Toxicological profi le of deltamethrin in Triatoma brasiliensis ( Hemiptera : Reduviidae ) in State of Ceará , Northeastern Brazil

Introduction: Triatoma brasiliensis is the species of greatest epidemiological relevance in the semi-arid region of Brazil. This species is predominantly found in domestic environments, and it has the ability to build large colonies with high levels of natural infection via Trypanosoma cruzi. Thus, T. brasiliensis is one of the most effi cient transmitters of Chagas disease (CD) to humans. Despite household spraying with residual insecticides, many areas report persistent reinfestations for reasons that remain poorly understood. Therefore, this study sought to characterize the toxicological profi le of deltamethrin in T. brasiliensis from areas with persistent reinfestation in State of Ceará, Brazil. Methods: The susceptibility reference lineage (SRL) was derived from Umari. Serial dilutions of deltamethrin were prepared and applied to the dorsal abdomen of fi rst instar nymphs. The control group received only pure acetone. Mortality was evaluated after 72h. Qualitative tests assessed mortality in response to a diagnostic dose of 1xLD99 (0.851 nanograms of active ingredient per treated nymph) of the SRL. Results: The susceptibility profi le characterization of the T. brasiliensis populations revealed 50% resistance ratios (RR50) that ranged from 0.32 to 1.21. The percentage of mortality in response to the diagnostic dose was 100%. Conclusions: We demonstrated that T. brasiliensis was highly susceptible to deltamethrin. The control diffi culties found might be related to the recolonization of the triatomines originating from neighboring environments and the possible operational failures related to the process of spraying that enabled specimens less susceptible to deltamethrin to survive.

Chagas disease (1) (CD) affects approximately 1.8 to 2.5 million people in Brazil.This disease is endemic to much of the country and targets entire populations with poor access to basic public health services (sanitation, housing, health, and so on).After many years of effort and investment in housing improvements and chemical control, home triatomine levels have been signifi cantly reduced in general, especially those of Triatoma infestans.However, studies of existing autochthonous species show that the effective entomological surveillance led by endemic/health agents, in partnership with the general population, must be conducted to maintain this result.
In many regions of Brazil, humans are in close contact with triatomines because of the proximity and overlap of domestic and wild environments, which often occurs in the Brazilian semi-arid region.The species of greatest epidemiological relevance in this region is Triatoma brasiliensis.Studies conducted in Bahia (2) , Piauí (3) and Ceará (4) have indicated that T. brasiliensis is the most common species in domestic environments, occasionally forming colonies of more than 100 specimens (5) and having high levels of natural infection via Trypanosoma cruzi; thus, this species is one of the most effi cient transmitters of CD to humans.Domestic infestation occurs more commonly during the dry season, and triatomines are usually attracted to light (6) .
In the wild, T. brasiliensis is associated with the Caatinga (a semi-arid scrub forest in northeastern Brazil) and is usually found in dens of various rodents (7) .Valença-Barbosa et al. (8) recently examined the T. brasiliensis colonization of the Pilosocereus gounellei cactus, popularly known as xique-xique in the Valley of Jaguaribe, Northeastern Brazil.The proximity of wild and domestic environments enables the dispersion of insects to housing units (9) .In peridomiciles, T. brasiliensis shows a preference for animal shelters built with bricks and covered with tiles (5) .Rodrigues et al. (10) have noted a strong tendency for T. brasiliensis to be located in poultry houses and near bird resting areas, especially in the lower, drier and warmer portions of walls.

