross River and Barmah Forest viruses in Brisbane, Australia. Am J Trop Med Hyg 76 :417–423.

    • Search Google Scholar
    • Export Citation
  • 9

    Muller MJ, Murray MD, Edwards JA, 1981. Blood-sucking midges and mosquitoes feeding on mammals at Beatrice Hill, N.T. Aust J Zool 29 :573–588.

    • Search Google Scholar
    • Export Citation
  • 10

    van den Hurk AF, Johansen CA, Zborowski P, Paru R, Foley PN, Beebe NW, Mackenzie JS, Ritchie SA, 2003. Mosquito host-feeding patterns and implications for Japanese encephalitis virus transmission in northern Australia and Papua New Guinea. Med Vet Entomol 17 :403–411.

    • Search Google Scholar
    • Export Citation
  • 11

    Australian Bureau of Statisitics, 2008. Regional Population Growth, Australia 2006–07, Catalog No. 3218.0 and National Regional Profile: Australia. Available at: www.abs.gov.au. Accessed November 12, 2008.

  • 12

    Ritchie SA, Fanning ID, Phillips DA, Standfast HA, McGinn D, Kay BH, 1997. Ross River virus in mosquitoes (Diptera: Culicidae) during the 1994 epidemic around Brisbane, Australia. J Med Entomol 34 :156–159.

    • Search Google Scholar
    • Export Citation
  • 13

    Harley D, Ritchie S, Phillips D, van den Hurk A, 2000. Mosquito isolates of Ross River virus from Cairns, Queensland, Australia. Am J Trop Med Hyg 62 :561–565.

    • Search Google Scholar
    • Export Citation
  • 14

    Lee DJ, Clinton KJ, O’Gower AK, 1954. The blood sources of some Australian mosquitoes. Aust J Biol Sci 7 :282–301.

  • 15

    Kay BH, Boreham PF, Fanning ID, 1985. Host-feeding patterns of Culex annulirostris and other mosquitoes (Diptera: Culicidae) at Charleville, southwestern Queensland, Australia. J Med Entomol 22 :529–535.

    • Search Google Scholar
    • Export Citation
  • 16

    Boyle DB, Dickerman RW, Marshall ID, 1983. Primary viraemia responses of herons to experimental infection with Murray Valley encephalitis, Kunjin and Japanese encephalitis viruses. Aust J Exp Biol Med Sci 61 :655–664.

    • Search Google Scholar
    • Export Citation
  • 17

    Komar N, Langevin S, Hinten S, Nemeth N, Edwards E, Hettler D, Davis B, Bowen R, Bunning M, 2003. Experimental infection of North American birds with the New York 1999 strain of West Nile virus. Emerg Infect Dis 9 :311–322.

    • Search Google Scholar
    • Export Citation
  • 18

    Boakye DA, Tang J, Truc P, Merriweather A, Unnasch TR, 1999. Identification of bloodmeals in haematophagous diptera by cytochrome B heteroduplex analysis. Med Vet Entomol 13 :282–287.

    • Search Google Scholar
    • Export Citation
  • 19

    Apperson CS, Harrison BA, Unnasch TR, Hassan HK, Irby WS, Savage HM, Aspen SE, Watson DW, Rueda LM, Engber BR, Nasci RS, 2002. Host-feeding habits of Culex and other mosquitoes (Diptera: Culicidae) in the Borough of Queens in New York City, with characters and techniques for identification of Culex mosquitoes. J Med Entomol 39 :777–785.

    • Search Google Scholar
    • Export Citation
  • 20

    van den Hurk AF, Smith IL, Smith GA, 2007. Development and evaluation of real-time polymerase chain reaction assays to identify mosquito (Diptera: Culicidae) bloodmeals originating from native Australian mammals. J Med Entomol 44 :85–92.

    • Search Google Scholar
    • Export Citation
  • 21

    Ngo KA, Kramer LD, 2003. Identification of mosquito bloodmeals using polymerase chain reaction (PCR) with order-specific primers. J Med Entomol 40 :215–222.

    • Search Google Scholar
    • Export Citation
  • 22

    Molaei G, Andreadis TG, Armstrong PM, Anderson JF, Vossbrinck CR, 2006. Host feeding patterns of Culex mosquitoes and West Nile virus transmission, northeastern United States. Emerg Infect Dis 12 :468–474.

    • Search Google Scholar
    • Export Citation
  • 23

    Williams CR, Long SA, Russell RC, Ritchie SA, 2006. Field efficacy of the BG-Sentinel compared with CDC Backpack Aspirators and CO2-baited EVS traps for collection of adult Aedes aegypti in Cairns, Queensland, Australia. J Am Mosq Control Assoc 22 :296–300.

    • Search Google Scholar
    • Export Citation
  • 24

    Blackwell A, Mordue AJ, Mordue W, 1994. Identification of blood-meals of the Scottish biting midge, Culicoides impunctatus, by indirect enzyme-linked immunosorbent assay (ELISA). Med Vet Entomol 8 :20–24.

    • Search Google Scholar
    • Export Citation
  • 25

    van den Hurk AF, Nisbet DJ, Johansen CA, Foley PN, Ritchie SA, Mackenzie JS, 2001. Japanese encephalitis on Badu Island, Australia: the first isolation of Japanese encephalitis virus from Culex gelidus in the Australasian region and the role of mosquito host-feeding patterns in virus transmission cycles. Trans R Soc Trop Med Hyg 95 :595–600.

    • Search Google Scholar
    • Export Citation
  • 26

    Cicero C, Johnson NK, 2001. Higher-level phylogeny of new world vireos (aves: vireonidae) based on sequences of multiple mitochondrial DNA genes. Mol Phylogenet Evol 20 :27–40.

    • Search Google Scholar
    • Export Citation
  • 27

    Tamura K, Dudley J, Nei M, Kumar S, 2007. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24 :1596–1599.

    • Search Google Scholar
    • Export Citation
  • 28

    Simpson D, Day N, 2004. Field Guide to the Birds of Australia. London: Christopher Helm.

  • 29

    Johnson KP, Sorenson MD, 1999. Phylogeny and biogeography of dabbling ducks (Genus: Anas): a comparison of molecular and morphological evidence. Auk 116 :792–805.

    • Search Google Scholar
    • Export Citation
  • 30

    Joseph L, Wilke T, Ten Have J, Terry Chesser R, 2006. Implications of mitochondrial DNA polyphyly in two ecologically undifferentiated but morphologically distinct migratory birds, the masked and white-browed woodswallows Artamus spp. of inland Australia. J Avian Biol 37 :625–636.

    • Search Google Scholar
    • Export Citation
  • 31

    Driskell AC, Christidis L, 2004. Phylogeny and evolution of the Australo-Papuan honeyeaters (Passeriformes, Meliphagidae). Mol Phylogenet Evol 31 :943–960.

    • Search Google Scholar
    • Export Citation
  • 32

    Cupp EW, Zhang D, Yue X, Cupp MS, Guyer C, Sprenger TR, Unnasch TR, 2004. Identification of reptilian and amphibian blood meals from mosquitoes in an eastern equine encephalomyelitis virus focus in central Alabama. Am J Trop Med Hyg 71 :272–276.

    • Search Google Scholar
    • Export Citation
  • 33

    Kerr KC, Stoeckle MY, Dove CJ, Weigt LA, Francis CM, Hebert PD, 2007. Comprehensive DNA barcode coverage of North American birds. Mol Ecol Notes 7 :535–543.

