
 insects - resistant population - samples from the el mal field 
 population of t. infestans were collected in november 2010 from 
 infested houses in the chaco province of argentina ( s2556.077 w6027.105 ) , where 
 vector control using pyrethroid insecticides is considered ineffective by the 
 authorities responsible for the chagas program of chaco province .
field - collected 
 insects were transported to the research center of pest and insecticides ( cipein ) 
 laboratory and further generations of these insects were bred in the laboratory .
the 50% 
 lethal dose ( ld50 ) obtained for deltamethrin for this population is 134 
 ng / insect , with a high resistance ratio of 1,031 ( carvajal et al .
a study performed at cipein has shown that eight years 
 after the most recent exposure to deltamethrin , the ld50 did not vary in a 
 population from northern argentina ( germano 
 2013 ) . 

susceptible population - for comparison , we used a susceptible 
 reference colony , nfs , derived from a domestic field population collected in december 
 2004 from santiago del estero , argentina .
this laboratory colony has been maintained without the 
 introduction of new insects from external sources .
a laboratory test with nfs has shown 
 an ld50 of 0.13 ( 0.11 - 0.15 ) ng / i for deltamethrin .
this value did not differ 
 statistically from that of the traditional laboratory - susceptible strain maintained at 
 cipein ( roca acevedo et al .
, each population was kept in enclosed boxes ( 30 x 30 x 30 cm ) at 28  1c 
 and 50 - 60% relative humidity with a photoperiod of 12:12 h ( l : d ) .
a pigeon was provided 
 weekly as a blood meal source ( who 1994 ) . 

chemicals - technical grade imidacloprid ( 98% ) provided by dr 
 ehrrenstorfer ( augsburg , germany ) was used in topical application bioassays . 

analytical grade acetone was purchased from jt baker ( san pedro xalostoc , 
 mexico ) . in the surface assays and spot - on bioassays ,
the following formulations of imidacloprid 
 were used : 35% emulsifiable concentrates ( ec ) ( mamboret confi and 
 ec - chemotcnica ( argentina ) , 70% wettable granule ( wg ) ( bayer 
 confidor , argentina ) and 10% spot - on ( bayer advantage g , 
 argentina ) .
moreover , 2.5% ec deltamethrin ( bayer k - othrine , argentina ) was 
 used only in surface bioassays . 

