Deltamethrin pyrethroid susceptibility characterization of Triatoma sordida Stål , 1859 ( Hemiptera : Reduviidae ) populations in the Northern Region of Minas Gerais , Brazil

Introduction: Triatoma sordida is the most captured Triatomine species in the Brazilian artifi cial environment. In 2008, the discovery of three Triatomine populations with altered susceptibilities to deltamethrin highlighted the importance of investigating the genetic potential for resistance in triatomines. The purpose of this study was to characterize the susceptibility to deltamethrin of peridomestic T. sordida populations in Minas Gerais, Brazil. Methods: A susceptibility reference lineage derived from Uberaba, Minas Gerais, Brazil was used. 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 of the susceptibility reference lineage. Results: Susceptibility profi le characterization of T. sordida populations revealed resistance ratios (RR50s) ranging from 0.42 to 3.94. The percentage mortality in response to the diagnostic dose varied from 70% to 100%. A comparison of the results obtained in the quantitative and qualitative assays demonstrated a lack of correspondence for some populations. Conclusions: We demonstrated that only T. sordida populations that present a RR50>1.0 have altered susceptibility, and the execution of simultaneous fi eld and laboratory tests is required to understand the actual effect of vector control. A possible cause of the observed resistance ratios might be the continuous use of pyrethroids in Brazil since the 1980s.

The Triatominae subfamily comprises vectors for Trypanosoma cruzi 1 , whose importance in human parasite transmission, within the classic epidemiological model, is associated to the colonization capacity of the domicile 2 .Triatoma sordida is the most captured Triatomine species in the Brazilian artifi cial environment.It is a local native species and has been very diffi cult to eradicate.T. sordida is frequently found in peridomiciliary areas in association with hens and low rates of infection by T. cruzi.Its marked ornithophily makes it a vector with less epidemiological importance than Panstrongylus megistus or Triatoma brasiliensi 3 .
According to a survey performed in 1940 in Minas Gerais, Panstrongylus megistus represented 79.8% of the captured triatomines, Triatoma infestans represented 9.3%, and T. sordida represented 9.7% 4 .Through control activities in 1989, Triatoma infestans was nearly eliminated in Minas Gerais.At that time, the number of captured T. sordida specimens demonstrated a marked increase 5 .In 2006, Brazil was awarded the International Certifi cation of Transmission Elimination of the Chagas Disease by Triatoma infestans 6 .Considering the diversity of the Brazilian triatomine fauna with native species of attested epidemiological importance, epidemiological surveillance assumed a critical role.
It is possible to conclude that triatomine chemical control has been feasible and effi cient when performed with technical, methodological and operational accuracy.Contradicting the expectations of some researchers, in 2008, three T. sordida populations with altered susceptibilities to deltamethrin were found in Minas Gerais 7 .This fact highlighted the necessity of developing studies concerning the genetic potential of triatomines for resistance to insecticides with the purposes of determining the occurrence frequency of this phenomenon, identifying the affected areas and identifying the relevant mechanisms.
The purpose of this work was to characterize the deltamethrin susceptibility of peridomestic T. sordida populations in the northern region of Minas Gerais, Brazil.

METHODS
The study populations were manually collected, without using a dislodging agent, from peridomiciles in endemic areas of the northern region of Minas Gerais (Cônego Marinho, Montalvânia, Monte Azul e Porteirinha) in which the Chagas  Disease Control Program performed continuous and systematic applications of insecticides with residual action in the last 30 years.

DISCUSSION
A susceptibility reference lineage (SRL) was used; breeding of the SRL started in 1992 at the insectary of the Laboratório de Triatomíneos e Epidemiologia da Doença de Chagas (LATEC) from Uberaba/MG in accordance with the criteria adopted by the Panamerican Health Organization (PAHO) 8 .
