culex mosquitoes are vectors of pathogens including the human filarial nematode , wuchereria bancrofti , and encephalitis - causing viruses , such as st . louis , japanese , venezuela equine , western equine encephalitis , and west nile virus ( nasci and miller , 1996).1 given the resistance of culex populations to modern insecticides , alternative methods of controls are sorely needed . larval development is a particularly vulnerable phase in the life cycle of culex mosquitoes , as eggs are laid in rafts from which hundreds of larvae emerge in confined areas  thus facilitating management . skatole , a natural product found in animal excreta and also a product of fermentation of organic material , has been identified as an oviposition attractant for the southern house mosquito , culex quinquefasciatus ( millar et al . , 1992 ) . field studies have demonstrated that traps baited with optimal doses of skatole collected significantly more eggs ( mboera et al . , 2000 ) and gravid females ( leal et al . , 2008 ) than control traps , thus suggesting that in combination with a biological agent , bacillus thuringiensis var . israelensis ( barbosa et al . , 2010 ) oviposition attractants may be used in  attract - and - kill  strategies . chemical ecology and olfaction are the pillars of these semiochemical - based , environmentally friendly strategies . therefore , identification of olfactory proteins involved in the reception of these semiochemicals may open the door for development of better oviposition attractants . recently , we demonstrated by rna interference that an odorant - binding protein ( obp ) from cx . quinquefasciatus , cquiobp1 , is involved directly in the reception of skatole and other oviposition attractants ( pelletier et al . , 2010a ) . we also have characterized an odorant receptor ( or ) from this mosquito species , cquior2 , which is highly sensitive to indole and moderately sensitive to skatole ( pelletier et al . , 2010b ) . here , we characterize cquior10 and show this or to be highly sensitive and narrowly tuned to skatole . expression of cquior10 in the xenopus oocyte system oocytes were prepared as previously described ( pelletier et al . , 2010b ) . cquior10 and cquior7 , initially cloned into pbluescript ( pelletier et al . , 2010b ) , were transferred to pgemhe for use as templates for synthesis of capped crna by using mmessage mmachine kits ( ambion ) . twenty - five ng of crna encoding each or subunit were injected into stage v  vi xenopus oocytes . oocytes were incubated at 18c in barth s saline ( in mm : 88 nacl , 1 kcl , 2.4 nahco3 , 0.3 cano3 , 0.41 cacl2 , 0.82 mgso4 , 15 hepes , ph 7.6 , and 100  g / ml amikacin ) for 25 d prior to electrophysiological recording . electrophysiology and data analysis odorant - induced currents were recorded under two - electrode voltage clamp from oocytes expressing ors , by using an automated parallel electrophysiology system ( opusxpress 6000a ; molecular devices ) . oocytes were perfused with nd96 ( in mm : 96 nacl , 2 kcl , 1 cacl2 , 1 mgcl2 , 5 hepes , ph 7.5 ) . odorants were diluted in nd96 and applied for 20  sec at a flow rate of 1.65  ml / min with extensive washing in nd96 ( 520  min at 4.6  ml / min ) between applications . current responses approached a plateau during the 20  sec application ( pelletier et al . , 2010b ) . micropipettes were filled with 3  m kcl and had resistances of 0.22.0  m. the holding potential was 70  mv . current responses were filtered ( 4-pole , bessel , low pass ) at 20  hz ( 3  db ) , sampled at 100  hz , and were captured and stored with opusxpress 1.1 software ( molecular devices ) . initial analysis of electrophysiological data was done with clampfit 9.1 software ( molecular devices ) . curve fitting of concentration - response data was done with prism 4 ( graphpad ) . in our search for molecular targets that may be used in a reverse chemical ecology approach for the development of better oviposition attractants ( leal et al . , 2008 ) , we recently have mined the genome of cx . quinquefasciatus and identified an or sensitive to indole , cquior2 , which also responded to methylindoles , including skatole ( iupac name , 3-methylindole ) . by mapping the antennae of female cx . quinquefasciatus , we previously observed that a skatole - detecting orn also responded to geranylacetone and ethyl hexanoate , but not indole ( syed and leal , 2009 ) . these observations prompted us to examine the odorant response profile of cquior10 , an or closely related to cquior2 ( pelletier et al . . full - length coding sequence of cquior10 and the obligatory co - receptor cquior7 ( pelletier et al . , 2010b ) were cloned in pgemhe for heterologous expression in xenopus oocytes . to identify the best ligand for this receptor , oocytes expressing cquior10 + cquior7 were screened first with a panel of odorants ( fig . 1a ) , each applied for 20  sec at a concentration of 10  m with extensive washing between applications . skatole ( 3-methylindole ) elicited the largest current responses , but the receptor also responded with lower sensitivity to indole , other methylindoles , and 2-methylphenol . interestingly , cquior10 was unresponsive to many compounds in the test panel of 23 odorants , including other oviposition attractants such as trimethylamine , nonanal , and the mosquito oviposition pheromone ( mop ) ( leal et al . , 2008 ) . each odorant was applied at a concentration of 10  m for 20  sec with 10  min washes between applications . all responses are normalized to the response of the same oocyte to 10  m indole ( mean  sem , n  =  45 ) . b oocytes expressing cquior10 + cquior7 were challenged with a range of concentrations of 3-methylindole ( top trace ) , indole ( middle trace ) or 2-methylphenol ( bottom trace ) . each odorant was applied for 20  sec with 520  min washes between applications . note different scales : from top to bottom 500 , 200 and 400  na . c oocytes expressing cquior10 + cquior7 were challenged with a range of concentrations of 3-methylindole , indole , and 2-methylphenol . responses were normalized to the response of each oocyte to 10  m indole and are presented as mean  sem ( n  =  35 for each odorant tested ) . data were fit to the equation : \documentclass[12pt]{minimal } 
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				 \begin{document}$$ { \hbox{i } } = { { \hbox{i } } _ { { \max } } } /\left ( { { 1 } + { { \left ( { { \hbox{e}}{{\hbox{c}}_{{5}0}}/{\hbox{x } } } \right)}^{\rm{n } } } } \right ) $ $ \end{document } where i represents the current response at a given concentration of odorant ( x ) , imax is the maximal response , ec50 is the concentration of odorant yielding a half maximal response , and n is the apparent hill coefficient . derived values are : 3-methylindole , ec50  =  90    17  nm , n  =  1.0    0.1 ; indole , ec50  =  2.4    0.3  m , n  =  1.1    0.2 ; 2-methylphenol , ec50  =  41    7  m , n  =  1.0    0.1 odorant receptor cquior10 is highly sensitive to skatole ( 3-methylindole ) . each odorant was applied at a concentration of 10  m for 20  sec with 10  min washes between applications . all responses are normalized to the response of the same oocyte to 10  m indole ( mean  sem , n  =  45 ) . b oocytes expressing cquior10 + cquior7 were challenged with a range of concentrations of 3-methylindole ( top trace ) , indole ( middle trace ) or 2-methylphenol ( bottom trace ) . each odorant was applied for 20  sec with 520  min washes between applications . note different scales : from top to bottom 500 , 200 and 400  na . c oocytes expressing cquior10 + cquior7 were challenged with a range of concentrations of 3-methylindole , indole , and 2-methylphenol . responses were normalized to the response of each oocyte to 10  m indole and are presented as mean  sem ( n  =  35 for each odorant tested ) . data were fit to the equation : \documentclass[12pt]{minimal } 