topical application bioassays - t. infestans first instars ( 5 - 7 days 
 old ) that had been starved since eclosion were selected for toxicity tests according to 
 the world health organization protocol ( who 1994 ) . the bioassays consisted of the 
 topical application of 0.2 l of the insecticide diluted in acetone on the dorsal 
 abdomen of the first instar using a 10 l hamilton syringe equipped with an automatic 
 dispenser . in the evaluation of blood feeding ,
nymphs were previously fed to repletion 
 on a pigeon and the insecticide was applied immediately after feeding .
the final concentrations of imidacloprid tested 
 ranged from 0.0025 - 0.5 mg / ml . all concentrations were replicated at least three times 
 with a minimum of 10 insects per replicate . to calculate the ld values at p < 0.05 ,
a 
 minimum of 30 insects per concentration was required and mortality values between 10 - 90% 
 were observed ( robertson et al .
mortality was evaluated after 24 h and also 
 after 48 h and 72 h in the delayed toxicity assays , by placing the insects on a circular 
 piece of filter paper ( 11 cm diameter ) and observing their ability to walk . only nymphs 
 that were able to walk from the centre of the filter paper to the border
evaluation of formulated insecticides - 
 on glass - insecticides were applied to a square area ( 96 
 cm ) using a 1 ml pipette and a constant flow to achieve uniform 
 impregnation .
the treated surface was dried for 24 h. each replicate consisted of a 
 negative control group ( water ) , a positive control group [ ec 2.5% deltamethrin in water 
 at 25 mg active ingredient ( ai)/m ] and one or more doses of formulated 
 imidacloprid , ranging from 1,000 - 5,000 mg ai / m .
groups of 10 nymphs ( 3rd - instar nymphs aged 10 - 20 days , starved since last moult ) were 
 confined in glass rings and exposed for 1 h to the treated surfaces . after exposure
, the 
 insects were placed in clean flasks with filter paper and were maintained under the 
 laboratory conditions described previously .
mortality was recorded after 24 h ( germano et al . 2014 ) . 
 on filter paper - circular disks of whatman n 1 filter paper ( 5.5 cm 
 diameter )
the papers 
 ( area 23.75 cm ) were homogeneously impregnated with 0.1 ml of the formulated 
 insecticide .
the control groups were exposed to filter paper 
 homogeneously impregnated with 0.1 ml of pure water .
after 1 h , when the solvent had 
 evaporated , the insects ( 10 1st - instar nymphs per group selected as in topical 
 application bioassays ) were held in contact with the treated surface for 1 h. after this 
 period , live insects were placed in clean flasks and were maintained under the 
 laboratory conditions described earlier .
mortality was recorded after 24 h ( rojas de 
 arias & fournet 2002 ) . 
 on pigeons
- selected pigeons were treated with various doses of 
 imidacloprid advantage g. the average weight of the pigeons was 252.7  39.4 
 g. twenty - one pigeons were used .
the experimental design included five groups ( 4 treated 
 and 1 control ) with a minimum of three pigeons per group .
prior to the application of 
 the spot - on , we removed several feathers from the pigeon to produce a  blind spot  or 
 arena where the insects could feed .
the insecticide was applied to the base of the neck 
 with a needle - less syringe .
the insects were then exposed in the area where the spot - on 
 formulation was applied . we have previously determined that this area was the most 
 feasible site because the insects were removed easily without any additional disturbance 
 to the treated pigeon .
the pigeon groups were treated with 1 , 5 , 20 and 40 mg / ai of the formulation ( i.e. , = 4 , 
 20 , 80 and 160 mg / ai per kg of animal ) .
the control group was manipulated similarly , 
 without the addition of any insecticide , but water .
synchronised insects ( 1st and 3rd - instar ) , previously starved for 15 - 20 days , were 
 allowed to feed for 30 min on the treated area of the pigeons .
the residual effect of 
 the drug was studied on feeding nymphs at different intervals of time ( 1 , 7 , 14 and 21 
 days post spot - on application ) after the administration of the drug .
the tests were performed 
 by allowing the insects housed in jars containing 10 - 30 insects to feed on the pigeon .
a 
 total of 1,054 and 717 first and third - instar nymphs were used , respectively .
the fed 
 insects were transferred to clean flasks with filter paper and kept under the laboratory 
 conditions described previously .
mortality was recorded after 24 h. 
 statistical analysis - in the topical application and surface 
 bioassays , mortality data were analysed using polo plus software v.2.0 .
dose - mortality 
 data were subjected to a probit analysis to estimate the ld ( ng / insect ) required to kill 
 50% of the treated individuals ( ld50 ) . in the spot - on bioassays ,
all mortality data were corrected for control mortality with 
 abbott s equation ( abbott 1925 ) .
the percentage 
 mortality was determined and transformed to arcsine square - root values for an anova . 

a 0.05 
 significance level was chosen as the criterion for biological significance among related 
 treatments .

 evaluation of delayed toxicity and influence of the blood feeding state 
 - the ld50 values for the susceptible ( s ) and resistant ( r ) 
 populations were 5.2 ( 3.4 - 7.8 ) and 9.2 ( 7.4 - 11.2 ) ng / insect , respectively , and did not 
 differ up to 72 h after the initial topical application .
we also studied the variation in the toxic effects of imidacloprid relative to the blood 
 feeding condition of the insect because the cuticular distention resulting from blood 
 feeding facilitates the penetration of the insecticide .
the blood feeding condition 
 ( starvation / feeding ) of the insects had no significant influence on the insecticidal 
 activity of the imidacloprid in either population ( table ) . 

table insecticidal activity of imidacloprid over starved and fed nymphs i of 
 triatoma infestans at 24 hpopulationblood feedingld50 
 confidencestate(ng / insect)limits 
 ( ng / insect)susceptiblestarved5.23.4 - 7.8fed42.4 - 7resistantstarved9.27.4 - 11.2fed10.86.4 - 19ld : lethal dose . 
 

formulation - surfaces - four formulations of imidacloprid were tested 
 against first and third - instar t. infestans on two different surfaces : 
 filter paper and glass .
the ecs chemotcnica and mamboret confi and the wg 
 confidor showed no effects , either on glass against third - instar nymphs 
 ( at 1,000 - 5,000 mg ai / m ) or on filter paper against first - instar nymphs ( at 
 a 100 mg / ml dose ) .
the spot - on advantage g could not be tested on glass because it did not form 
 a film after 24 h of drying . on filter paper ,
advantage g was effective , 
 with a mortality of 100% with a 100 mg / ml dose in both susceptible and resistant 
 populations .
the lc50 obtained for advantage g on filter paper 
 was 22.84 ( 14.94 - 36.89)mg / ml . in contrast , a 10 mg / ml dose of deltamethrin on filter paper caused 100% mortality in 
 the susceptible population , whereas no mortality ( 0% ) was found in the resistant 
 population . 

spot - on on pigeons - a first evaluation of formulations of imidacloprid 
 on glass and filter paper showed that only the spot - on formulation was effective . 