    • Search Google Scholar
    • Export Citation
  • 34

    Edman JD, Kale HW, 1971. Host behavior: its influence on the feeding success of mosquitoes. Ann Entomol Soc Am 64 :513–516.

  • 35

    Edman JD, Webber LA, Schmid AA, 1974. Effect of host defenses on the feeding pattern of Culex nigripalpus when offered a choice of blood sources. J Parasitol 60 :874–883.

    • Search Google Scholar
    • Export Citation
  • 36

    Webber LA, Edman JD, 1972. Anti-mosquito behaviour of ciconiiform birds. Anim Behav 20 :228–232.

  • 37

    Darbro JM, Harrington LC, 2007. Avian defensive behaviour and blood-feeding success of the West Nile vector mosqutio, Culex pipiens. Behav Ecol 18 :750–757.

    • Search Google Scholar
    • Export Citation
  • 38

    Scott TW, Chow E, Strickman D, Kittayapong P, Wirtz RA, Lorenz LH, Edman JD, 1993. Blood-feeding patterns of Aedes aegypti (Diptera: Culicidae) collected in a rural Thai village. J Med Entomol 30 :922–927.

    • Search Google Scholar
    • Export Citation
  • 39

    Scott TW, Morrison AC, Lorenz LH, Clark GG, Strickman D, Kittayapong P, Zhou H, Edman JD, 2000. Longitudinal studies of Aedes aegypti (Diptera: Culicidae) in Thailand and Puerto Rico: population dynamics. J Med Entomol 37 :77–88.

    • Search Google Scholar
    • Export Citation
  • 40

    Ponlawat A, Harrington LC, 2005. Blood feeding patterns of Aedes aegypti and Aedes albopictus in Thailand. J Med Entomol 42 :844–849.

  • 41

    Marshall ID, 1988. Murray Valley and Kunjin encephalitis. Monath TP, ed. The Arboviruses: Epidemiology and Ecology. Boca Raton, FL: CRC Press, 151–189.

  • 42

    Boyle DB, Marshall ID, Dickerman RW, 1983. Primary antibody responses of herons to experimental infection with Murray Valley encephalitis, Kunjin and Japanese encephalitis viruses. Aust J Exp Biol Med Sci 61 :665–674.

    • Search Google Scholar
    • Export Citation
  • 43

    Russell RC, 1995. Arboviruses and their vectors in Australia: an update on the ecology and epidemiology of some mosquito-borne arboviruses. Rev Med Vet Entomol 83 :141–158.

    • Search Google Scholar
    • Export Citation
  • 44

    Ritchie SA, Phillips D, Broom A, Mackenzie J, Poidinger M, van den Hurk A, 1997. Isolation of Japanese encephalitis virus from Culex annulirostris in Australia. Am J Trop Med Hyg 56 :80–84.

    • Search Google Scholar
    • Export Citation
  • 45

    Johansen CA, van den Hurk AF, Pyke AT, Zborowski P, Phillips DA, Mackenzie JS, Ritchie SA, 2001. Entomological investigations of an outbreak of Japanese encephalitis virus in the Torres Strait, Australia, in 1998. J Med Entomol 38 :581–588.

    • Search Google Scholar
    • Export Citation
  • 46

    van den Hurk AF, Nisbet DJ, Hall RA, Kay BH, MacKenzie JS, Ritchie SA, 2003. Vector competence of Australian mosquitoes (Diptera: Culicidae) for Japanese encephalitis virus. J Med Entomol 40 :82–90.

    • Search Google Scholar
    • Export Citation
  • 47

    Doherty RL, Carley JG, Kay BH, Filippich C, Marks EN, Frazier CL, 1979. Isolation of virus strains from mosquitoes collected in Queensland, 1972–1976. Aust J Exp Biol Med Sci 57 :509–520.

    • Search Google Scholar
    • Export Citation
  • 48

    Liehne PF, Anderson S, Stanley NF, Liehne CG, Wright AE, Chan KH, Leivers S, Britten DK, Hamilton NP, 1981. Isolation of Murray Valley encephalitis virus and other arboviruses in the Ord River Valley 1972–1976. Aust J Exp Biol Med Sci 59 :347–356.

    • Search Google Scholar
    • Export Citation
  • 49

    van den Hurk AF, Johansen CA, Zborowski P, Phillips DA, Pyke AT, Mackenzie JS, Ritchie SA, 2001. Flaviviruses isolated from mosquitoes collected during the first recorded outbreak of Japanese encephalitis virus on Cape York Peninsula, Australia. Am J Trop Med Hyg 64 :125–130.

    • Search Google Scholar
    • Export Citation
  • 50

    Lee DJ, Hicks MM, Debenham ML, Griffiths M, Russell RC, Bryan JH, Russell RC, Marks EN, 1989. The Culicidae of the Australasian Region. Entomology Monograph No. 2. Canberra: Australian Government Publishing Service Press.

  • 51

    Russell RC, Kay BH, 2004. Medical entomology: changes in the spectrum of mosquito-borne disease in Australia and other vector threats and risks, 1972–2004. Aust J Entomol 43 :271–282.

    • Search Google Scholar
    • Export Citation
  • 52

    Russell RC, 1987. The mosquito fauna of Conjola state forest on the South coast of New South Wales. Part 2. Female feeding behaviour and flight activity. Gen Appl Entomol 19 :17–24.

    • Search Google Scholar
    • Export Citation
  • 53

    Kilpatrick AM, Kramer LD, Jones MJ, Marra PP, Daszak P, 2006. West Nile virus epidemics in North America are driven by shifts in mosquito feeding behavior. PLoS Biol 4 :606–610.

    • Search Google Scholar
    • Export Citation
  • 54

    Edman JD, Taylor DJ, 1968. Culex nigripalpus: seasonal shift in the bird-mammal feeding ratio in a mosquito vector of human encephalitis. Science 161 :67–68.

    • Search Google Scholar
    • Export Citation
  • 55

    Christidis L, Boles WE, 2008. Systematics and Taxonomy of Australian Birds. Collingwood, Australia: Commonwealth Scientific and Industrial Research Organisation Publishing.

  • 56

    Groth JG, 1998. Molecular phylogenetics of finches and sparrows: consequences of character state removal in cytochrome b sequences. Mol Phylogenet Evol 10 :377–390.

    • Search Google Scholar
    • Export Citation
  • 57

    Kennedy M, Gray RD, Spencer HG, 2000. The phylogenetic relationships of the shags and cormorants: can sequence data resolve a disagreement between behavior and morphology? Mol Phylogenet Evol 17 :345–359.

    • Search Google Scholar
    • Export Citation
 
 
 
 

 

 

 

 

 

 

 

 

 

Blood Sources of Mosquitoes Collected from Urban and Peri-Urban Environments in Eastern Australia with Species-Specific Molecular Analysis of Avian Blood Meals

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  • 1 Australian Biosecurity Cooperative Research Centre for Emerging Infectious Disease, and School of Molecular and Microbial Sciences, University of Queensland, St. Lucia, Queensland, Australia; Department of Medical Entomology, University of Sydney, Westmead Hospital, New South Wales, Australia; Organic Chemistry, and Virology, Communicable Diseases Unit, Queensland Health Forensic and Scientific Services, Queensland, Australia; World Health Organization/World Health Organisation for Animal Health/Food and Agricultural Organization Collaborating Centre for Reference and Research on Leptospirosis, Communicable Diseases Unit, Queensland Health Forensic and Scientific Services, Coopers Plains, Australia; School of Public Health, Tropical Medicine and Rehabilitation Sciences, James Cook University, Smithfield, Queensland, Australia; Tropical Population Health Network, Queensland Health, Cairns, Queensland, Australia

To identify the hosts of mosquitoes collected from urban and peri-urban habitats in eastern Australia, 1,180 blood fed mosquitoes representing 15 species were analyzed by enzyme-linked immunosorbent assay and molecular techniques. Four common and epidemiologically important species could be classified according to their host-feeding patterns: Aedes aegypti was anthropophilic, Ae. vigilax was mammalophilic, Culex quinquefasciatus was ornithophilic, and Cx. annulirostris was opportunistic, readily feeding on birds and mammals. Mitochondrial cytochrome b DNA sequence data showed that more than 75% of avian blood meals identified from Cx. annulirostris collected from Brisbane, Newcastle, and Sydney originated from ducks (Order Anseriformes, Family Anatidae). More than 75% of avian blood meals from Cx. quinquefasciatus from Cairns belonged to one of three Passerine species, namely Sphecotheres vieilloti (figbird), Sturnus tristis (common myna), and Philemon buceroides (helmeted friarbird). This study demonstrates associations between vectors in Australia and vertebrate hosts of endemic and exotic arboviruses.

