Quantification of enzymes associated to insecticide resistance in different populations of Plutella xylostella L. (Lepidoptera: Plutelliidae) from the State of Guanajuato, Mexico.

E. Cerna Chávez1; J. F. Rodríguez Rodríguez2; A. Hernández Juárez1; L. A. Aguirre Uribe1; J. Landeros Flores1; F. Cervantes Ortiz3; L. P. Guevara Acevedo3; Y. M. Ochoa Fuentes1*

1. Universidad Autónoma Agraria Antonio Narro., Universidad Autónoma Agraria Antonio Narro, Universidad Autónoma Agraria Antonio Narro, Mexico , 2. Universidad Autónoma Agraria Antonio Narro. Maestría en Ciencias en Parasitología Agrícola. Calzada Antonio Narro 1923, C.P. 25315. Buenavista, Saltillo; Coahuila, México., Universidad Autónoma Agraria Antonio Narro, Universidad Autónoma Agraria Antonio Narro,

<postal-code>25315</postal-code>
<city>Saltillo</city>
<state>Coahuila</state>
, Mexico , 3. Instituto Tecnológico de Roque, Km 8 Carretera Celaya-Juventino Rosas, Apartado Postal 508, C.P. 38110, Celaya; Guanajuato, México., Instituto Tecnológico de Roque, Instituto Tecnológico de Roque,
<postal-code>38110</postal-code>
<city>Celaya</city>
<state>Guanajuato</state>
, Mexico

Correspondence: *. Corresponding Author: Ochoa Funetes Yisa María. Universidad Autónoma Agraria Antonio Narro, Calzada Antonio Narro 1923, C.P. 25315. Buenavista, Saltillo, Coahuila, México. Phone: +52(844) 411 0326. E-mail: E-mail: .


Abstract

The diamondback moth is one of the main pests that affect cruciferous plants, due to the importance of these crops and the damage generated has become a serious problem for producers. Its control is mainly based on the chemical method, in which only insecticides compatible with the environment and human health are used. Nevertheless, the high pressure of selection of insecticides induced the development of resistance to the different active materials used for its control. Therefore, biochemical tests were performed to quantify α and β esterase, glutathione S-transferase, acetylcholinesterase and oxidases, enzymes related to resistance to insecticides, in four populations of Plutella xylostella from the state of Guanajuato, Mexico (Abasolo, Celaya, San Luis de la Paz and Valle de Santiago) and a “susceptible line” as a reference. The results indicate that the enzymes with the highest presence were the α and β esterases in Celaya and Valle de Santiago, as well as oxidases in populations of San Luis de la Paz. Moreover, we can mention that the enzymes responsible for the lack of effectiveness in products applied for the control of this pest as pyrethroids, organophosphates and benzoylureas are the esterase enzymes. On the other hand, the acetylcholinesterase and glutathione S-transferase enzymes were not relevant as a detoxification mechanism.

Received: 2017 December 19; Accepted: 2018 June 13

revbio. 2020 Mar 23; 5(spe1): e424
doi: 10.15741/revbio.05.nesp.e424

Keywords: Key words: Detoxification enzymes, diamondback moth, insecticide tolerance.

