**2. Materials and methods**

#### **2.1. Insect populations**

This study was conducted at the Laboratory of Integrated Pests Management at Universidade Federal de Viçosa, Rio Paranaíba Campus (UFV-CRP). We selected six municipalities with coffee cultivation of the species *C. arabica* and *C. canephora*, located in producing regions of the Brazilian states of Minas Gerais, Espírito Santo, São Paulo, and Pernambuco. These areas were selected because they are the largest coffee producing regions in Brazil. In these regions, we collected leaves from the middle third of plants randomly selected in commercial crops during the 2012-2013 crop season, with active mines (live caterpillars) of *L. coffeella*. These crops were georeferenced with the help of a portable Garmin E-trex Summit Hc GPS (Figure 1).

The first documented case of resistance was in 1914, in San Jose Scale (*Quadraspidiotus perniciosus)* (Comstock, 1881) (Hemiptera: Diaspididae) exposed to repeated doses of sulfur powder [8]. Reports of insect resistance began to increase in the 1940s as insecticides and miticides emerged. There are over 7740 reported cases of resistance, involving 331 compounds and more than 540 species of insects and mite pests [9]. From 1914 to 2007, the vast majority

Lepidopteran species such as *Alabama argillacea* (Lepidoptera: Noctuidae) [10], *Plutella xylostella* (Lepidoptera: Plutellidae) [11], and *Tuta absoluta* (Lepidoptera: Gelechiidae) [12] have shown resistance to several groups of insecticides. These authors studied insect populations from different locations, using different groups of insecticides with varying mechanisms of action. Studies with *L. coffeella*, however, have focused only on the organophosphate group with no studies on other chemical groups [13,14]. As such, studies of different populations and

Among the insecticides used, most are neurotoxins, and it is this group that presents the most problems of insect resistance [9]. These neurotoxic insecticides (e.g., organophosphates and pyrethroids) cause rapid death of susceptible insects, and abamectin, neonicotinoids, and

It is therefore possible to detect resistance to a particular active ingredient by comparing the time of death of each population to different neurotoxic insecticides. Similar experiments have been done with other insects, such as the mosquito *Culex tarsalis* (Diptera: Culicidae) [16]. Slower deaths may indicate the population is beginning to become resistant. Delayed mortality could be compared to the effect of sublethal doses, which put the insects in a state of stress and reduce their metabolism before death [17]. One way to detect resistance using the lethal time of death (LT) is to collect geographically distant populations to obtain more precise information and compare populations across regions since the resistance is relative. Thus, based on the mechanism of action of each insecticide group, it is possible to compare resistance by meas‐

There are two studies focusing on the detection of insecticide resistance among populations of *L. coffeella* and just with organophosphate insecticides. Our proposal is to study different groups and regions. This study aimed to recognize populations of *L. coffeella* in different regions

This study was conducted at the Laboratory of Integrated Pests Management at Universidade Federal de Viçosa, Rio Paranaíba Campus (UFV-CRP). We selected six municipalities with coffee cultivation of the species *C. arabica* and *C. canephora*, located in producing regions of the Brazilian states of Minas Gerais, Espírito Santo, São Paulo, and Pernambuco. These areas were selected because they are the largest coffee producing regions in Brazil. In these regions, we

of Brazil that were resistant to neurotoxic insecticides by comparing the lethal time.

of cases of resistance occurred in Lepidoptera, with 1799 confirmed cases.

various insecticide groups are needed.

4 Insecticides Resistance

diamides are slower in causing death of insects [15].

uring how quickly the insecticides act on a population.

**2. Materials and methods**

**2.1. Insect populations**

**Figure 1.** Location and characterization of *Leucoptera coffeella* collection in coffee-producing regions. Dark spots repre‐ sent coffee-producing regions. White spherical symbols within the dark spots represent collection sites of leafminer populations.