INTRODUCTION

Analysis of the host-feeding patterns of mosquitoes provides valuable insight into the relative roles of mosquito species in arbovirus transmission cycles and can describe the extent of contact with vertebrate amplifying hosts. Coupled with data from vector competence experiments and virus isolation studies, this information is essential for the development and implementation of surveillance and control strategies. Australia possesses numerous endemic arboviruses that circulate between a variety of vertebrate hosts and mosquitoes. Endemic flaviviruses including Kunjin virus (KUNV) and Murray Valley encephalitis virus (MVEV) are transmitted between birds and Culex mosquitoes,1 and marsupials are hosts of the most common alphavirus, Ross River virus.2

In addition to endemic arboviruses, Australia is receptive to the introduction of exotic arboviruses. Dengue viruses are introduced into northern Queensland by viremic travelers, causing periodic outbreaks, 3,4 and annual Japanese encephalitis virus (JEV) activity occurs in the Torres Strait.5 The presence of competent vectors of exotic arboviruses, including West Nile virus (WNV)6 and chikungunya virus (van den Hurk AF and others, unpublished data) in Australia highlights the biosecurity threat from the potential introduction of exotic viruses.

A detailed knowledge of the host-feeding patterns of Australian mosquitoes is essential for a thorough understanding of the ecology of arboviruses. Previous studies have examined the host-feeding patterns and host preference of Australian mosquitoes.710 However, most of these studies have examined mosquitoes collected from remote and/or rural environments, and only a few have investigated common mosquitoes in the urban environment.8 If one considers that 68.5% of the Australian population resides in major urban cities (with most persons concentrated on the east coast), 11 it is pertinent to examine host-feeding behavior in these habitats/environments. This fact is especially important because arbovirus activity is frequently documented in urban centers of eastern Australia, with epidemic activity occurring in some years. 12,13 Finally, the urban centers of eastern Australia also represent major transport and tourism hubs in Australia, suggesting that they may be at risk to the introduction of exotic arboviruses.

Previous studies of host-feeding patterns in Australia have used immunologic assays, including gel diffusion 8,10 and precipitin tests. 7,14,15 Although these studies have generated critical epidemiologic information, serologic techniques are limited by availability of antisera against target species and the cross-reactivity between serum proteins from closely-related species. These factors can limit the capacity of the assay to resolve the identity of blood meals between taxonomically related hosts. However, species-specific data can be particularly informative because vertebrate species involved in transmission cycles may be limited to a precise host range. For instance, the primary maintenance hosts of MVEV, KUNV, and JEV are ardeid birds, 16 and the most important hosts of other flaviviruses, including WNV in North America, are passerine birds. 17

The development of molecular technologies specific for the mitochondrial cytochrome b gene has circumvented limitations of current serologic assays. Polymerase chain reaction (PCR) assays, such as heteroduplex analysis, 18,19 real-time PCR, 20 use of order-specific primers with restriction enzyme digestion, 21 and DNA sequencing 22 have provided valuable species-specific identification of mosquito blood meals.

In this study, we identify the host sources of blood meals in mosquitoes collected from urban and peri-urban habitats in eastern Australia by conducting an initial assessment using an enzyme-linked immunosorbent assay (ELISA), followed by a detailed molecular analysis of blood meals that were identified as avian in origin. We identified common bird species that are fed upon by Australian mosquitoes in urban habitats.

MATERIALS AND METHODS

Mosquitoes.

From January 2005 through April 2008, adult mosquitoes were collected opportunely from urban and peri-urban areas of Cairns (16°55′S, 145°46′E), Brisbane (27°28′S, 153°01′E), Newcastle (32°55′S, 151°46′E), and Sydney (33°52′S, 151°12′E). Various collection methods were used, including modified Centers for Disease Control miniature light traps (model 512; John W. Hock, Gainesville, FL) and encephalitis virus surveillance traps (Australian Entomological Supplies, Bangalow, New South Wales, Australia) baited with CO2 (as dry ice). Unbaited BG-Sentinel traps (Biogents AG, Regensburg, Germany) were also used to target domestic species including Aedes aegypti and Culex quinquefasciatus.23 Mosquitoes were frozen and sorted on a chilled table according to collection site, species, and engorgement status. The abdomens of engorged mosquitoes were removed and homogenized in 150 μL of phosphate-buffered saline (PBS). After centrifugation, the supernatant was removed and stored at −20°C until analysis.

Bird samples.

Reference bird tissue was obtained from various sources including Currumbin Wildlife Sanctuary, the National Parks and Wildlife Service, Queensland Museum, and Sydney Olympic Park Authority. Samples were supplied as muscle or skin tissue from deceased birds.

Serologic assay.

Homogenized blood meals were initially screened using an indirect ELISA (adapted from Blackwell and others) 24 that used commercially produced antisera. Each blood meal was individually tested using 11 antisera: anti-horse, anti-rabbit, anti-rat, anti-human, anti-dog, anti-chicken, anti-cat (Cappal Laboratories, ICN Pharmaceuticals, Aurora, OH), anti-bird, anti-kangaroo (Bethyl Labora tories, Montgomery, TX), anti-cow, and anti-pig (Dako, Kingsgrove, New South Wales, Australia). Specificity of the antisera was tested using diluted serum (1:200,000) from each target animal. Cross-reactivity was minimized by pre-absorbing the antisera by mixing (10:1) with serum from cross-reacting non-target animals and incubating at 4°C for 12 hours before use.

Previous work demonstrated that the kangaroo antiserum cross-reacts with most marsupials including wallabies and possums. Likewise, the chicken antiserum is cross-reactive with most birds and the rat antiserum is cross-reactive with mouse. 25 Thus, blood meals that reacted with kangaroo, chicken or rat antiserum were classified as marsupial, avian, or rodent, respectively.

Wells of 96-well microtiter plates were coated with antigen (mosquito homogenate diluted 1:1,000 or vertebrate control serum diluted 1:200,000) and incubated overnight at 4°C. The plates were washed twice with PBS-Tween before blocking with 100 μL of 5% milk diluent buffer. After incubation at room temperature for up to 2 hours, 50 μL of target antiserum (at a dilution of 1:1,000–1:2,000 in blocking buffer, depending on optimal concentration for each antiserum preparation) was added, and plates were incubated for one hour at room temperature. After four washes with PBS-Tween, 50 μL of horseradish peroxidase–conjugated secondary antibody (diluted 1:1,000–1:8,000 in blocking buffer; Dako) was added and incubated for one hour at room temperature. Plates were then washed six times with PBS-Tween and 50 μL of 3,3′,5,5′-tetramethylbenzidine substrate and hydrogen peroxide (ELISA Systems, Windsor, New South Wales, Australia) was added to each well. After incubation for 10 minutes in darkness at room temperature, the reaction was stopped with 50 μL/well of 1N H2SO4. Absorbance at 450 nm was measured using a TECAN Minilyser Spectra II plate reader (Tecan, Mannedorf, Switzerland).