accordingly , we analysed the effect of the spot - on formulation of imidacloprid by 
 applying different doses of the insecticide to pigeons
. the applied dose of 1 mg / ai showed no lethal effects against first and third - instar 
 t. infestans ( p > 0.05 ) .
twenty - four hours after the application of 5 mg / ai to pigeons , nymphs that had fed on 
 the pigeons showed a higher mortality rate ( 49.8  1% and 40.5  18% for first and 
 third - instar , respectively ) than the control group ( p < 0.01 ) .
nymphs fed seven days 
 after a spot - on application did not show significant differences in mortality between 
 the treated and control groups ( p > 0.05 ) .
nymphs fed 14 and 21 days after spot - on application did not show significant differences 
 in mortality between the treated and control groups at any studied dose or nymphal stage 
 ( p > 0.05 ) .
both first and third - instar nymphs fed on pigeons that had been treated with 20 mg or 40 
 mg of the formulation showed a higher mortality rate than the control group one and 
 seven days post - treatment ( p < 0.01 ) . the residual effect ( 7 days 
 after treatment ) was higher for 40 mg than for 20 mg ( p < 0.01 )
the lethal effect was similar against first and third - instar nymphs at all doses and 
 time intervals ( a , b in figure ) . 

mortality of triatoma infestans fed at different intervals 
 after the spot - on application .
bars with different letters are significantly different ( p  0.05 ) within each 
 day . 

although the pyrethroids deltamethrin and -cia - lothrin are available , the only 
 insecticides approved by the health service of argentina for use in the field control of 
 t. infestans are the organophosphates fenitrothion and malathion . 

these insecticides were used to control t. infestans in the 70 s , but 
 because of their high toxicity in mammals , strong odour and tendency to leave stains on 
 the walls after application they were replaced by pyrethroids in the 80 s ( schofield & dias 1999 ) .
fenitrothion has been 
 shown to be effective against several deltamethrin - resistant populations under 
 laboratory and field conditions ( picollo et al . 

, the development of pyrethroid - resistant populations 
 has led to the re - utilisation of either malathion or fenitrothion against triatomines by 
 the health authorities of argentina and bolivia .
this is the first study of the efficacy of several formulations of imidacloprid on 
 different surfaces against susceptible and pyrethroid - resistant t. 
 infestans . in an attempt to characterise the toxicology of imidacloprid against t. 
 infestans
, we studied the variation in mortality through time after topical 
 application and the influence of the blood feeding condition of the insect on this 
 toxicity .
our results showed that the ld50 did not vary significantly up to 
 72 h after the initial topical application .
this result could indicate that the 
 toxicological effects of the imidacloprid remain stable through time .
( 2001 ) , who found a decrease in the toxicity of imidacloprid through time in the 
 ground beetle h. pennsylvanicus ( coleoptera : carabidae ) following 
 contact exposure .
the blood feeding condition ( starvation / feeding ) of the insects had no 
 significant influence on the insecticidal activity of the imidacloprid .
thus , the rate 
 of penetration associated with physicochemical modifications of the cuticle after blood 
 feeding appears not to alter the toxic effects after topical application .
we also analysed various types of commercial formulations of imidacloprid on two 
 different surfaces .
thus , the first step , focusing on the field application of the 
 insecticide , is to find a correct formulation of the insecticide .
we found that neither 
 the ec nor the wg formulations were effective against t. infestans 
 nymphs .
although most surfactants and polymers are biologically inert when applied to insects , 
 these chemicals can profoundly affect the biological activity of the pesticide when used 
 as part of a formulation ( scher 1988 ) .
traditional spraying is highly effective inside domiciles , but it usually leaves a 
 number of residual individuals of the vector in the peridomestic environment , as 
 reported in the southern part of the chaco region ( porcasi et al .
these 
 residual populations eventually re - colonise the domestic sites , re - establishing the 
 domestic transmission cycle of t. cruzi ( grtler et al .
1995 ) , 
 pyrethroid - impregnated curtains ( ferral et al . 
 2010 ) and a residual paint formulated as a micro - encapsulate containing an 
 organophosphate and a juvenile hormone analogue ( alarico 
 et al .
although t. cruzi is transmitted by several species of triatomines , 
 animals such as dogs , cats and chickens are the main domestic reservoirs of t. 
 cruzi in the endemic areas of chagas disease ( grtler et al .
thus , reithinger et al . ( 2006 ) found that deltamethrin - impregnated dog collars 
 reduced the survival and fecundity of exposed kissing bugs on dogs .
similarly , a spot - on 
 formulation of fipronil applied on dogs and a pour - on formulation of cypermethrin 
 applied on chickens have been successfully tested against t. infestans 
 ( rojas de arias & fournet 2002 , amelotti et al . 

the imidacloprid advantage g spot - on formulation is recommended for treating 
 cat fleas . the recommended dose ranges from 10 - 40
mg ai / kg . in this study , we tested 
 doses from 4 - 160 mg ai / kg on pigeons . at a dose of 20
mg ai / kg , 50% of the nymphs were 
 killed 24 h after the application .
the doses of 80 and 160 mg ai / kg produced 100% 
 mortality and had a high residual effect until seven days post - treatment . 