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				 \begin{document}$$ { \hbox{i } } = { { \hbox{i } } _ { { \max } } } /\left ( { { 1 } + { { \left ( { { \hbox{e}}{{\hbox{c}}_{{5}0}}/{\hbox{x } } } \right)}^{\rm{n } } } } \right ) $ $ \end{document } where i represents the current response at a given concentration of odorant ( x ) , imax is the maximal response , ec50 is the concentration of odorant yielding a half maximal response , and n is the apparent hill coefficient . derived values are : 3-methylindole , ec50  =  90    17  nm , n  =  1.0    0.1 ; indole , ec50  =  2.4    0.3  m , n  =  1.1    0.2 ; 2-methylphenol , ec50  =  41    7  m , n  =  1.0    0.1 next , we performed concentration - response analyses for skatole and two other ligands , indole and 2-methylphenol , which were identified as the best ligands among the indoles and phenols , respectively , for the related receptor cquior2 ( pelletier et al . , 2010b ) . skatole was the most potent of these compounds , activating the cquior10 + cquior7 receptor with an ec50 of 90  nm . indole and 2-methylphenol were less potent , activating cquior10 + cquior7 with ec50 values of 2.4  m and 41  m , respectively . interestingly , indole and 2-methylphenol also displayed lower efficacy ( maximal response ) than skatole ( 40    2% and 69    3% of skatole , respectively ) . in addition , the response threshold for skatole was two to three orders of magnitude higher than that observed for indole and 2-methylphenol ( fig . thus , we found that heterologously expressed cquior10 is highly sensitive and narrowly tuned to the oviposition attractant skatole . in female antennae of the southern house mosquito , skatole is detected by a small - spike - amplitude orn housed in a1 sensilla ( syed and leal , 2009 ) , which is also sensitive to a lower degree to geranylacetone and ethyl hexanoate , but does not respond to indole or 2-methylphenol [ see figs . s5 , s6 , supporting information in ( syed and leal , 2009 ) ] . in the xenopus oocyte system , although expression of ors in heterologous systems , such as the xenopus oocyte system , is an invaluable tool for de - orphanizing and characterizing receptors , it does not completely mimic the insect olfactory system . typically , these systems are devoid of obps , odorant - degrading enzymes , sensory neuron membrane proteins , and other olfactory proteins that may play a part in the selectivity and sensitivity of the olfactory system . thus , it is conceivable that heterologously expressed cquior10 and the receptor in its native environment differ in the detection of geranylacetone because the former is devoid of obps . however , one can not rule out the possibility that a separate orn responding to geranylacetone has the same spike amplitude as the skatole - detecting orn ( syed and leal , 2009 ) , thus rendering them indistinguishable by single unit recordings . alternatively , the same small - spike neuron sensitive to skatole may express another or along with cquior10 . in marked contrast to the mammalian olfactory system , co - expression of ors has been documented in drosophila melanogaster . co - expression of cquior10 and another or would not be entirely surprising given the number of putative odorant receptors in the southern house mosquito genome ( pelletier et al . , 2010b ) and the number of orns in their sensory system ( syed and leal , 2007 , 2008 ) . future research aimed at testing these three hypotheses might lead to deeper understanding of odorant reception in mosquitoes .
oviposition attractants are environmental cues that allow culex gravid female mosquitoes to locate suitable sites for egg - laying and , therefore , may be exploited for environmentally friendly strategies for controlling mosquito populations . 
 naturally occurring skatole has been identified as an oviposition attractant for the southern house mosquito , culex quinquefasciatus . 
 previously , we identified in cx . 
 quinquefasciatus female antennae an olfactory receptor neuron ( orn ) highly sensitive to skatole and an odorant - binding protein involved in the detection of this semiochemical . here 
 , we describe the characterization of an odorant receptor ( or ) , cquior10 , which is narrowly tuned to skatole when expressed in the xenopus oocyte system . 
 odorant - induced response profiles generated by heterologously expressed cquior10 suggest that this or is expressed in the mosquito orn sensitive to skatole . 
 however , geranylacetone , which stimulates the antennal orn , was not detected by cquior10-expressing oocytes , thus raising interesting questions about reception of oviposition attractants in mosquitoes .