Each sample was tested against each antiserum in duplicate. Samples were considered positive if they displayed an absorbance greater than the mean + 2× SD of the observed background absorbance measured in wells coated with homogenized unfed mosquitoes.

Molecular identification of avian blood meals.

DNA from blood meal samples that were identified as avian in origin or a subsample of those negative in the ELISA was extracted using the Qiagen DNeasy kit (Qiagen, Doncaster, Victoria, Australia) according to manufacturer’s instructions. A portion of the cytochrome b gene (approximately 508 basepairs) was amplified by PCR using avian-specific primers (5′-GAC TGT GAC AAA ATC CCN TTC CA-3′ (forward) and 5′-GGT CTT CAT CTY HGG YTT ACA AGA C-3′ (reverse)). 26 Briefly, a 50-μL PCR volume was prepared containing 10 μL of extracted template DNA, 1× PCR buffer II, 0.24 mM of each dNTP, 1.5 mM MgCl2, 2 units of AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA), and each primer at a concentration of 0.4 μM. Thermal cycling conditions consisted of incubation at 95°C for 10 minutes, 45 cycles at 95°C for 1 minute, 57°C for 1 minute, and 72°C for 1.5 minute, followed by a final elongation at 72°C for 10 minutes.

The PCR product was purified using the MultiScreen® 96-well filtration platform (Millipore, Bedford, MA). Samples were diluted (2:5) before transfer to the filter plate. Samples were washed with 100 μL of water and eluted in 100 μL of water. The sequencing reaction was performed using BigDye® Terminator version 3.1 chemistry (Applied Biosystems) according to manufacturer’s protocol and used 0.32 μM primer and approximately 20 ng of purified PCR product.

Sequencing products were purified using the Montage® platform (Millipore). Briefly, samples were diluted in 20 μL of injection solution (Millipore) and transferred to the filter plate. After a second wash with 20 μL of injection solution and an additional wash with 50 μL of water, the sample was resus-pended in 20 μL of water and dried in a vacuum centrifuge. Sequence data were obtained using the Applied Biosystems 3130xl Genetic Analyser at the Griffith University DNA Sequencing Facility (Griffith University, Nathan, Queensland, Australia). Sequences were aligned using the MEGA version 4 program 27 and compared with the GenBank DNA sequence database (National Center for Biotechnology Information, Bethesda, MD; http://www.ncbi.nlm.nih.gov/Genbank/index.html). Percentage sequence homology was used to determine avian sequence identity, commensurate with expected variation for each bird family, as previously described in the literature. Samples were classified to species level only if observed sequence homology was ≥ 99%.

Molecular identification of avian reference tissue.

In addition, reference bird tissue samples (muscle or skin) were extracted using the Qiagen DNeasy kit as described above, but with the addition of a lysis step. Extracted DNA was diluted 1:20 before addition to the avian PCR. DNA sequencing was conducted as described above.

Molecular identification of mammalian blood meals.

Using a similar protocol, sequence data obtained with mammalian specific primers were used to further resolve a selection of samples that were unidentified in the ELISA because of multiple reactivity with more than one mammalian host antisera. Briefly, a 772-basepair portion of extracted DNA was amplified using mammalian specific primers described by Ngo and Kramer 21 (5′-CGA AGC TTG ATA TGA AAA ACC ATC GTT G-3′ [forward] and 5′-TGT AGT TRT CWG GGT CHC CTA-3′ [reverse]. Fifty-microliter reactions consisted of 10 μL of extracted template DNA, 1× PCR buffer II, 0.24 mM of each dNTP, 1.5 mM MgCl2, 3 units of AmpliTaq Gold DNA polymerase, and each primer at a concentration of 0.6 μM. Thermal cycling conditions consisted of incubation at 95°C for 15 minutes, 45 cycles of 95°C for 1 minute, 51°C for 30 seconds and 72°C for 1.5 minutes, and finally 72°C for 10 minutes. Amplified product was purified and sequenced as described above.

RESULTS

A total of 1,180 blood fed mosquitoes representing 21 species was analyzed (Table 1). Overall, 74.3% (877) reacted to one or more antisera in the ELISA. An additional 25 mammalian and 19 avian samples were identified using PCR, resulting in a total detection rate of 78.1%. Four species, Ae. aegypti, Ae. vigilax, Culex annulirostris, and Cx. quinquefasciatus, comprised most samples analyzed (83.6%). Avian blood meals accounted for 31.6% of total blood meals identified, and human, marsupial, and dog blood meals comprised 22.0%, 12.5%, and 9.2%, respectively.

Most (75.3%) blood meals identified from Ae. aegypti were human in origin (n = 174). The remaining blood meals originated from dogs (13.2%), birds (5.7%), cats and rabbits (both < 1.1%). Multiple blood meals on humans and dogs were observed in 4.0% of the Ae. aegypti blood meals identified.

Most Cx. quinquefasciatus processed were from Cairns (86.7%, 163 of 188) and, of those identified (149), 95.3% of the blood meals were avian in origin. Of the 20 blood meals identified from Sydney, 50% originated from birds. Overall, humans accounted for only 3.5% of total Cx. quinquefasciatus blood meals identified.

Alternatively, Ae. vigilax fed mostly on mammals (94.9% of total blood meals identified, n = 117), with cows (26.5%), rabbits (25.6%), and humans (13.7%) commonly fed upon by this species. Horses and dogs comprised 7.7% and 5.1% of total blood meals identified for Ae. vigilax, respectively.

Culex annulirostris appeared to be an opportunistic species, with birds and mammals accounting for 22.0% and 78.0% of blood meals identified, respectively (n = 300). Of the mammals fed upon by Cx. annulirostris, marsupials comprised 22.7% of identified blood meals, and rodents, dogs (each comprising 11.0%) and humans (10.7%) were also commonly identified.

Molecular identification of avian reference tissue.

DNA was amplified and sequenced from a total of 68 birds, comprising 50 species. Bird species examined and associated GenBank accession numbers are shown in Table 2.

Molecular identification of avian blood meals.

Avian DNA was successfully amplified from 219 mosquitoes. DNA was sequenced from 212 of these mosquitoes and avian sequence data was successfully obtained from 177 mosquitoes. A variety of bird species was identified from mosquito blood meals (Table 3), with most samples identified from Cx. quinquefasciatus and Cx. annulirostris.

More than 96% of avian blood meals examined from Cx. quinquefasciatus collected from Cairns were identified as originating from passeriform birds. The most common bird families identified were Oriolidae and Meliphagidae, comprising 40.4% and 28.4%, respectively, of sequenced avian blood meals. Of these blood meals identified to species level, three bird species were repeatedly identified with ≥ 99% sequence homology: Sphecotheres vieilloti (figbird, 44 of 109 identified), Sturnus tristis (common myna, 21 of 109), and Philemon buceroides (helmeted friarbird, 19 of 109). Two Cx. quinquefasciatus from Cairns were found to have fed on members of the cockatoo family (Cacatuidae, order Psittaciformes). Sequences from both mosquitoes were most similar to the sequence data from the cockatiel, Nymphicus hollandicus (97% sequence homology). Avian sequence data from two Cx. quinquefasciatus from Sydney shared 96% sequence homology with a number of Zosterops spp., indicating probable origins from a member of the genus Zosterops and, considering the distribution of this genus in Australia, 28 most likely Zosterops lateralis, the gray-backed silvereye.

