Variability of susceptibility to deltamethrin in peridomestic Triatoma sordida from Triângulo Mineiro , State of Minas Gerais , Brazil

Introduction: Despite chemical and physical vector control strategies, persistent infestations of Triatoma sordida have been reported in a large part of Minas Gerais, Brazil, and the cause for this is little investigated. We aimed to characterize the deltamethrin toxicological profi le in peridomestic T. sordida populations from Triângulo Mineiro area of Minas Gerais. Methods: Susceptibility to deltamethrin was assessed in seventeen peridomestic T. sordida populations. Serial dilutions of deltamethrin in acetone (0.2μL) were topically applied on the fi rst instar nymphs (F1; fi ve days old; fasting weight, 1.2 ± 0.2mg). Dose response results were analyzed using Probit software, and the lethal doses, slope and resistance ratios were determined. Qualitative tests were also performed. Results: The deltamethrin susceptibility profi le of T. sordida populations revealed resistance ratios ranging from 0.84 to 2.8. The percentage mortality in response to a diagnostic dose was 100.0% in all populations. Conclusions: From our results, the lack of resistance to insecticides but persistent T. sordida infestations in the Triângulo Mineiro area may be because of: 1) environmental degradation facilitating dispersion of T. sordida, allowing colonization in artifi cial ecotopes; 2) operational failures; and 3) complexity of the peridomicile in the study area.These variables are being investigated.

Triângulo Mineiro, State of Minas Gerais, Brazil, is an old colonization area, cleared by pioneers at the end of the past century during their search for gold and precious stones.The region took on great economic importance with the introduction of the Zebu cattle, which also resulted in a drastic reduction of the savanna areas, by about 70% (1) .Agriculture became important only in the beginning of the 1970s, with the development of techniques that promoted soybean culture in the savanna.By the end of the 1980s, the region was characterized by a small strip of land, with a small population that owned large properties.Currently, this region is the center for large agroindustrial projects, such as livestock, represented by large cattle-producers, the main economic activity of the region (2) .
Triatoma sordida is an endemic triatomine of the cerrado, living mainly under the bark of trees that are preserved during the conversion of forests to fields and pastures.Because of its habitat, which sustains few vertebrates, it frequently encounters long periods without feeding.A recent ecological niche modeling study revealed the possibility that the T. sordida distribution area could be larger than that initially known, i.e., this triatomine could also inhabit other biomes such as Caatinga and Pantanal (4) .Such biological characteristics, including their ability to resist fasting, ease of adapting to different hosts, and relative mobility (mainly the adults) facilitate dispersion and colonization in artifi cial ecotopes (5) .This, T. sordida can be considered a semidomicile species, given the frequency with which it has been found in peridomicile and intradomicile environments (6) .Currently, four Triatominae species deserve special attention in Trypanosoma cruzi transmission to man: Triatoma brasiliensis, P. megistus, Triatoma pseudomaculata, and T. sordida (7) .
Although CD is endemic to a large part of Minas Gerais, significant reductions in the occurrence of new cases has been noted in recent decades, possibly due to 1) the chemical activities of the National Program of Chagas Disease Control, 2) epidemiological vigilance employed in municipalities, and 3) socio-economic factors of rural areas, such as rural exodus, income and habitation improvement, electricity, and access to education and health services, among others (8) .However, despite these efforts, reports show persistent T. sordida infestation in Minas Gerais, and the causes are little investigated (9) .This could slowly and progressively cause the resurgence of this endemic disease mainly in poverty-stricken areas and less politically represented regions, such as the Triângulo Mineiro.
To verify the involvement of insecticide resistance in the difficulty of vector control strategies in the field, we characterized the toxicological profile for deltamethrin in peridomestic T. sordida populations from Triângulo Mineiro that has persistent infestation.

Triatoma sordida populations
Twenty populations were manually collected in the peridomiciles of eight endemic areas of Triângulo Mineiro (Carneirinho, Frutal, Gurinhatã, Iturama, Limeiro do Oeste, São Francisco de Sales, Tupaciquara, and União de Minas), where the NPCDC was employed for the past 30 years, with continuous and systematic applications of insecticides with residual action (Figure 1).No dislodging agent was used in the collection of the triatomines.toxicological profi les when exposed to the same chemical class of insecticide (10) .We emphasize that laboratory studies are only indicative of resistance in the fi eld, and they cannot suggest another pyrethroid as a replacement.In case of indication of resistance, fi eld trials must be carried out by using the insecticide (formulated product) used by the Ministry of Health.