( 2009 ) studied the efficacy in 
 chickens of a pour - on formulation containing cypermethrin .
they found that after a week 
 of initial exposure to the insecticide at a dose of 120 mg / chicken , 53% of the treated 
 third - instar nymphs were killed , whereas at day 14 , mortality had values similar to the 
 controls ( 4.9% ) .
this finding is similar to our results because a dose of 160 mg / kg 
 between days 7 - 14 after initial exposure produced a mortality of 58% and 8% , 
 respectively .
rojas de arias and fournet ( 2002 ) studied the residual activity of fipronil with a 
 contact test of the insecticide on filter paper against fifth - instar t. 
 infestans .
the authors reported a value of lc50 of 106 
 mg / m . despite the subtle differences in methodology , the lc50 
 value of 960 mg / m of imidacloprid suggest that this compound has a 
 high contact activity that depends on the type of formulation .
thus , an approach 
 involving the use of the spot - on formulation might complement traditional pyrethroid 
 spraying , which has shown a low efficacy in the elimination of t. 
 infestans peridomestic populations .
the effectiveness of imidacloprid against t. infestans is reinforced by 
 its lower oral and dermal mammalian toxicity than fenitrothion , the current alternative 
 to pyrethroids .
for instance , the oral and dermal ld50 in rats for 
 imidacloprid are 450 mg / kg and > 5,000 mg / kg , whereas the values for fenitrothion are 
 250 mg / kg and 2,500 mg / kg , respectively . additionally , the no - observed effect level in 
 rats is higher for a diet containing 300 mg / kg imidacloprid [ based on a unit of 1 kg of 
 body weight than for a diet containing 10 mg / kg fenitrothion ( tomlin 1997 ) ] .
this concern is highly important if an insecticide must 
 be used indoors and in a domestic environment .
because of the increasing number of populations resistant to pyrethroids and the high 
 mammalian toxicity of fenitrothion , it is essential that other insecticidal compounds , 
 especially those with alternative modes of action to pyrethroids and organophosphates , 
 are rapidly made available for t. infestans control programmes in south 
 america .
this study has indicated the potential of imidacloprid in the control of chagas disease 
 vectors .
however , imidacloprid should be incorporated into an integrated pest management 
 programme because its effectiveness is primarily restricted to domestic and peridomestic 
 animals .
moreover , the type of formulation selected is essential in the function of the 
 toxicokinetic and toxicodynamic processes by which the active ingredient ( i.e. , 
 imidacloprid ) affects the insect ( t. infestans )
. a spot - on formulation 
 appears to improve this interaction , resulting in increased mortality of the triatomine 
 vector in the laboratory .
further studies are needed to test this type of formulation 
 under semi - field or field conditions and to incorporate this formulation as a 
 complementary strategy for triatomine control .
 the prevention of chagas disease is based primarily on the chemical control of 
 triatoma infestans ( klug ) using pyrethroid insecticides . 
 however , 
 high resistance levels , correlated with control failures , have been detected in 
 argentina and bolivia . 
 a previous study at our laboratory found that imidacloprid 
 could serve as an alternative to pyrethroid insecticides . 
 we studied the delayed 
 toxicity of imidacloprid and the influence of the blood feeding condition of the 
 insect on the toxicity of this insecticide ; we also studied the effectiveness of 
 various commercial imidacloprid formulations against a pyrethroid - resistant 
 t. infestans population from the gran chaco ecoregion . 
 variations 
 in the toxic effects of imidacloprid were not observed up to 72 h after exposure and 
 were not found to depend on the blood feeding condition of susceptible and resistant 
 individuals . 
 of the three different studied formulations of imidacloprid on glass and 
 filter paper , only the spot - on formulation was effective . 
 this formulation was 
 applied to pigeons at doses of 1 , 5 , 20 and 40 mg / bird . 
 the nymphs that fed on 
 pigeons treated with 20 mg or 40 mg of the formulation showed a higher mortality rate 
 than the control group one day and seven days post - treatment ( p < 0.01 ) . 
 a spot - on 
 formulation of imidacloprid was effective against pyrethroid - resistant t. 
 infestans populations at the laboratory level . 