Bioassays were performed according to the methods of Pessoa 7 .Serial dilutions (0.01 -4.0ng/μL) of deltamethrin (98.2% purity, Bayer -São Paulo, SP -Brazil) were prepared and applied to the abdomen of fi rst instar nymphs from the F1 generation (fi ve days of age, fasting weight 1.2 ± 0.2mg) using a Hamilton micro-syringe (0.2µL per insect).The insecticide was diluted in acetone.At least six doses surrounding the lethal dose of 50% (LD 50 ) and producing between 10 and 90% mortality were administered.Acetone alone was applied to the insects in the control group.Three replicates of ten nymphs were conducted for each dose.The mortality was recorded at 72h.The criterion for mortality was the inability of a nymph to walk out of a fi lter paper disc 7cm in diameter 9 .
The mortality data were analyzed using Basic Probit Analysis 10 software, estimating the LD 50 the active ingredient per treated nymph (a.i./nymph) as well as the slope in nanograms.Fifty percent resistance ratios (RR 50 ) were calculated by dividing the LD 50 of each fi eld population by the corresponding SRL.
The susceptibility status classifi cation was performed according to Zerba and Picollo 11 and PAHO 8 .
After setting the base susceptibility line of the T. sordida reference population, 30 nymphs from each fi eld population were subjected to a diagnostic dose of 1xLD 99 based on the SRL.The survival of at least two insects in three replicates, was interpreted as a resistance indicator 11 .
The susceptibility reference lineage presented an LD 50 and LD 95 of 0.064 and 0.255ng a.i./nymph treated, respectively.The susceptibility profi le characterization of T. sordida populations revealed RR 50 values ranging from 0.42 to 3.4.Only four populations from Cônego Marinho/Cruz dos Araújos e Sapé, Montalvânia/ Quilômetro and Porteirinha/Furado da Onça presented slopes lower than the slope of the SRL, revealing higher frequencies of individuals with resistant alleles.The mortality percentage in response to the diagnostic dose ranged from 70% to 100% (Table 1).
A growing number of reports of triatomine populations with increased resistance ratios have had a major effect, stimulating scientists and sanitarians to search for new options for vector control.Accordingly, studies concerning susceptibility to insecticides have taken priority in the fi eld of Chagas disease.There are two major problems in the interpretation of the obtained results: the selection of the susceptibility reference population and the actual meaning of the resistance ratios for fi eld vector control.
Considering that resistance ratio (RR) is calculated from SRL lethal doses, its choice directly interferes in the results obtained and consequently, in future developments to be adopted by the managers involved with Triatominae control the fi eld.According to Obara et al. 12 , there are no records of Brazilian T. sordida populations resistant to deltamethrin.A susceptibility study of 11 populations from the center-west region of Brazil revealed RR 50 values ranging from 1.05 to 1.48.The LD 50 estimated for the reference population from the municipality of Cordeiros (State of Bahia) was 0.585ng a.i./nymph.Redefi ning the RR 50 for the populations of Obara et al. 12 using the LD 50 from the SRL adopted in this work, the RR 50 values range from 9.9 to 13.3, which results in all of the populations being designated as resistant and suggests that the insecticide used in the fi eld should be changed.Additionally, the reference population could be considered resistant.
These results emphasize in a practical manner that the utilization of different SRLs hinders comparisons and the interpretation of RRs, highlighting the importance of adopting a single population of each species for all groups investigating the susceptibility of triatomines to insecticides.In this context, another concern involves the maintenance of this SRL in the laboratory ad infi nitum, considering the diffi culties of keeping viable colonies for extended periods inside an insectary without a supply of external materials.
Another issue focuses on the concept of the susceptibility reference lineage -a population with more than fi ve generations in the laboratory, without contact with insecticide or supply of external material and collected in locations where there was no treatment with insecticide 8 .To comply with these criteria, this study selected a lineage from Uberaba, State of Minas Gerais, Brazil.However, three fi eld populations were found to be more susceptible than the SRL (RR 50 <1.0).
The SRL used in this study was established in 1992 in an insectary without contact with insecticide and without a supply of external materials.Since the 1970s, the region of Uberaba has been subjected to intense environmental degradation because of the implementation of pasture areas intended for livestock.Additionally, in the past, the same area was subjected to strong pressure from insecticides for triatomine and anopheline control (organochlorine and organophosphate insecticides).In this context, the discovery of populations more susceptible than the SRL suggests that this population already harbored resistant alleles when it was collected in the fi eld and that these alleles have been retained.