Bioassays
The susceptibility reference lineage (SRL) of T. sordida was obtained from Uberaba (19° 44′ 52′′ S 47° 55′ 55′′ O), where it has been maintained in the laboratory for more 20 years, without contact with insecticide or inclusion of external material (11) .
Serial of in acetone were prepared.For each concentration, three repetitions were carried out with ten fi rst instar nymphs of the F1 generation (5 days-old, fasting, weight 1.2 ± 0.2mg).The treatment consisted of the application of 0.2µL of insecticide dilution on the dorsal abdomen by using a Hamilton microsyringe mounted on a repeating dispenser, according to the procedures of the World Health Organization (12) and Pessoa (13) .For each populations, a minimum of eight doses of insecticide active ingredient (a.i.) ranging from 0.01 to 4.0ng to cause mortalities between >0% to <100% of the individuals, were applied per insect.Acetone was applied to the control group.Mortality was assessed 72h after application, and it was determined by the inability or lack of coordination of the nymphs to move from the center to the edge of a fi lter paper (7cm diameter ).Other signs such as paralysis and lack of response to external stimuli were also considered.During and after the experiment, the insects were maintained under controlled conditions of temperature and humidity (25°C ± 1°C; 60% ± 10% RH).

Diagnostic dose
After setting the base susceptibility dose for the T. sordida reference population, 30 nymphs from each peridomestic populations were subjected to a diagnostic dose of 1× LD 99 (0.45ng a.i./nymph.)based on the SRL.The survival of at least two insects in three replicates was interpreted as a resistance indicator (12) (13) .

Data analysis
Data from the dose-response tests of each population were analyzed using the Probit program (14) Thus, we estimated the slope and lethal doses required to kill 50% of the treated individual (LD 50 ), as well as the resistance ratio 50% (RR 50 ), which was calculated by dividing the LD 50 of each field population by the LD 50 of the SRL.

