Gottdenker NL, Streicker DG, Faust CL, Carroll CR, 2014. Anthropogenic land use change and infectious diseases: a review of the evidence. Ecohealth 11: 619–632.
Hassell JM, Begon M, Ward MJ, Fèvre EM, 2017. Urbanization and disease emergence: dynamics at the wildlife–livestock–human interface. Trends Ecol Evol 32: 55–67.
Bradley CA, Altizer S, 2007. Urbanization and the ecology of wildlife diseases. Trends Ecol Evol 22: 95–102.
Gong P et al. 2013. Finer resolution observation and monitoring of global land cover: first mapping results with Landsat TM and ETM+ data. Int J Remote Sens 34: 2607–2654.
Richards EE, Masuoka P, Brett-Major D, Smith M, Klein TA, Kim HC, Anyamba A, Grieco J, 2010. The relationship between mosquito abundance and rice field density in the Republic of Korea. Int J Health Geogr 9: 32.
Diuk-Wasser MA, Touré MB, Dolo G, Bagayoko M, Sogoba N, Sissoko I, Traoré SF, Taylor CE, 2007. Effect of rice cultivation patterns on malaria vector abundance in rice-growing villages in Mali. Am J Trop Med Hyg 76: 869–874.
Centers for Disease Control and Prevention (CDC), 2016. West Nile Virus- Total Human Disease Cases. Available at: https://diseasemaps.usgs.gov/mapviewer/. Accessed March 17, 2017.
Kilpatrick AM, 2011. Globalization, land use, and the invasion of West Nile virus. Science 334: 323–327.
LaDeau SL, Kilpatrick AM, Marra PP, 2007. West Nile virus emergence and large-scale declines of North American bird populations. Nature 447: 710–713.
Kilpatrick AM, Peters RJ, Dupuis AP 2nd, Jones MJ, Marra PP, Kramer LD, 2013. Predicted and observed mortality from vector-borne disease in small songbirds. Biol Conserv 165: 79–85.
Wheeler SS, Barker CM, Fang Y, Armijos MV, Carroll BD, Husted S, Johnson WO, Reisen WK, 2009. Differential impact of West Nile virus on California birds. Condor 111: 1–20.
Kilpatrick AM, Kramer LD, Campbell SR, Alleyne EO, Dobson AP, Daszak P, 2005. West Nile virus risk assessment and the bridge vector paradigm. Emerg Infect Dis 11: 425–429.
Kramer LD, Styer LM, Ebel GD, 2008. A global perspective on the epidemiology of West Nile virus. Annu Rev Entomol 53: 61–81.
Kilpatrick AM, Pape WJ, 2013. Predicting human West Nile virus infections with mosquito surveillance data. Am J Epidemiol 178: 829–835.
Paull SH, Horton DE, Ashfaq M, Rastogi D, Kramer LD, Diffenbaugh NS, Kilpatrick AM, 2017. Drought and immunity determine the intensity of West Nile virus epidemics and climate change impacts. Proc Biol Sci 284: pii: 20162078.
Bradley CA, Gibbs SE, Altizer S, 2008. Urban land use predicts West Nile virus exposure in songbirds. Ecol Appl 18: 1083–1092.
Gomez A, Kilpatrick A, Kramer LD, Ii APD, Maffei JG, Goetz SJ, Marra PP, Daszak P, Aguirre AA, 2008. Land use and West Nile virus seroprevalence in wild mammals. Emerg Infect Dis 14: 962–965.
Ruiz MO, Walker ED, Foster ES, Haramis LD, Kitron UD, 2007. Association of West Nile virus illness and urban landscapes in Chicago and Detroit. Int J Health Geogr 6: 10.
Bowden SE, Magori K, Drake JM, 2011. Regional differences in the association between land cover and West Nile virus disease incidence in humans in the United States. Am J Trop Med Hyg 84: 234–238.
Degroote J, Sugumaran R, 2012. National and regional associations between human West Nile virus incidence and demographic, landscape, and land use conditions in the coterminous United States. Vector Borne Zoonotic Dis 12: 657–665.
Degroote J, Degroote JP, Sugumaran R, Ecker M, 2014. Landscape, demographic and climatic associations with human West Nile virus occurrence regionally in 2012 in the United States of America. Geospat Health 9: 153–168.