Introduction

In Guanajuato, Mexico, 20,590.50 hectares of broccoli are annually sown with a production of 292,345.21 t (SIAP, 2014); its production is mainly destined for the export market, which is why cultivation represents an important source of money and significant benefits for producers (Bujanos et al., 2013). The main pest of economic significance in cruciferous plants is the “diamondback month” Plutella xylostela (P. xylostella) (INIFAP, 2013), which is a highly destructive cosmopolitan pest (Hecket, 2006), that affects the quality of the product due to the contamination caused by their eggs and larvae, causing its rejection for exportation (INIFAP, 2013). This specialist of cruciferous plants may have its origin in Europe (Sarfraz et al., 2006), or eastern Asia (Liu et al., 2003), nonetheless, it can exist in any place where its host plants are found (Torres et al., 2006), causing severe damages on them, since the diamondback moth interferes with plant growth, and it may even cause death or total loss (Da Silva, 2008). The study of moths has become a research topic in all producing regions, with the objective of obtaining technically appropriate, economically satisfactory and ecofriendly control measures (Thüler, 2006). P xylostella is considered to be one of the most difficult pests to control, so far insecticides are the main method for controlling them, diamides, avermectins, pyrethrins and Bacillus thuringiensis (Bt) being the main groups of insecticides used for eradicating this pest (Xia et al., 2014). Alongside the consequences for the environment such as elimination of natural enemies and the emergence of secondary pests, the increase of risks, as for the presence of residues in the edible product as for farmers (Brujanos, 2013), the inadequate and continuous use of the same active materials generated populations of P. xylostella resistant to insecticides (Attique et al., 2006, Khaliq et al., 2007). In many countries P. xylostella developed resistance to almost every insecticide used against it, (Furlong et al., 2013). According to the Arthropod Pesticide Resistance Database (APRD), for the year 2015, the diamondback moth had developed resistance to approximately 91 compounds with different ways of action, including organochloride, organophosphates, carbamates, pyrethroids, nereistoxin analogue, benzoylurea, Bt, avermectins, spinosyn, fipronil, indoxacarb, diacylhydrazines and diamides (APRD, 2015). However, the most important aspect when handling resistance to insecticides is the understanding of mechanisms that lead to the resistance of pests to them. Previous research studies, indicate that the mechanisms of resistance of insects to insecticides involved mutation of amino acids, the overexpression or mutation of detoxification, detoxifying enzymes, resistance to penetration and behavioral resistance (Ahmad et al., 2006; Bass et al., 2015). Nevertheless, most of common resistance mechanisms is metabolic resistance, with an increase in esterase, glutathione S-transferase and oxidases activities (Li et al., 2007; Bass et al., 2011). In P. xylostella, high levels of esterases correlate with resistance to organophosphates, carbamates, pyrethroids, indoxacarb, avermectins and benzoyl ureas (Sayyed et al., 2006; Eziah et al., 2009; Furlong et al., 2013); and the overexpression of glutathione S-transferase is responsible for resistance to organophosphates, pyrethroids and diamides, as well as indoxacarb (Furlong et al., 2013; Hu et al., 2014a). Also, the increase of oxidase activities contributes to resistance to carbamates, pyrethroids, nereistoxin analogue and diamides (Bautista et al., 2009; Furlong et al., 2013; Hu et al., 2014b). Insect metabolism plays an important role in the resistance of insecticides and, the knowledge about it can be used to improve the toxicity of insecticides, by mixing them with other insecticides that do not possess the same metabolic pathways (Mohan & Gujar, 2003). In relation to the afore mentioned, the objective of this research paper was to generate data about enzymatic mechanisms of resistance in different populations of P. xylostella in Guanajuato, Mexico, and their quantification through biochemical tests.

Material and Methods

Five populations of P. xylostella from Guanajuato, Mexico, were evaluated, which are: Valle de Santiago, San Luis de la Paz, Abasolo, Celaya, and for population known as “susceptible line,” individuals provided by the Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP), Campo Experimental Bajío in Celaya, Guanajuato, Mexico, were used; reproduced since 1996 with no selection pressure to insecticides. Considering they are the populations with the largest surface of sowing broccoli in Guanajuato, Mexico. Sampling was performed manually, in commercial batch of the populations mentioned earlier, pupa larvae and adults of P. xylostella were collected, which were placed in plastic containers inside coolers for their transportation into entomological boxes in laboratory at 27 °C, 50 % of relative humidity and 16:8 h light: dark, this for their reproduction until F1 and for having enough individuals of the same age for further study.

For each population of P. xylostella, five biochemical tests were used for determining the enzymatic levels of α-esterasas (α-Est), β-esterases (β-Est), oxidases (Oxid), glutathione S-transferases (GST) and acetylcholinesterases (AChE). Every test was run by triplicate in 96-wells plates and were read using the microplate reader.

Biochemical tests

For determining protein in P. xylostella larvae, the methodology described by Bradford (1976) and modified by Brogdon (1984), and Brogdon & Barber (1987) was used. Eight samples were placed in eppendorf tubes with 0.25, 0.50, 0.75, 1, 1.25, 1.50, 1.75, and 2 larvae of P. xylostella with four repetitions, 500 μL of buffer solution (KPO4) were added to 0.05 M and 7.2 pH, they were crushed and gauged to 1 mL in order to use it as an enzyme source. A 96-wells microplate was used, where 20 μL of homogenate were placed into each well, then 80 μL of buffer solution were added, plus 200 μL of diluted dye; this was done by triplicate for each repetition. Absorbance was read, using a 630 nm filter and the values of μg m·L-1 of protein were calculated, ranging from 80 to 140 μg.

Enzymatic levels of β and α-esterases were determined by means of Brogdon & Dickinson method (1983). In summary, 100 μL of the homogenate and 100 μL of β o α-naphthyl acetate were added into each well, it was incubated for 10 min and 100 μL of O-dianisidine were added, later, it was incubated for 2 min and were read with a 540 nm filter. As for oxidases, the methodology of Brogdon et al. (1997) was used, 100 μL of the homogenate, 200 μL of 5,5’ Tetramethylbenzidine dihydrochloride (TMB) and 25 μL of H2O2 at 3 % were added, it was incubated for 5 min and absorbance was read using a 620 nm filter. For GSTs, the method of Brogdon & Barber (1990) was used, in summary, 100 µL of the homogenate, 100 µL of reduced glutathione and 100 µL of 1-chloro 2,4’ dinitrobenzene (CDNB) were added, it was read at time zero (T0) and 5 min later (T5) using a 340 nm filter, the difference between both times were taken for the analysis of results. Lastly, following the methodology of Brogdon (1988), AChE levels were determined, placing 100 µL of the homogenate, 100 µL of acetylcholine iodide 3.0 mM and 100 µL of 5,5’-ditio-bis-2-nitrobenzoic acid (DTNB), the first reading (T0) and after 10 min (T10) were taken, using the 414 nm filter, the difference between both times were taken for the analysis of results.