Ethical considerations
This study was approved by the Animal Ethics Committee of Fundação Oswaldo Cruz (number 29/14-1).
The SRL showed an LD 50 of 0.064ng a.i./nymph.The susceptibility profi le characterization of T. sordida populations revealed RR 50 values ranging from 0.84 to 2.8.All populations, except that from União de Minas (Três Barras), showed slopes lower or equal to that of the SRL.We noted 100% mortality in all populations in response to the diagnostic dose (Table 1).
Despite reports of persistent T. sordida infestation, only two studies have determined the susceptibility of Brazilian populations of this triatomine to insecticides (13) (15) .Pessoa (13) showed the deltamethrin toxicological profile in eleven peridomestic T. sordida populations collected from areas with reports of persistent infestation, in the North of Minas Gerais.The resistance ratios (RR 50 2.6-6.8) in that study were the highest for T. sordida.In accordance with the Pan-American Health Organization (PAHO) (11) , fi ve (45.0%) populations with RR 50 > 5 were considered resistant to deltamethrin.Following this, Obara et al. (15) determined the toxicological profile for eleven T. sordida populations from Goiás, Mato Grosso, and Mato Grosso do Sul, showing discrete alterations of susceptibility to deltamethrin (RR 50 1.19-2.36).All populations were classifi ed as susceptible to the tested insecticide.
The susceptibility profi le of T. sordida in our study is similar to that by Obara et al. (15) but unlike that by Pessoa et al. (13) .However, the different toxicological profi les of deltamethrin in T. sordida populations from distinct locations in the same municipality (e.g., Frutal/Guilherme F. Correa and Frutal/ Aureliano N. Cruz), showed the complexity of the resistance phenotype, at both macro-and microgeographical levels, as observed by Pessoa et al. (16) in their studies of Brazilian T. infestans populations from Rio Grande do Sul.They observed that deltamethrin does not present homogenous effects over different populations from the same locality, suggesting independent selection pressure.Such results prove that the results of one population cannot be transposed to another, even if these populations are geographically close.
Of 18 populations, 17 (95%) presented a slope equal or less than the SRL, suggesting a small degree of intrapopulational heterogeneity and reduced possibility of change in the toxicological profi le before selection pressure occurs with continued use of insecticides.This could be because of the triatomine population structure with small groups of limited dispersal and reduced genetic fl ow.In addition, molecular studies have shown that genetic diversity is signifi cantly lower in chemically treated areas than in untreated areas, indicating active chemical pressure on populations (17) .
According to Pan American Health Organization (PAHO) (11) , the alteration of susceptibility observed in the T. sordida populations in this study is by individual variations among triatomines.Therefore, the fi eld chemical control activities with the insecticide currently in use may be continued but resistance must be monitored.
Despite insecticide spraying diffi culties and low permanence in the peridomicile, the lack of resistance and only one annual cycle of T. sordida, which means slow reconstruction of the original population, suggest that one annual spraying is suffi cient for the control of this triatomine (18) .Thus, the persistent T. sordida infestation in Triângulo Mineiro may be because of the following: 1) environmental degradation that facilitate the dispersion of T. sordida, allowing colonization in artifi cial ecotopes (5) (19) (20) ; 2) operational failures (9) ; and 3) complexity of the peridomicile in the Triângulo Mineiro region (18) .Regarding environmental degradation, studies have shown that profound modifi cations to the natural environment of the Triângulo Mineiro region to promote agriculture and increase livestock caused displacement or disappearance of the refuges and natural food sources of T. sordida, which then sought these in the alternative artifi cial environment in order to survive.It seems the change in vegetable coverage, hair least to some extent, provide T. sordida dispersion.High infestation in houses closest to the wild environment suggests recolonization of triatomines (nymphs to adults) in the artifi cial environment from their natural ecotopes (5) (19) (20) , contributing to the persistent infestation in the region.
Control and operational failures (11) can contribute to the persistence of T. sordida in the Triângulo Mineiro area.Such failures may be because of lack of insecticide effi cacy owing to poor quality of the active ingredient and/or inadequate formulation.Operational failures due to insecticide dilution errors, inadequate application, and spraying machine problems.Discontinuity of spraying cycles due to administrative, budgetary, and logistical reasons must also be considered.We recommend ensuring 1) the use of good quality and effective formulations of insecticides, proven by control programs of triatomines and 2) good quality work by thorough spraying of endemic agents proven in the vector control of Chagas disease.Currently, in Brazil, triatomines control is maintained using alphacypermethrin.Similar to other pyrethroids, the lethal dose of this insecticide leaves little residual effect and does not remain on the treated surfaces, but the recommended doses produce an initial high impact on the populations.The environmental conditions that promote the persistence of triatomines are ultraviolet radiation, rain, alkaline pH of the insecticide formulation, characteristics of the substrate on which the insecticide is applied, and/or other favorable physical conditions favorable to permanence of triatomine (e.g.complexity of peridomestic structures) (21) .
Finally, the persistent T. sordida infestation in the study area can be related to the complexity of the peridomicile.The Triângulo Mineiro area has a complex peridomicile, with high triatomine densities (about 13 triatomines per peridomicile).The large variety of ecotopes in these peridomiciles makes insecticide spraying an exhaustive job, which requires shifting of materials accumulated in these ecotypes, presenting an operationally impossible situation for the responsible fi eld agent.Consequently, T. sordida (eggs, nymphs, and adults) persist even after application of the insecticide, deep in piles of fi rewood, on the roofs of barns, and in a variety of other nearly inaccessible hiding places, free from contact with the active ingredient and/or in contact with sub-lethal doses, favoring their multiplication in these ecotopes (18) .Considering that T. sordida is a native species of Brazil, and may not be eradicable, we recommended the following: 1) development and fortifi cation of effective epidemiological surveillance, entomological surveillance, and environmental surveillance in the Triangulo Mineiro area and 2) investigation of possible operational failures that could compromise the effectiveness of actions in the field, in order to prevent intradomiciliary colonization of T. sordida and possible transmission of T. cruzi to human beings.
The T. sordida populations from Triângulo Mineiro region showed high susceptibility to the tested insecticide.Low resistance ratios (RR 50 0.84-2.8)and 100.0%mortality were noted in all populations in response to diagnostic dose were observed.The persistent T. sordida infestation in the Triângulo Mineiro may be caused by 1) environmental degradation associated with the facilitated dispersion of T. sordida, allowing colonization in artifi cial ecotopes by triatomines originating wild environments; 2) operational failures; and 3) complexity of the peridomicile in the Triângulo Mineiro area.