The leaves collected in each region were transported to the laboratory in separate plastic bags for visual selection of mines that did not present any harm (e.g., open or with signs of para‐ sitism/predation). Selected mined leaves were combined for insect rearing in a greenhouse (20 × 10 m). These leaves were placed in vials with water (25 mL) inside wooden cages covered with organza. The larvae were fed seedlings coffee of Catuaí cultivar grown in a greenhouse without insecticide application. Only larvae with at least one generation in the laboratory were used in bioassays to prevent the expression of insecticide tolerance due to differing environ‐ mental conditions at the different sampling sites (i.e., differences without any genetic basis).

#### **2.2. Insecticides**

Six neurotoxic insecticides were selected for bioassays of *L. coffeella* resistance to the concen‐ trated active ingredients abamectin 18 g l-1 EC (emulsifiable concentrate) (Syngenta, São Paulo, Brazil), chlorantraniliprole 350 g l-1 WG (water-dispersible granules) (DuPont, Paulínia, Brazil), chlorpyrifos 480 g l-1 EC (Fersol, Mairinque, Brazil), deltamethrin 25 g l-1 EC (Bayer SA, São Paulo, Brazil), profenofos 550 g l-1 EC (Syngenta, São Paulo, Brazil), and thiamethoxam 250 g l -1 WG (Syngenta, São Paulo, Brazil) (Table 1).



a LT50 = time (h) lethal to kill 50% of the population.

b CI = confidence interval of 95%.

c RT50 = ratio of lethal time to kill 50% of the population.

<sup>d</sup>χ<sup>2</sup> = chi-square.

**2.2. Insecticides**

6 Insecticides Resistance


**Insecticide Population LT50 <sup>a</sup>**

Carmo do Paranaíba-MG Santa

l

Abamectin

Chlorpyrifos

Chlorantraniliprole

Deltamethrin

Six neurotoxic insecticides were selected for bioassays of *L. coffeella* resistance to the concen‐ trated active ingredients abamectin 18 g l-1 EC (emulsifiable concentrate) (Syngenta, São Paulo, Brazil), chlorantraniliprole 350 g l-1 WG (water-dispersible granules) (DuPont, Paulínia, Brazil), chlorpyrifos 480 g l-1 EC (Fersol, Mairinque, Brazil), deltamethrin 25 g l-1 EC (Bayer SA, São Paulo, Brazil), profenofos 550 g l-1 EC (Syngenta, São Paulo, Brazil), and thiamethoxam 250 g

 **(CI95%)**

Rio Paranaíba-MG 13.29 (11.29–15.34) 40 1.74 2.87 (3) 0.59 Abaeté dos Mendes-MG 14.70 (12.59–16.53) 40 1.92 6.95 (3) 0.07 Carmo do Paranaíba-MG 36.75 (33.63–40.97) 40 4.80 4.05 (3) 0.26 Santa Teresa-ES 9.11 (6.03–11.52) 40 1.19 2.89 (3) 0.59 Guaranhuns-PE 17.85 (15.87–19.71) 40 2.33 4.97 (3) 0.17 Franca-SP 12.41 (10.99–15.12) 40 1.62 3.42 (3) 1.12 Guaraciaba-MG 7.65 (6.85–10.11) 40 1.00 5.63 (3) 3.11

Rio Paranaíba-MG 8.16 (7.02–9.20) 40 8.08 7.35 (4) 0.12 Abaeté dos Mendes-MG 17.18 (15.68–18.75) 40 17.01 9.07 (4) 0.06 Carmo do Paranaíba-MG 16.39 (15.12–17.76) 40 16.23 1.66 (3) 0.65 Santa Teresa-ES 4.58 (3.62–5.54) 40 4.53 7.56 (5) 0.18 Guaranhuns-PE 8.59 (6.70–10.21) 40 8.50 2.39 (3) 0.50 Franca-SP 18.82 (17.54–20.15) 40 18.63 8.20 (4) 0.08 Guaraciaba-MG 1.01 (0.35–2.07) 40 1.00 6.32 (7) 0.06

Rio Paranaíba-MG 27.70 (24.70–31.56) 40 1.98 3.66 (3) 0.30 Abaeté dos Mendes-MG 26.30 (22.15–34.79) 40 1.88 1.57 (2) 0.54