A high proportion of Cx. annulirostris and Culex sitiens collected in Sydney, Newcastle, and Brisbane were found to have fed on ducks (order Anseriformes, family Anatidae). Collectively, 75.7% (28 of 37) of avian blood meals of Cx. annulirostris from Sydney, Newcastle, and Brisbane were identified as originating from ducks, and 50.0% (5 of 10) of the avian blood meals examined from Cx. sitiens from Sydney and Newcastle also originated from ducks. Of these mosquitoes, four Cx. annulirostris and one Cx. sitiens from Sydney were identified to have fed on the Australian wood (maned) duck, Chenonetta jubata, and a single Cx. annulirostris had fed on the domestic goose, Anser anser. Because of low sequence variation observed within the cytochrome b region of some members of the Anatidae, 29 it was not possible to further resolve which species had been targeted by the remaining mosquitoes. However, based on the distribution of Australian ducks 28 and observed sequence homologies, it is likely that these remaining blood meals identified as originating from members of the family Anatidae were from Anas superciliosa (Pacific black duck), Anas platyrhynchos (northern mallard), Anas castanea (chestnut teal), or Anas gracilis (gray teal).

Six mosquitoes fed on either magpies or currawongs (Artamidae). These mosquitoes comprised three Cx. quinquefasciatus and one Ae. aegypti from Cairns, and one of each Cx. quinquefasciatus and Cx. annulirostris from Sydney. It was not possible to further resolve the origin of the blood meal to species level because of low sequence variation in the cytochrome b gene between artamid species. 30

Blood meals from two Coquillettidia xanthogaster from Brisbane shared 96% sequence homology with a fairy wren (genus Malurus ). Because cytochrome b sequence divergence ≤ 19% is documented in the sister taxon Meliphagidae, 31 it is most likely that the blood sources for these mosquitoes belonged to the family Maluridae, and probably to the genus Malurus (fairy wrens). The remaining Cq. xanthogaster examined fed upon Corvus orru (Corvidae, 100% sequence homology).

Molecular identification of mammalian blood meals.

Mammalian DNA was successfully sequenced from 25 of 32 samples tested. Twenty-three of the 25 samples identified using the mammalian PCR were identified as human in origin (100% sequence homology) (Table 1), highlighting possible low specificity of the human antisera used in the ELISA. Sequence data from the remaining two samples indicated that the samples were either dog or cat in origin (Table 1), each with 100% sequence homology.

DISCUSSION

We investigated host sources of blood meals from common urban mosquitoes, with an emphasis on identifying species that obtain blood meals from birds. This report describes the application of DNA sequencing for the analysis of the vertebrate origin of blood meals of mosquitoes in Australia. It highlights the utility of mitochondrial DNA sequence data for the successful identification of host blood meals, particularly at the species level for avian blood meals.

Because ELISAs are limited by availability and sensitivity of commercially produced antisera, molecular techniques were used to supplement the ELISA in an effort to resolve ambiguous or unidentified samples. However, 21.9% of samples in the current study remained unidentified, despite the use of ELISA and PCR. This was probably attributable to small or partial blood meals or degradation of the blood meal (and target vertebrate DNA) during storage or while exposed to digestive enzymes in the mosquito gut. Alternatively, some unidentified blood meals may be attributed to blood meals acquired from hosts not considered in this study, including amphibians and reptiles. 32

Avian sequence data was used to determine the species origin of most avian blood meals examined. However, in some instances, lack of cytochrome b sequence data for Australian bird species on GenBank limited the capacity of molecular analysis to resolve avian host identity. This limitation may be overcome by inclusion of a larger collection of reference birds or embracing an alternative, standardized DNA-based identification system, as has been described for North American birds. 33 However, much effort is still required to enhance the library of accessible sequence data for birds from Australia.

The inability to obtain sequence data from some samples, despite successful amplification with avian-specific primers, can be explained in a number of ways. First, anti-mosquito behavior of various birds is well documented, 3437 and may interrupt mosquito feeding, thus potentially inducing mosquitoes to feed on multiple bird hosts. Because successful DNA sequencing requires a single DNA template, this method cannot resolve mixed blood meals. Also, as previously discussed, template DNA may have degraded by exposure to digestive enzymes in the mosquito gut or prolonged storage in traps under field conditions.

Aedes aegypti, the only vector of dengue virus on mainland Australia, is known to be highly anthropophilic and previous studies abroad have demonstrated almost exclusive feeding on humans. 3840 The lower proportion of human blood meals we observed may be attributed to limited sensitivity of the human antisera used in the ELISA, and molecular analysis of the unidentified Ae. aegypti samples (42.8% of the total tested) may have increased the observed proportion of blood meals that were identified as human in origin. However, even if the unidentified samples are included in analysis, dog and bird blood meals account for 7.6% (23 of 304) and 3.3% (10 of 304) of the total Ae. aegypti tested, respectively. This finding clearly demonstrates that the Cairns population of Ae. aegypti, although primarily anthropophilic, will feed on other vertebrates, particularly domestic animals. Perhaps this behavior is reflective of host availability because many houses are vacant during daytime hours, inducing Ae. aegypti to feed on alternative hosts.

Of particular interest, this study demonstrates a field association of what is considered a primary Australian flavivirus vector with a suspected key amplifying host species. In three instances, Cx. annulirostris from Sydney was shown to have fed upon Nycticorax caledonicus, the Nankeen (Rufous) night heron. This species (in addition to other Ardeid wading birds) has repeatedly been implicated/incriminated in the transmission cycle of a number of flaviviruses, including KUNV, JEV, and MVEV. 1,16,41,42 Accordingly, Cx. annulirostris is an important arbovirus vector in Australia and is regarded as the major KUNV and MVEV vector across most of Australia. 1,43 Furthermore, this species has been implicated as the primary JEV vector in northern Australia. 4446 An additional blood meal from N. caledonicus was identified from Cx. quinquefasciatus collected in Cairns. This species is also a competent vector of exotic flaviviruses, including JEV and WNV 6,46 and has yielded some field isolates of endemic flaviviruses. 47,48

Catholic feeding behavior has been commonly documented in Cx. annulirostris. Although considered primarily a mammalian feeder, 7,8,10,25,49 bird feeding has been repeatedly reported. Recently, Kay and others8 showed that in some urban localities in Brisbane, ≤ 16% of blood meals from Cx. annulirostris were avian in origin. This finding is comparable to the avian feeding data observed in this study for Cx. annulirostris from Brisbane (15.5%), Cairns (17.6%), and Newcastle (10.3%), although a slightly greater proportion of avian blood meals (27.8% of those identified) was recorded from this species from Sydney.

Sequence data confirmed that a high proportion (75.7%) of the avian blood meals examined from Cx. annulirostris from Sydney, Newcastle, and Brisbane originated from ducks. It is not surprising that a high proportion of the avian blood meals tested from Cx. annulirostris were found to be from waterfowl. Waterfowl are highly abundant near permanent and semi-permanent freshwater, which is the same habitat shared by larval Cx. annulirostris.50 Thus, waterfowl offer a plentiful host source for newly emerged adult mosquitoes. Second, these data may be influenced by the choice of collection sites; most traps were intentionally set near potentially productive larval habitats to ensure adult collections, but placement of mosquito traps near such water bodies will likely increase the probability of collecting mosquitoes that had recently fed upon nearby water birds.