Rochlin I, Faraji A, Ninivaggi DV, Barker CM, Kilpatrick AM, 2016. Anthropogenic impacts on mosquito populations in North America over the past century. Nat Commun 7: 13604.
Trawinski PR, Mackay DS, 2010. Identification of environmental covariates of West Nile virus vector mosquito population abundance. Vector Borne Zoonotic Dis 10: 515–526.
Landau KI, van Leeuwen WJ, 2012. Fine scale spatial urban land cover factors associated with adult mosquito abundance and risk in Tucson, Arizona. J Vector Ecol 37: 407–418.
Chuang T-W, Hockett CW, Kightlinger L, Wimberly M, 2012. Landscape-level spatial patterns of West Nile virus risk in the northern Great Plains. Am J Trop Med Hyg 86: 734–731.
Eisen L, Barker CM, Moore CG, Pape WJ, Winters AM, Cheronis N, 2010. Irrigated agriculture is an important risk factor for West Nile virus disease in the hyperendemic Larimer-Boulder-Weld area of north central Colorado. J Med Entomol 47: 939–951.
Crowder DW, Dykstra EA, Brauner JM, Duffy A, Reed C, Martin E, Dutilleul P, Owen JP, Peterson W, Carrie Y, 2013. West Nile virus prevalence across landscapes is mediated by local effects of agriculture on vector and host communities. PLoS One 8: e55006.
Schurich JA, Kumar S, Eisen L, Moore CG, 2014. Modeling Culex tarsalis abundance on the northern Colorado front range using a landscape-level approach. J Am Mosq Control Assoc 30: 7–20.
Skaff NK, Cheruvelil KS, 2016. Fine-scale wetland features mediate vector and climate- dependent macroscale patterns in human West Nile virus incidence. Landsc Ecol 31: 1615–1628.
USDA National Agricultural Statistics Service (NASS), 2014. CropScape and Cropland Data Layer. Available at: http://www.nass.usda.gov/Research_and_Science/Cropland/SARS1a.php. Accessed January 11, 2017.
USGS, 2012. Moderate Resolution Imaging Spectroradiometer (MODIS) Irrigated Agriculture Dataset for the United States. Available at: http://earlywarning.usgs.gov/USirrigation. Accessed March 17, 2017.
North America Land Data Assimilation System (NLDAS), 2011. Daily Air Temperatures and Heat Index. Available at: http://wonder.cdc.gov/nasa-nldas.html. Accessed March 1, 2017.
US Census Bureau, 2010. County Human Population. Available at: https://www.census.gov/2010census/data/. Accessed March 17, 2017.
Reisen WK, Lothrop HD, Lothrop B, 2003. Factors influencing the outcome of mark-release-recapture studies with Culex tarsalis (Diptera: Culicidae). J Med Entomol 40: 820–829.
Muggeo MVMR, 2017. Package “segmented”. Biometrika 58: 525–534.
Reisen W, 2012. The contrasting bionomics of Culex mosquitoes in western North America. J Am Mosq Control Assoc 28: 82–91.
Calhoun LM, Avery M, Jones LA, Gunarto K, King R, Roberts J, Burkot TR, Fox M, 2007. Combined sewage overflows (CSO) are major urban breeding sites for Culex quinquefasciatus in Atlanta, Georgia. Am J Trop Med Hyg 77: 478–484.
Chaves LF, Keogh CL, Vazquez-Prokopec GM, Kitron UD, 2009. Combined sewage overflow enhances oviposition of Culex quinquefasciatus (Diptera: Culicidae) in urban areas. J Med Entomol 46: 220–226.
Deichmeister JM, Telang A, 2011. Abundance of West Nile virus mosquito vectors in relation to climate and landscape variables abundance of West Nile virus mosquito vectors in relation to climate. J Vector Ecol 36: 75–85.
Andreadis TG, Anderson JF, Vossbrinck CR, Main AJ, 2004. Epidemiology of West Nile virus in Connecticut: a five-year analysis of mosquito data 1999–2003. Vector Borne Zoonotic Dis 4: 360–378.
Pitcairn M, Wilson L, 1994. Spatial patterns of Anopheles freeborni and Culex tarsalis (Diptera: Culicidae) larvae in California rice fields. J Med Entomol 31: 545–553.