Teresa-ES 14.01 (11.87–16.47) 40 1.00 7.51 (5) 0.18 Santa Teresa-ES 31.53 (28.44–35.74) 40 2.25 5.50 (3) 0.14 Guaranhuns-PE 18.82 (17.54–20.15) 40 3.23 8.20 (4) 0.08 Franca-SP 14.28 (11.00–18.23) 40 1.02 6.30 (5) 1.22 Guaraciaba-MG 8.59 (6.70–10.21) 40 1.88 2.39 (3) 0.50

Rio Paranaíba-MG 31.12 (27.59–36.20) 40 5.35 4.96 (4) 0.17 Abaeté dos Mendes-MG 25.73 (23.34–28.56) 40 4.42 3.83 (3) 0.28 Carmo do Paranaíba-MG 28.18 (24.46–34.29) 40 4.84 2.22 (3) 0.53 Santa Teresa-ES 5.82 (4.23–7.65) 40 1.00 5.99 (4) 0.07 Guaranhuns-PE 20.38 (17.53–23.23) 40 3.50 6.04 (3) 0.11

**<sup>b</sup> n RT50 <sup>c</sup> χ2 d(df)e** *Pf*

e *df* = degrees of freedom.

f *P* = probability.

**Table 1.** Time and mortality curves (LT50) of Brazilian populations of *Leucoptera coffeella* under the effect of seven insecticides at the recommended doses.

The registered label rates of the respective active ingredients in Brazil were 0.18 mg mL-1 (0.026 mg a.i. mL-1) for abamectin, 0.072 mg mL-1 and 0.078 mg a.i. mL-1 for chlorantraniliprole, 0.05 mg mL-1 (4.800 mg a.i. mL-1) for chlorpyrifos, 0.032 mg mL-1 (0.013 mg a.i. mL-1) for deltameth‐ rin, 0.4 mg mL-1 (1.100 mg a.i. mL-1) for profenofos, and 0.024 mg mL-1 (2.000 mg a.i. mL-1) for thiamethoxam.

#### **2.3. Time-mortality bioassay**

For time-mortality analysis, circular discs (diameter 90 mm) of filter paper were dipped into the insecticide solutions diluted in distilled water, using the recommended doses to control *L. coffeella*. The control used embedded disks with distilled water. The discs containing the insecticides and the water were fixed on a clothesline to dry in the shade and then placed separately into Petri dishes (9.0 × 1.5 cm). Ten larvae of *L. coffeella* reared in the lab were transferred to each Petri dish using a fine-tipped brush. The Petri dishes with the larvae were kept in the BOD incubator (model SP-500) at 25°C ± 1°C until the time of evaluation. The experiments were conducted in a completely randomized design with four replications.

Preliminary tests using only discs soaked in water were carried out to observe caterpillar mortality over a 48-h period. This was necessary to estimate the maximum evaluation time after bioassay assembly that causes 20% lower mortality in the control [18]. Thus, to have a mortality range from 0% to 100%, evaluations were made at 2, 6, 12, 16, 24, 32 and 48 h (treatments) after bioassay assembly. The time intervals were assessed in independent experimental units, to avoid pseudoreplicates. We considered insects dead when they did not move after being touched with the fine-tipped brush.

#### **2.4. Spatial dependence of insecticide resistance**

To determine the spatial dependence of *L. coffeella* insecticide resistance, the semivariance statistical model of LT50 values to *L. coffeella* populations for each insecticide and the distance between sampling locations of each population were used. The distance between the sampling sites of each insect population was determined using geographic coordinates with a global positioning system (GPS 12, Garmin International, Olathe, KS). The semivariograms were estimated from the semivariance data of the LTs50 of each population for each insecticide and used as dependent variables in regression analysis, with the distance between the sampling sites as an independent variable. The first inflection point of the semivariogram curve represents the maximum distance of interference between the populations of *L. coffeella* in relation to susceptibility to a given insecticide.