The remaining 78.0% (234 of 300) of blood meals from Cx. annulirostris were identified from a range of mammals, and this species did not show any clear affiliation with any particular mammal species tested. This finding is consistent with findings of previous studies in residential areas of Brisbane that found that this species commonly fed upon a range of available mammals, but with a higher proportion feeding on dogs (42.9%).8 Because mammals, particularly native macropods and possibly other marsupials, are considered amplifying hosts of Ross River virus, 51 this finding highlights the potential for this species to be involved in urban transmission of this virus.

It is pertinent to note that Russell 52 recorded a seasonal shift in feeding behavior of Cx. annulirostris from birds to mammals in a study conducted on the south coast of New South Wales. It would be of particular interest to determine if a similar seasonal shift is observed elsewhere, particularly in urban environments. Similarly, seasonal shifts in the feeding patterns have been documented for other species abroad, including Cx. pipiens53 and Cx. nigripalpus.54 If Australian mosquito species are shown to exhibit seasonal changes in feeding behavior, this finding may markedly influence the ecology of Australian arboviruses in the urban environment, as has been described for WNV in North America. 53

Culex quinquefasciatus is widely distributed across Australia, and previous studies have also demonstrated ornithophilic host-feeding behavior. 7,14,15 Avian feeding rates of up to 80% have been recorded in the rural township of Charleville in southwest Queensland. 15 Using a precipitin test, Kay and others showed that Cx. quinquefasciatus fed predominantly on domestic poultry (Galliformes) within the township. However, the current study shows a marked tendency to feed upon passeriform birds (96% of avian blood meals identified) in major urban areas. This discrepancy probably reflects the relative availability of different bird groups in these regions. However, Cx. quinquefasciatus showed affinity for some bird species in the current study. A total of 77.1% of identified avian blood meals from Cx. quinquefasciatus from Cairns belonged to one of three avian species, namely S. vieilloti (figbird), S. tristis (common myna), and P. buceroides (helmeted friarbird). Each of these species is considered abundant or common within their distribution around the Cairns area. 28 Although the current study did not consider the relative abundance of different bird species, such a high incidence of feeding on these three bird species potentially suggests that Cx. quinquefasciatus may be exhibiting some degree of host preference between avian species.

We have shown which bird species are fed upon by mosquito species in urban environments in Australia. Coupled with the knowledge of amplifying vertebrate hosts, this data can provide important insight into the epidemiology and transmission dynamics of arboviruses in Australia. This study establishes that the ornithophilic mosquito species in Australia examined obtain a high proportion of avian blood meals from only a few species. However, in the absence of bird species abundance data, accurate host preference profiles cannot be implied. Nevertheless, this data may incriminate epidemiologically important vertebrate species that should be considered in future virological host studies.

Table 1

Blood meals identified from mosquitoes collected in urban and peri-urban habitats of Brisbane, Cairns, Newcastle, and Sydney, Australia, using an enzyme-linked immunosorbent assay and/or avian and mammalian group-specific polymerase chain reaction (PCR)

Table 1
Table 2

Bird species from Australia from which reference cytochrome b sequence data were obtained with relevant GenBank accession numbers

Table 2
Table 3

Avian hosts from Australia identified from mosquito blood meals by using cytochrome b sequence analysis

Table 3

*

Address correspondence to Cassie C. Jansen, Australian Biosecurity Cooperative Research Centre for Emerging Infectious Disease and School of Molecular and Microbial Sciences, University of Queensland, St. Lucia, Queensland, 4067, Australia, c/o Virology, Queensland Health Forensic and Scientific Services, 39 Kessels Road, Coopers Plains, Queensland 4108, Australia. E-mail: cassie.jansen@csiro.au

Authors’ addresses: Cassie C. Jansen, Australian Biosecurity Cooperative Research Centre for Emerging Infectious Disease and School of Molecular and Microbial Sciences, University of Queensland, St. Lucia, Queensland, 4067, Australia, c/o Virology, Queensland Health Forensic and Scientific Services, 39 Kessels Road, Coopers Plains, Queensland 4108, Australia, E-mail: cassie.jansen@csiro.au. Cameron E. Webb and Richard C. Russell, Department of Medical Entomology, University of Sydney, Westmead Hospital, Westmead, New South Wales, 2145, Australia, E-mails: Cameron.Webb@swahs.health.nsw.gov.au and rrussell@usyd.edu.au. Glenn C. Graham, Organic Chemistry, Forensic and Scientific Services, Queensland Health, 39 Kessels Road, Coopers Plains, Queensland, 4108, Australia, E-mail: Glenn_Graham@health.qld.gov.au. Scott B. Craig, World Health Organization/World Health Organisation for Animal Health/Food and Agricultural Organization Collaborating Centre for Reference and Research on Leptospirosis, Communicable Diseases Unit, Forensic and Scientific Services, Queensland Health, Coopers Plains, Queensland 4108, Australia, E-mail: Scott_Craig@health.qld.gov.au. Paul Zborowski, School of Molecular and Microbial Sciences, University of Queensland, St. Lucia, Queensland, Australia, E-mail: p.zborowski@uq.edu.au. Scott A. Ritchie, School of Public Health, Tropical Medicine and Rehabilitation Sciences, James Cook University, Smithfield, Queensland, 4878, and Australia and Tropical Population Health Network, Queensland Health, Cairns, Queensland, 4870, Australia, E-mail: Scott_Ritchie@health.qld.gov.au. Andrew F. van den Hurk, Virology, Communicable Diseases Unit, Queensland Health Forensic and Scientific Services, 39 Kessels Road, Coopers Plains, Queensland, 4108, Australia, E-mail: andrew_hurk@health.qld.gov.au.

Acknowledgments: We thank the members of Queensland Health’s Dengue Action Response Team, especially Sharron Long, for field assistance in Cairns; Petrina Johnson for coordinating collection of Ae. aegypti samples; Ian Northcott and Anabelle Olson for supplying reference bird tissue from Cairns; Allan McKinnon (National Parks and Wildlife Service), Michael Pyne (Currumbin Sanctuary), and Heather Janetzski (Queensland Museum) for bird tissue from Brisbane; the Sydney Olympic Park Authority for both reference bird tissue from Sydney and assistance with adult mosquito collections; and Alyssa Pyke and Sonja Hall-Mendelin for technical advice.

Financial support: This study was supported by the Australian Biosecurity Cooperative Research Centre for Emerging Infectious Disease.

REFERENCES

  • 1

    Mackenzie JS, Lindsay MD, Coelen RJ, Broom AK, Hall RA, Smith DW, 1994. Arboviruses causing human disease in the Australasian zoogeographic region. Arch Virol 136 :447–467.

    • Search Google Scholar
    • Export Citation
  • 2

    Russell RC, 2002. Ross River virus: ecology and distribution. Annu Rev Entomol 47 :1–31.

  • 3

    Hanna JN, Ritchie SA, Phillips DA, Serafin IL, Hills SL, van den Hurk AF, Pyke AT, McBride WJ, Amadio MG, Spark RL, 2001. An epidemic of dengue 3 in far north Queensland, 1997–1999. Med J Aust 174 :178–182.

    • Search Google Scholar
    • Export Citation
  • 4

    Ritchie SA, Long S, Smith G, Pyke A, Knox TB, 2004. Entomological investigations in a focus of dengue transmission in Cairns, Queensland, Australia, by using the sticky ovitraps. J Med Entomol 41 :1–4.