Barker CCM, Eldridge BBF, Reisen WWK, 2010. Seasonal abundance of Culex tarsalis and Culex pipiens complex mosquitoes (Diptera: Culicidae) in California. J Med 47: 759–768.
Thongsripong P, Green A, Kittayapong P, Kapan D, Wilcox B, Bennett S, 2013. Mosquito vector diversity across habitats in central Thailand endemic for dengue and other arthropod-borne diseases. PLoS Negl Trop Dis 7: e2507.
Bisanzio D, Giacobini M, Bertolotti L, Mosca A, Balbo L, Kitron U, Vazquez-Prokopec GM, 2011. Spatio-temporal patterns of distribution of West Nile virus vectors in eastern Piedmont Region, Italy. Parasit Vectors 4: 230.
Reisen W, Lothrop H, 1995. Population ecology and dispersal of Culex tarsalis (Diptera: Culicidae) in the Coachella Valley of California. J Med Entomol 32: 490–502.
Wood B, Washino R, Beck L, Hibbard K, 1991. Distinguishing high and low anopheline-producing rice fields using remote sensing and GIS technologies. Prev Vet Med 11: 277–288.
McDermott E, Mullens B, 2018. The dark side of light traps. J Med Entomol 55: 251–261.
Erlanger TE, Weiss S, Keiser J, Utzinger J, Wiedenmayer K, 2009. Past, present, and future of Japanese encephalitis. Emerg Infect Dis 15: 1–7.
Keiser J, Maltese MF, Erlanger TE, Bos R, Tanner M, Singer BH, Utzinger J, 2005. Effect of irrigated rice agriculture on Japanese encephalitis, including challenges and opportunities for integrated vector management. Acta Trop 95: 40–57.
Amerasinghe FP, Ariyasena TG, 1991. Survey of adult mosquitoes (Diptera: Culicidae) during irrigation development in the Mahaweli project, Sri Lanka. J Med Entomol 28: 387–393.
Samuel P, Ramesh D, Thenmozhi V, Nagaraj J, Muniaraj M, Arunachalam N, 2016. Japanese encephalitis vector abundance and infection frequency in Cuddalore district, Tamil Nadu, India: a five-year longitudinal study. J Entomol Acarol Res 48: 366.
Miller RH, Masuoka P, Klein TA, Kim HC, Somer T, Grieco J, 2012. Ecological niche modeling to estimate the distribution of Japanese encephalitis virus in Asia. PLoS Negl Trop Dis 6: e1678.
Tian HY et al. 2015. How environmental conditions impact mosquito ecology and Japanese encephalitis: an eco-epidemiological approach. Environ Int 79: 17–24.
Guo S, Ling F, Hou J, Wang J, Fu G, Gong Z, 2014. Mosquito surveillance revealed lagged effects of mosquito abundance on mosquito-borne disease transmission: a retrospective study in Zhejiang, China. PLoS One 9: e112975.
FAO, 2017. Food and Agriculture Organization of the United Nations. Available at: http://www.fao.org/faostat/en/#home. Accessed March 17, 2017.
Kilpatrick AM, Salkeld DJ, Titcomb G, Hahn MB, 2017. Conservation of biodiversity as a strategy for improving human health and well-being. Philos Trans R Soc Lond B Biol Sci 372: pii: 20160131.
Past two years | Past Year | Past 30 Days | |
---|---|---|---|
Abstract Views | 238 | 194 | 18 |
Full Text Views | 832 | 20 | 3 |
PDF Downloads | 174 | 21 | 2 |
Anthropogenic land use change, including agriculture, can alter mosquito larval habitat quality, increase mosquito abundance, and increase incidence of vector-borne disease. Rice is a staple food crop for more than half of the world’s population, with ∼1% of global production occurring within the United States (US). Flooded rice fields provide enormous areas of larval habitat for mosquito species and may be hotspots for mosquito-borne pathogens, including West Nile virus (WNV). West Nile virus was introduced into the Americas in 1999 and causes yearly epidemics in the US with an average of approximately 1,400 neuroinvasive cases and 130 deaths per year. We examined correlations between rice cultivation and WNV disease incidence in rice-growing regions within the US. Incidence of WNV disease increased with the fraction of each county under rice cultivation in California but not in the southern US. We show that this is likely due to regional variation in the mosquitoes transmitting WNV. Culex tarsalis was an important vector of WNV in California, and its abundance increased with rice cultivation, whereas in rice-growing areas of the southern US, the dominant WNV vector was Culex quinquefasciatus, which rarely breeds in rice fields. These results illustrate how cultivation of particular crops can increase disease risk and how spatial variation in vector ecology can alter the relationship between land cover and disease.