DISCUSSION METHODS
The proximity of wild and domestic environments in the northeastern semi-arid region enables an intense fl ow of T. brasiliensis between these environments.As such, reinfestation processes are frequently reported (11) (12) .In this sense, the use of residual insecticides is extremely relevant to prevent new specimens from invading housing units.However, despite systematic and continuous spraying activities in accordance with the recommendations of the Ministry of Health (13) , controlling triatomines has been diffi cult in many areas.Diotaiuti et al. (12) examined households in Independência four months after deltamethrin spraying and found triatomines of all development phases in 9.7% of the sampled units.This fi nding suggests that triatomine recolonization from neighboring locations occurs via specimens surviving the spraying process.This result might be related to operational failures in the spraying activity, T. brasiliensis resistance to the insecticides used for vector control, or both.
To better understand the factors that hinder the control of T. brasiliensis in the households of Ceará, Brazil, this study characterized the toxicological profi le of deltamethrin pyrethroid in T. brasiliensis populatio across six municipalities using laboratory bioassays.
Bioassays were performed using the methods of Pessoa (14) and the World Health Organization (WHO) (15) .Serial dilutions (0.1 -8.0ng/μL) of deltamethrin (98.2% purity, Bayer: São Paulo, Brazil) were prepared and applied to the abdomen of the fi rst instar nymphs from the F1 generation (fi ve days old, fasting weight 1.2 ± 0.2mg) using a Hamilton micro-syringe (0.5µL per insect).The insecticide was diluted in acetone.At least six doses surrounding the lethal dose of 50% (LD 50 ) producing mortality rates between 10% and 90% were administered.Acetone alone was applied to the insects in the control group.Three replicates of ten nymphs were performed for each dose.Mortality was recorded at 72h.The mortality criterion was the inability of the nymphs to walk out of a fi lter paper disc (7cm in diameter (15) ).
The mortality data were analyzed using Basic Probit Analysis (16) software to estimate slope and the LD 50 in nanograms of active ingredient per treated nymph (ng a.i./nymph).Fifty percent resistance ratios (RR 50 ) were calculated by dividing the LD 50 of each fi eld population by its corresponding SRL.A susceptibility status classifi cation was performed according to the Pan American Health Organization (PAHO) (17) .
After setting the base susceptibility line of the T. brasiliensis reference population, 30 nymphs from all of the fi eld samples received a diagnostic dose of 1xLD 99 (0.851ng a.i./nymph) based on the SRL.The survival of at least two insects in three replicates was interpreted as an indicator of resistance (15) .The susceptibility reference lineage presented an LD 50 of 0.293 ng a.i./nymph treated.The susceptibility profile characterization of the T. brasiliensis samples revealed RR 50 values ranging from 0.32 to 1.21.The mortality percentage in response to the diagnostic dose for all samples was 100%.Only six samples from Jaguaruana (Cipriano Lopes), Independência (Pajeú), Jaguaribe (Serrote), Quixeré (Boqueirão), Ipaporanga (Lagoa de Dentro, Casa 6) presented a slope less than the SRL, revealing higher frequencies of individuals with resistant alleles (Table 1).
The virtual control of T. infestans (a native species of Bolivia, introduced in the late 18 th century in Brazil) (18) , accompanied by the International Elimination of the Transmission of Chagas Disease Certifi cate in 2006 (19) , strengthened the spurious idea that CD had been eliminated in Brazil and disregarded the epidemiological importance of native triatominic species in the Northeast region, such as T. brasiliensis.The signifi cance of these triatomines with regard to maintaining the cycle of T. cruzi near human homes should not be neglected, hence the importance of control activities, epidemiological surveillance and health awareness.Despite systematic and continuous spraying activity, reinfestation has often been reported because of operational failures, resistance to the insecticides used in vector control strategies, or both.
Sonoda et al. (20) found low levels of T. brasiliensis resistance to deltamethrin in populations in Tauá, Ceará.Intradomicile populations (Cachoeira dos Pedrosas RR 50 =1.16 Obara (21) characterized the toxicological profile of 16 populations of T. brasiliensis with regard to deltamethrin in the States of Paraíba, Pernambuco, Piauí and Rio Grande do Norte.All of these populations presented low RR 50 values ranging from 1.0 to 2.17.Field trials to assess the effect of WG250 deltamethrin at 25 ng a.i./nymph on different substrates (as recommended by the Ministry of Health) have confi rmed the susceptibility of all populations, with 100% mortality rates for all trials.This work assessed 15 populations of T. brasiliensis from Ceará and identifi ed low RR 50 values (0.32-1.21).According to the WHO (1994), all populations are susceptible to deltamethrin.In the present study, the qualitative assays confi rmed this susceptibility and showed 100% mortality rates for all of the samples in response to diagnostic dose.Importantly, populations from different locations within the same municipality presented different toxicological profi les.This fi nding suggests that the selection process for pesticides can occur independently, even in neighboring houses (22) (23) .
The existence of populations with RR 50 <1.0(i.e., those more susceptible than the susceptibility reference strain used) suggests that the SRL already had resistant alleles when it was collected in the fi eld in 1984 and that they remain to the present day.A similar fi nding was reported for Triatoma sordida (23) (24) , which led us to refl ect on the weak criteria recommended by the PAHO 17 for the choice of SRLs: over fi ve generations in the laboratory without insecticide contact, external material contribution, or originating from places that have never been treated with insecticide.
Of the 15 populations studied, 9 presented slopes equal to or greater than the SRL, suggesting little intrapopulational heterogeneity.This fi nding corroborates molecular studies showing that genetic diversity is lower in areas with chemical treatment than untreated areas (25) (26) (27) .However, the other 6 populations showed lower slopes than the SRL, indicating greater heterogeneity most likely because of the high dispersal rates of the T. brasiliensis specimens in Ceará that invade houses by fl ying (12) .This fact shows the signifi cant recolonization potential of artifi cial environments from wild and peridomicile sources.Importantly, one cannot exclude the possible development of resistance among populations with lower slopes than that of the SRL when pressured by insecticides.This possibility justifi es the need to monitor timeline susceptibility changes.
The results obtained from qualitative and quantitative bioassays did not prove that T. brasiliensis is resistant to deltamethrin.Despite successive insecticide spraying, the persistence of triatomines might be associated with their behavior.Peridomicile (and even intradomicile) infestations present a wide range of environments, corresponding to an infi nite number of hiding spots.Spraying these environments requires extensive work; unstacking all of the material accumulated inside is often impossible for field agents.Consequently, triatomines (eggs, nymphs and adults) might remain in the area after insecticide application, having no or limited contact with the chemical, thereby encouraging the selection of those that are least susceptible and enabling their survival (28) (29) .Moreover, the high temperatures and ultraviolet (UV) radiation of northeastern Brazil can degrade pyrethroids faster, thereby reducing their duration, availability on treated surfaces, and their consequent triatomicide action.
Importantly, the small changes in susceptibility found in this study are unlikely to have contributed to the emergence of CD cases in the study area; however, they serve as a warning to improve the quality and methodological rigor of the chemical control activities associated with the spatial reordering of households to avoid refractory T. brasiliensis.

Pessoa GCD et al. -
Toxicological profi le of deltamethrin in T. brasiliensis