    • Search Google Scholar
    • Export Citation
  • 5

    Mackenzie JS, Johansen CA, Ritchie SA, van den Hurk AF, Hall RA, 2002. Japanese encephalitis as an emerging virus: the emergence and spread of Japanese encephalitis virus in Australasia. Curr Top Microbiol Immunol 267 :49–73.

    • Search Google Scholar
    • Export Citation
  • 6

    Jansen CC, Webb CE, Northill JA, Ritchie SA, Russell RC, van den Hurk AF, 2008. Vector competence of Australian mosquito species for a North American strain of West Nile virus. Vector Borne Zoonotic Dis 8 :805–811.

    • Search Google Scholar
    • Export Citation
  • 7

    Kay BH, Boreham PFL, Williams GM, 1979. Host preferences and feeding patterns of mosquitoes at Kowanyama, Cape York Peninsula, northern Queensland. Bull Entomol Res 69 :441–457.

    • Search Google Scholar
    • Export Citation
  • 8

    Kay BH, Boyd AM, Ryan PA, Hall RA, 2007. Mosquito feeding patterns and natural infection of vertebrates with Ross River and Barmah Forest viruses in Brisbane, Australia. Am J Trop Med Hyg 76 :417–423.

    • Search Google Scholar
    • Export Citation
  • 9

    Muller MJ, Murray MD, Edwards JA, 1981. Blood-sucking midges and mosquitoes feeding on mammals at Beatrice Hill, N.T. Aust J Zool 29 :573–588.

    • Search Google Scholar
    • Export Citation
  • 10

    van den Hurk AF, Johansen CA, Zborowski P, Paru R, Foley PN, Beebe NW, Mackenzie JS, Ritchie SA, 2003. Mosquito host-feeding patterns and implications for Japanese encephalitis virus transmission in northern Australia and Papua New Guinea. Med Vet Entomol 17 :403–411.

    • Search Google Scholar
    • Export Citation
  • 11

    Australian Bureau of Statisitics, 2008. Regional Population Growth, Australia 2006–07, Catalog No. 3218.0 and National Regional Profile: Australia. Available at: www.abs.gov.au. Accessed November 12, 2008.

  • 12

    Ritchie SA, Fanning ID, Phillips DA, Standfast HA, McGinn D, Kay BH, 1997. Ross River virus in mosquitoes (Diptera: Culicidae) during the 1994 epidemic around Brisbane, Australia. J Med Entomol 34 :156–159.

    • Search Google Scholar
    • Export Citation
  • 13

    Harley D, Ritchie S, Phillips D, van den Hurk A, 2000. Mosquito isolates of Ross River virus from Cairns, Queensland, Australia. Am J Trop Med Hyg 62 :561–565.

    • Search Google Scholar
    • Export Citation
  • 14

    Lee DJ, Clinton KJ, O’Gower AK, 1954. The blood sources of some Australian mosquitoes. Aust J Biol Sci 7 :282–301.

  • 15

    Kay BH, Boreham PF, Fanning ID, 1985. Host-feeding patterns of Culex annulirostris and other mosquitoes (Diptera: Culicidae) at Charleville, southwestern Queensland, Australia. J Med Entomol 22 :529–535.

    • Search Google Scholar
    • Export Citation
  • 16

    Boyle DB, Dickerman RW, Marshall ID, 1983. Primary viraemia responses of herons to experimental infection with Murray Valley encephalitis, Kunjin and Japanese encephalitis viruses. Aust J Exp Biol Med Sci 61 :655–664.

    • Search Google Scholar
    • Export Citation
  • 17

    Komar N, Langevin S, Hinten S, Nemeth N, Edwards E, Hettler D, Davis B, Bowen R, Bunning M, 2003. Experimental infection of North American birds with the New York 1999 strain of West Nile virus. Emerg Infect Dis 9 :311–322.

    • Search Google Scholar
    • Export Citation
  • 18

    Boakye DA, Tang J, Truc P, Merriweather A, Unnasch TR, 1999. Identification of bloodmeals in haematophagous diptera by cytochrome B heteroduplex analysis. Med Vet Entomol 13 :282–287.

    • Search Google Scholar
    • Export Citation
  • 19

    Apperson CS, Harrison BA, Unnasch TR, Hassan HK, Irby WS, Savage HM, Aspen SE, Watson DW, Rueda LM, Engber BR, Nasci RS, 2002. Host-feeding habits of Culex and other mosquitoes (Diptera: Culicidae) in the Borough of Queens in New York City, with characters and techniques for identification of Culex mosquitoes. J Med Entomol 39 :777–785.

    • Search Google Scholar
    • Export Citation
  • 20

    van den Hurk AF, Smith IL, Smith GA, 2007. Development and evaluation of real-time polymerase chain reaction assays to identify mosquito (Diptera: Culicidae) bloodmeals originating from native Australian mammals. J Med Entomol 44 :85–92.

    • Search Google Scholar
    • Export Citation
  • 21

    Ngo KA, Kramer LD, 2003. Identification of mosquito bloodmeals using polymerase chain reaction (PCR) with order-specific primers. J Med Entomol 40 :215–222.

    • Search Google Scholar
    • Export Citation
  • 22

    Molaei G, Andreadis TG, Armstrong PM, Anderson JF, Vossbrinck CR, 2006. Host feeding patterns of Culex mosquitoes and West Nile virus transmission, northeastern United States. Emerg Infect Dis 12 :468–474.

    • Search Google Scholar
    • Export Citation
  • 23

    Williams CR, Long SA, Russell RC, Ritchie SA, 2006. Field efficacy of the BG-Sentinel compared with CDC Backpack Aspirators and CO2-baited EVS traps for collection of adult Aedes aegypti in Cairns, Queensland, Australia. J Am Mosq Control Assoc 22 :296–300.

    • Search Google Scholar
    • Export Citation
  • 24

    Blackwell A, Mordue AJ, Mordue W, 1994. Identification of blood-meals of the Scottish biting midge, Culicoides impunctatus, by indirect enzyme-linked immunosorbent assay (ELISA). Med Vet Entomol 8 :20–24.

    • Search Google Scholar
    • Export Citation
  • 25

    van den Hurk AF, Nisbet DJ, Johansen CA, Foley PN, Ritchie SA, Mackenzie JS, 2001. Japanese encephalitis on Badu Island, Australia: the first isolation of Japanese encephalitis virus from Culex gelidus in the Australasian region and the role of mosquito host-feeding patterns in virus transmission cycles. Trans R Soc Trop Med Hyg 95 :595–600.

    • Search Google Scholar
    • Export Citation
  • 26

    Cicero C, Johnson NK, 2001. Higher-level phylogeny of new world vireos (aves: vireonidae) based on sequences of multiple mitochondrial DNA genes. Mol Phylogenet Evol 20 :27–40.

    • Search Google Scholar
    • Export Citation
  • 27

    Tamura K, Dudley J, Nei M, Kumar S, 2007. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24 :1596–1599.

    • Search Google Scholar
    • Export Citation
  • 28

    Simpson D, Day N, 2004. Field Guide to the Birds of Australia. London: Christopher Helm.

  • 29

    Johnson KP, Sorenson MD, 1999. Phylogeny and biogeography of dabbling ducks (Genus: Anas): a comparison of molecular and morphological evidence. Auk 116 :792–805.

    • Search Google Scholar
    • Export Citation
  • 30

    Joseph L, Wilke T, Ten Have J, Terry Chesser R, 2006. Implications of mitochondrial DNA polyphyly in two ecologically undifferentiated but morphologically distinct migratory birds, the masked and white-browed woodswallows Artamus spp. of inland Australia. J Avian Biol 37 :625–636.