Financial support: Funding was provided by National Science Foundation grants DEB-1115069 and EF-0914866, National Institutes of Health grant 1R01AI090159, and a postgraduate doctoral scholarship from National Sciences and Engineering Research Council of Canada.
Authors’ addresses: Tony J. Kovach and A. Marm Kilpatrick, Department of Ecology and Evolutionary Biology, University of California Santa Cruz, Santa Cruz, CA, E-mails: tokovach@gmail.com and akilpatr@ucsc.edu.
Gottdenker NL, Streicker DG, Faust CL, Carroll CR, 2014. Anthropogenic land use change and infectious diseases: a review of the evidence. Ecohealth 11: 619–632.
Hassell JM, Begon M, Ward MJ, Fèvre EM, 2017. Urbanization and disease emergence: dynamics at the wildlife–livestock–human interface. Trends Ecol Evol 32: 55–67.
Bradley CA, Altizer S, 2007. Urbanization and the ecology of wildlife diseases. Trends Ecol Evol 22: 95–102.
Gong P et al. 2013. Finer resolution observation and monitoring of global land cover: first mapping results with Landsat TM and ETM+ data. Int J Remote Sens 34: 2607–2654.
Richards EE, Masuoka P, Brett-Major D, Smith M, Klein TA, Kim HC, Anyamba A, Grieco J, 2010. The relationship between mosquito abundance and rice field density in the Republic of Korea. Int J Health Geogr 9: 32.
Diuk-Wasser MA, Touré MB, Dolo G, Bagayoko M, Sogoba N, Sissoko I, Traoré SF, Taylor CE, 2007. Effect of rice cultivation patterns on malaria vector abundance in rice-growing villages in Mali. Am J Trop Med Hyg 76: 869–874.
Centers for Disease Control and Prevention (CDC), 2016. West Nile Virus- Total Human Disease Cases. Available at: https://diseasemaps.usgs.gov/mapviewer/. Accessed March 17, 2017.
Kilpatrick AM, 2011. Globalization, land use, and the invasion of West Nile virus. Science 334: 323–327.
LaDeau SL, Kilpatrick AM, Marra PP, 2007. West Nile virus emergence and large-scale declines of North American bird populations. Nature 447: 710–713.
Kilpatrick AM, Peters RJ, Dupuis AP 2nd, Jones MJ, Marra PP, Kramer LD, 2013. Predicted and observed mortality from vector-borne disease in small songbirds. Biol Conserv 165: 79–85.
Wheeler SS, Barker CM, Fang Y, Armijos MV, Carroll BD, Husted S, Johnson WO, Reisen WK, 2009. Differential impact of West Nile virus on California birds. Condor 111: 1–20.
Kilpatrick AM, Kramer LD, Campbell SR, Alleyne EO, Dobson AP, Daszak P, 2005. West Nile virus risk assessment and the bridge vector paradigm. Emerg Infect Dis 11: 425–429.
Kramer LD, Styer LM, Ebel GD, 2008. A global perspective on the epidemiology of West Nile virus. Annu Rev Entomol 53: 61–81.
Kilpatrick AM, Pape WJ, 2013. Predicting human West Nile virus infections with mosquito surveillance data. Am J Epidemiol 178: 829–835.
Paull SH, Horton DE, Ashfaq M, Rastogi D, Kramer LD, Diffenbaugh NS, Kilpatrick AM, 2017. Drought and immunity determine the intensity of West Nile virus epidemics and climate change impacts. Proc Biol Sci 284: pii: 20162078.
Bradley CA, Gibbs SE, Altizer S, 2008. Urban land use predicts West Nile virus exposure in songbirds. Ecol Appl 18: 1083–1092.
Gomez A, Kilpatrick A, Kramer LD, Ii APD, Maffei JG, Goetz SJ, Marra PP, Daszak P, Aguirre AA, 2008. Land use and West Nile virus seroprevalence in wild mammals. Emerg Infect Dis 14: 962–965.