The existence of 11 populations with slopes equal to or higher than the slope of the SRL suggests a small degree of heterogeneity among the populations for the assessed characteristic.Populations from different localities within the same municipality, although geographically close to each other, presented distinct RR 50 s: Cônego Marinho (0.42 to 2.66), Montalvânia (0.42 to 2.02), Monte Azul (0.53 to 3.91) and Porteirinha (1.05 to 3.01).A possible explanation for the small intrapopulational heterogeneity observed and the different interpopulational susceptibility profi les within a single municipality is the distribution into small groups with limited dispersion and reduced genetic fl ow, as observed in some Triatominae species.Considering this possibility, a population in a certain location might undergo a selection process from insecticides regardless of what happens to triatomines in neighboring locations.
The small intrapopulational heterogeneity inferred from the slope analysis for the majority of the populations corresponds to molecular studies demonstrating that in areas with chemical treatment, the genetic diversity is lower than in non-treated areas [13][14][15] .Studies using allozymes of T. sordida from three different ecotopes in Minas Gerais revealed low levels of genetic variation in peridomestic populations 16 .For populations with slopes lower than that of the SRL, the possibility of resistance evolving because of insecticide exposure could not be discounted, which justifi es the need for follow-up regarding susceptibility changes over time.
There are two interpretations for the meaning of RR 50 values.According to Zerba and Picollo 10 , populations with RR >2 are considered resistant, whereas according to the PAHO 8 , only populations with RR >5 receive this classifi cation.This criterion should be better defi ned.The RR 50 values observed in this work range from 0.42 to 3.4.We affi rm that only the T. sordida populations that presented an RR 50 >1.0have altered susceptibility.Simultaneous field and laboratory tests are required to understand the actual effect of vector control.
In bioassays, the diagnostic dose mortality ranged from 70 to 100%.Using the criterion proposed by the World Health Organization (WHO) 9 , populations with mortality lower than 96.7% must be considered to be resistant.A comparison of the response-dose results obtained in this work with the diagnosticdose results revealed a lack of correspondence for some populations, i.e., possible resistance detected via DD was not confi rmed by the RR 50 value.A possible explanation for this discrepancy could be the reduced sampling number used in the qualitative tests, which might not represent the characteristics of the population.Amelotti et al. 17 studied a population of T. infestans from Argentina and assessed the susceptibility to deltamethrin of nymphs generated by females maintained individually throughout their lifecycle.While they were young, the females generated more resistant breeds; as they aged, the females generated more susceptible offspring.These results demonstrate how complex the issue of genetic variability is at the individual level, allowing us to refl ect on the effect of such variation in the context of the population.
A possible cause of the resistance ratios observed in this work could be attributed to the continuous use of pyrethroids in Brazil since the 1980s, as described by Vassena et al. 18 in studies performed with T. infestans populations from Rio Grande do Sul (RR 50 3.6 to 7.0).Pressure continues from insecticides used for multiple purposes (agricultural, domestic), even overlapping with control programs for other vectors.The northern region of Minas Gerais, where the populations studied were collected, was subjected to intense environmental changes in the 1970s to implement cotton cultivation, supporting the possibility of signifi cant insecticide pressure from agriculture 19 .
Additionally, the persistence of T. sordida, predominantly in the peridomicile environment, despite successive insecticide treatments over time might be related to behavioral aspects of this triatomine.The peridomicile presents a wide variety of ecotopes corresponding to an infi nite number of hidden loci associated with sources of food for T. sordida, such as chickens, dogs, pigs, and cats.Spraying the peridomicile is exhausting work in which unstacking all of the material accrued is operationally impossible for the fi eld agent in charge.After insecticide application, eggs and nymphs could remain nearly free of contact with active chemicals and/or could be in contact with sublethal doses, thus selecting for the specimens less susceptible to the chemicals and allowing for the survival of those insects in such ecotopes 19,20 .