    • Search Google Scholar
    • Export Citation
  • 31

    Driskell AC, Christidis L, 2004. Phylogeny and evolution of the Australo-Papuan honeyeaters (Passeriformes, Meliphagidae). Mol Phylogenet Evol 31 :943–960.

    • Search Google Scholar
    • Export Citation
  • 32

    Cupp EW, Zhang D, Yue X, Cupp MS, Guyer C, Sprenger TR, Unnasch TR, 2004. Identification of reptilian and amphibian blood meals from mosquitoes in an eastern equine encephalomyelitis virus focus in central Alabama. Am J Trop Med Hyg 71 :272–276.

    • Search Google Scholar
    • Export Citation
  • 33

    Kerr KC, Stoeckle MY, Dove CJ, Weigt LA, Francis CM, Hebert PD, 2007. Comprehensive DNA barcode coverage of North American birds. Mol Ecol Notes 7 :535–543.

    • Search Google Scholar
    • Export Citation
  • 34

    Edman JD, Kale HW, 1971. Host behavior: its influence on the feeding success of mosquitoes. Ann Entomol Soc Am 64 :513–516.

  • 35

    Edman JD, Webber LA, Schmid AA, 1974. Effect of host defenses on the feeding pattern of Culex nigripalpus when offered a choice of blood sources. J Parasitol 60 :874–883.

    • Search Google Scholar
    • Export Citation
  • 36

    Webber LA, Edman JD, 1972. Anti-mosquito behaviour of ciconiiform birds. Anim Behav 20 :228–232.

  • 37

    Darbro JM, Harrington LC, 2007. Avian defensive behaviour and blood-feeding success of the West Nile vector mosqutio, Culex pipiens. Behav Ecol 18 :750–757.

    • Search Google Scholar
    • Export Citation
  • 38

    Scott TW, Chow E, Strickman D, Kittayapong P, Wirtz RA, Lorenz LH, Edman JD, 1993. Blood-feeding patterns of Aedes aegypti (Diptera: Culicidae) collected in a rural Thai village. J Med Entomol 30 :922–927.

    • Search Google Scholar
    • Export Citation
  • 39

    Scott TW, Morrison AC, Lorenz LH, Clark GG, Strickman D, Kittayapong P, Zhou H, Edman JD, 2000. Longitudinal studies of Aedes aegypti (Diptera: Culicidae) in Thailand and Puerto Rico: population dynamics. J Med Entomol 37 :77–88.

    • Search Google Scholar
    • Export Citation
  • 40

    Ponlawat A, Harrington LC, 2005. Blood feeding patterns of Aedes aegypti and Aedes albopictus in Thailand. J Med Entomol 42 :844–849.

  • 41

    Marshall ID, 1988. Murray Valley and Kunjin encephalitis. Monath TP, ed. The Arboviruses: Epidemiology and Ecology. Boca Raton, FL: CRC Press, 151–189.

  • 42

    Boyle DB, Marshall ID, Dickerman RW, 1983. Primary antibody responses of herons to experimental infection with Murray Valley encephalitis, Kunjin and Japanese encephalitis viruses. Aust J Exp Biol Med Sci 61 :665–674.

    • Search Google Scholar
    • Export Citation
  • 43

    Russell RC, 1995. Arboviruses and their vectors in Australia: an update on the ecology and epidemiology of some mosquito-borne arboviruses. Rev Med Vet Entomol 83 :141–158.

    • Search Google Scholar
    • Export Citation
  • 44

    Ritchie SA, Phillips D, Broom A, Mackenzie J, Poidinger M, van den Hurk A, 1997. Isolation of Japanese encephalitis virus from Culex annulirostris in Australia. Am J Trop Med Hyg 56 :80–84.

    • Search Google Scholar
    • Export Citation
  • 45

    Johansen CA, van den Hurk AF, Pyke AT, Zborowski P, Phillips DA, Mackenzie JS, Ritchie SA, 2001. Entomological investigations of an outbreak of Japanese encephalitis virus in the Torres Strait, Australia, in 1998. J Med Entomol 38 :581–588.

    • Search Google Scholar
    • Export Citation
  • 46

    van den Hurk AF, Nisbet DJ, Hall RA, Kay BH, MacKenzie JS, Ritchie SA, 2003. Vector competence of Australian mosquitoes (Diptera: Culicidae) for Japanese encephalitis virus. J Med Entomol 40 :82–90.

    • Search Google Scholar
    • Export Citation
  • 47

    Doherty RL, Carley JG, Kay BH, Filippich C, Marks EN, Frazier CL, 1979. Isolation of virus strains from mosquitoes collected in Queensland, 1972–1976. Aust J Exp Biol Med Sci 57 :509–520.

    • Search Google Scholar
    • Export Citation
  • 48

    Liehne PF, Anderson S, Stanley NF, Liehne CG, Wright AE, Chan KH, Leivers S, Britten DK, Hamilton NP, 1981. Isolation of Murray Valley encephalitis virus and other arboviruses in the Ord River Valley 1972–1976. Aust J Exp Biol Med Sci 59 :347–356.

    • Search Google Scholar
    • Export Citation
  • 49

    van den Hurk AF, Johansen CA, Zborowski P, Phillips DA, Pyke AT, Mackenzie JS, Ritchie SA, 2001. Flaviviruses isolated from mosquitoes collected during the first recorded outbreak of Japanese encephalitis virus on Cape York Peninsula, Australia. Am J Trop Med Hyg 64 :125–130.

    • Search Google Scholar
    • Export Citation
  • 50

    Lee DJ, Hicks MM, Debenham ML, Griffiths M, Russell RC, Bryan JH, Russell RC, Marks EN, 1989. The Culicidae of the Australasian Region. Entomology Monograph No. 2. Canberra: Australian Government Publishing Service Press.

  • 51

    Russell RC, Kay BH, 2004. Medical entomology: changes in the spectrum of mosquito-borne disease in Australia and other vector threats and risks, 1972–2004. Aust J Entomol 43 :271–282.

    • Search Google Scholar
    • Export Citation
  • 52

    Russell RC, 1987. The mosquito fauna of Conjola state forest on the South coast of New South Wales. Part 2. Female feeding behaviour and flight activity. Gen Appl Entomol 19 :17–24.

    • Search Google Scholar
    • Export Citation
  • 53

    Kilpatrick AM, Kramer LD, Jones MJ, Marra PP, Daszak P, 2006. West Nile virus epidemics in North America are driven by shifts in mosquito feeding behavior. PLoS Biol 4 :606–610.

    • Search Google Scholar
    • Export Citation
  • 54

    Edman JD, Taylor DJ, 1968. Culex nigripalpus: seasonal shift in the bird-mammal feeding ratio in a mosquito vector of human encephalitis. Science 161 :67–68.

    • Search Google Scholar
    • Export Citation
  • 55

    Christidis L, Boles WE, 2008. Systematics and Taxonomy of Australian Birds. Collingwood, Australia: Commonwealth Scientific and Industrial Research Organisation Publishing.

  • 56

    Groth JG, 1998. Molecular phylogenetics of finches and sparrows: consequences of character state removal in cytochrome b sequences. Mol Phylogenet Evol 10 :377–390.

    • Search Google Scholar
    • Export Citation
  • 57

    Kennedy M, Gray RD, Spencer HG, 2000. The phylogenetic relationships of the shags and cormorants: can sequence data resolve a disagreement between behavior and morphology? Mol Phylogenet Evol 17 :345–359.

    • Search Google Scholar
    • Export Citation
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