Ruiz MO, Walker ED, Foster ES, Haramis LD, Kitron UD, 2007. Association of West Nile virus illness and urban landscapes in Chicago and Detroit. Int J Health Geogr 6: 10.
Bowden SE, Magori K, Drake JM, 2011. Regional differences in the association between land cover and West Nile virus disease incidence in humans in the United States. Am J Trop Med Hyg 84: 234–238.
Degroote J, Sugumaran R, 2012. National and regional associations between human West Nile virus incidence and demographic, landscape, and land use conditions in the coterminous United States. Vector Borne Zoonotic Dis 12: 657–665.
Degroote J, Degroote JP, Sugumaran R, Ecker M, 2014. Landscape, demographic and climatic associations with human West Nile virus occurrence regionally in 2012 in the United States of America. Geospat Health 9: 153–168.
Rochlin I, Faraji A, Ninivaggi DV, Barker CM, Kilpatrick AM, 2016. Anthropogenic impacts on mosquito populations in North America over the past century. Nat Commun 7: 13604.
Trawinski PR, Mackay DS, 2010. Identification of environmental covariates of West Nile virus vector mosquito population abundance. Vector Borne Zoonotic Dis 10: 515–526.
Landau KI, van Leeuwen WJ, 2012. Fine scale spatial urban land cover factors associated with adult mosquito abundance and risk in Tucson, Arizona. J Vector Ecol 37: 407–418.
Chuang T-W, Hockett CW, Kightlinger L, Wimberly M, 2012. Landscape-level spatial patterns of West Nile virus risk in the northern Great Plains. Am J Trop Med Hyg 86: 734–731.
Eisen L, Barker CM, Moore CG, Pape WJ, Winters AM, Cheronis N, 2010. Irrigated agriculture is an important risk factor for West Nile virus disease in the hyperendemic Larimer-Boulder-Weld area of north central Colorado. J Med Entomol 47: 939–951.
Crowder DW, Dykstra EA, Brauner JM, Duffy A, Reed C, Martin E, Dutilleul P, Owen JP, Peterson W, Carrie Y, 2013. West Nile virus prevalence across landscapes is mediated by local effects of agriculture on vector and host communities. PLoS One 8: e55006.
Schurich JA, Kumar S, Eisen L, Moore CG, 2014. Modeling Culex tarsalis abundance on the northern Colorado front range using a landscape-level approach. J Am Mosq Control Assoc 30: 7–20.
Skaff NK, Cheruvelil KS, 2016. Fine-scale wetland features mediate vector and climate- dependent macroscale patterns in human West Nile virus incidence. Landsc Ecol 31: 1615–1628.
USDA National Agricultural Statistics Service (NASS), 2014. CropScape and Cropland Data Layer. Available at: http://www.nass.usda.gov/Research_and_Science/Cropland/SARS1a.php. Accessed January 11, 2017.
USGS, 2012. Moderate Resolution Imaging Spectroradiometer (MODIS) Irrigated Agriculture Dataset for the United States. Available at: http://earlywarning.usgs.gov/USirrigation. Accessed March 17, 2017.
North America Land Data Assimilation System (NLDAS), 2011. Daily Air Temperatures and Heat Index. Available at: http://wonder.cdc.gov/nasa-nldas.html. Accessed March 1, 2017.
US Census Bureau, 2010. County Human Population. Available at: https://www.census.gov/2010census/data/. Accessed March 17, 2017.
Reisen WK, Lothrop HD, Lothrop B, 2003. Factors influencing the outcome of mark-release-recapture studies with Culex tarsalis (Diptera: Culicidae). J Med Entomol 40: 820–829.
Muggeo MVMR, 2017. Package “segmented”. Biometrika 58: 525–534.
Reisen W, 2012. The contrasting bionomics of Culex mosquitoes in western North America. J Am Mosq Control Assoc 28: 82–91.
Calhoun LM, Avery M, Jones LA, Gunarto K, King R, Roberts J, Burkot TR, Fox M, 2007. Combined sewage overflows (CSO) are major urban breeding sites for Culex quinquefasciatus in Atlanta, Georgia. Am J Trop Med Hyg 77: 478–484.
Chaves LF, Keogh CL, Vazquez-Prokopec GM, Kitron UD, 2009. Combined sewage overflow enhances oviposition of Culex quinquefasciatus (Diptera: Culicidae) in urban areas. J Med Entomol 46: 220–226.
Deichmeister JM, Telang A, 2011. Abundance of West Nile virus mosquito vectors in relation to climate and landscape variables abundance of West Nile virus mosquito vectors in relation to climate. J Vector Ecol 36: 75–85.
Andreadis TG, Anderson JF, Vossbrinck CR, Main AJ, 2004. Epidemiology of West Nile virus in Connecticut: a five-year analysis of mosquito data 1999–2003. Vector Borne Zoonotic Dis 4: 360–378.
Pitcairn M, Wilson L, 1994. Spatial patterns of Anopheles freeborni and Culex tarsalis (Diptera: Culicidae) larvae in California rice fields. J Med Entomol 31: 545–553.
Barker CCM, Eldridge BBF, Reisen WWK, 2010. Seasonal abundance of Culex tarsalis and Culex pipiens complex mosquitoes (Diptera: Culicidae) in California. J Med 47: 759–768.
Thongsripong P, Green A, Kittayapong P, Kapan D, Wilcox B, Bennett S, 2013. Mosquito vector diversity across habitats in central Thailand endemic for dengue and other arthropod-borne diseases. PLoS Negl Trop Dis 7: e2507.
Bisanzio D, Giacobini M, Bertolotti L, Mosca A, Balbo L, Kitron U, Vazquez-Prokopec GM, 2011. Spatio-temporal patterns of distribution of West Nile virus vectors in eastern Piedmont Region, Italy. Parasit Vectors 4: 230.
Reisen W, Lothrop H, 1995. Population ecology and dispersal of Culex tarsalis (Diptera: Culicidae) in the Coachella Valley of California. J Med Entomol 32: 490–502.
Wood B, Washino R, Beck L, Hibbard K, 1991. Distinguishing high and low anopheline-producing rice fields using remote sensing and GIS technologies. Prev Vet Med 11: 277–288.
McDermott E, Mullens B, 2018. The dark side of light traps. J Med Entomol 55: 251–261.
Erlanger TE, Weiss S, Keiser J, Utzinger J, Wiedenmayer K, 2009. Past, present, and future of Japanese encephalitis. Emerg Infect Dis 15: 1–7.
Keiser J, Maltese MF, Erlanger TE, Bos R, Tanner M, Singer BH, Utzinger J, 2005. Effect of irrigated rice agriculture on Japanese encephalitis, including challenges and opportunities for integrated vector management. Acta Trop 95: 40–57.
Amerasinghe FP, Ariyasena TG, 1991. Survey of adult mosquitoes (Diptera: Culicidae) during irrigation development in the Mahaweli project, Sri Lanka. J Med Entomol 28: 387–393.
Samuel P, Ramesh D, Thenmozhi V, Nagaraj J, Muniaraj M, Arunachalam N, 2016. Japanese encephalitis vector abundance and infection frequency in Cuddalore district, Tamil Nadu, India: a five-year longitudinal study. J Entomol Acarol Res 48: 366.
Miller RH, Masuoka P, Klein TA, Kim HC, Somer T, Grieco J, 2012. Ecological niche modeling to estimate the distribution of Japanese encephalitis virus in Asia. PLoS Negl Trop Dis 6: e1678.
Tian HY et al. 2015. How environmental conditions impact mosquito ecology and Japanese encephalitis: an eco-epidemiological approach. Environ Int 79: 17–24.
Guo S, Ling F, Hou J, Wang J, Fu G, Gong Z, 2014. Mosquito surveillance revealed lagged effects of mosquito abundance on mosquito-borne disease transmission: a retrospective study in Zhejiang, China. PLoS One 9: e112975.
FAO, 2017. Food and Agriculture Organization of the United Nations. Available at: http://www.fao.org/faostat/en/#home. Accessed March 17, 2017.
Kilpatrick AM, Salkeld DJ, Titcomb G, Hahn MB, 2017. Conservation of biodiversity as a strategy for improving human health and well-being. Philos Trans R Soc Lond B Biol Sci 372: pii: 20160131.
Past two years | Past Year | Past 30 Days | |
---|---|---|---|
Abstract Views | 238 | 194 | 18 |
Full Text Views | 832 | 20 | 3 |
PDF Downloads | 174 | 21 | 2 |