With the absorbance of each enzyme, a frequency distribution was performed and a threshold of resistance was established. Finally, an analysis of variance (ANOVA) and a Tukey’s test (p=0.05) were performed, using the statistics software, version R 3.3.1.

Results and Discussion

For determining enzymatic levels, firstly, the quantity of proteins contained in P. xylostella larvae was calculated, and therefore obtaining the number of insects per sample, where from 0.25 to 1 larvae, protein content was lower than the required interval (from 80 to 120 µg); while from 1.25 to 2 larvae, protein content was within the limit allowed; choosing 1.75 larvae as the number of insects for the enzyme source (Figure 1). In relation to the enzyme source, Bradford (1976) mentions that the out-of-range values are not reliable for the quantification of proteins in tissues. While Dary et al. (1990) report that there exists a close relationship between sample size and protein quantity, which is why differences may be observed in the obtained results.


[Figure ID: f1] Figure 1.

Absorbance of proteins in Plutella xylostella homogenates in Phosphate Buffer (pH: 7.2).


To determine enzymatic levels, we will mention that the studied populations were under management with pyrethroid, macrocycle lactones, spinosads, diamides, phosphates and carbamic insecticides. In Table 1, absorbances per enzyme can be observed for different populations of P. xylostella; where significant differences were observed among studied populations for each enzyme. Where β and α-Est are the enzymes that were expressed in a higher quantity for all of the populations, followed by Oxid, while GST and AChE were the ones that presented a lower content. β-Est was expressed in a higher quantity for the population of Celaya, Guanajuato, Mexico, with an average of 2.337, while San Luis de la Paz, Guanajuato, Mexico and the susceptible line presented the lowest values for this enzyme, with absorbances of 1.722 and 1.847 respectively. While α-Est highest content was reported in Celaya, followed by Valle de Santiago with average values of 2.589 and 2.068 respectively; the susceptible line along with Abasolo, Guanajuato, Mexico presented the lowest absorbances for α-Est with a average of 1.401 and 1.582 respectively. In the case of Oxid, its highest content was observed to be expressed for the population of San Luis de la Paz, Guanajuato, Mexico, with an average value of 2.003, and its lowest content was observed for the population of Celaya, Guanajuato, Mexico, with an absorbance of 0.207. On the other hand, San Luis de la Paz reported the maximum value of absorbances for GST with a mean of 0.016, while Abasolo and Valle de Santiago were the ones that presented the lowest means for this enzyme with values of 0.001 and 0.004 respectively. Regarding AChE, the population corresponding to San Luis de la Paz was the one that presented a higher expression for this enzyme with a mean of 0.130, while the susceptible line reported a null expression for AChE.

Table 1.

Absorbance average of enzyme for different populations of Plutella xylostella in the state of Guanajuato, Mexico.


Population n β -Est α -Est GST AChE Oxid
Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD
Susceptible line 12 1.847±0.516bc 1.401±0.124d 0.006±0.006bc 0.000±0.000c 0.236±0.081c
Celaya 12 2.337±0.295a 2.589±0.142a 0.012±0.007ab 0.042±0.036b 0.207±0.021c
V. de Santiago 12 1.995±0.282b 2.068±0.075b 0.004±0.003c 0.065±0.035b 0.349±0.095b
Abasolo 12 1.974±0.408b 1.582±0.264cd 0.001±0.002c 0.008±0.016c 0.275±0.076bc
Sn. Luis de la Paz 12 1.722±0.018b 1.625±0.093c 0.016±0.004a 0.130±0.010a 2.003±0.145a

TFN1β-Est: β-Esterases, α-Est: α-Esterases, GST: Glutathione S-transferases, AChE: Acetylcholinesterase, Oxid: Oxidases.


Previous studies report that the main mechanism of resistance to pyrethroids, organochlorides Ponce et al. (2004), organophosphates (Bisset et al., 2001), indoxacarb Toshio et al. (2004), avermectin, and benzoylurea (Sayyed et al., 2006; Eziah et al., 2009; Furlong et al., 2013) is the cause of the high esterase content. For oxidases, which was the second detoxification mechanism, Pimentel et al. (2008) mention that the oxidases play an essential role in detoxification of several pesticides, directly participating in the disabling of the product or rusting for others enzymatic systems to enter and to be detoxified. Oxidase high values might be possibly due to the repetitive applications of abamectin in some of the locations; according to Clark et al. (1994), oxidative enzymes are the main physiological mechanism of resistance to abamectin. Finally, GSTs, in the production zone of the state of Guanajuato, are not a determinant factor for the presence of resistance, the obtained results are in agreement with the ones reported by Diaz et al. (2004) and Landeros et al. (2010), who used this same methodology, they report a low presence of GST in mosquitoes and red spider mites, respectively. One of the possible reasons to find low levels of GST, is that these enzymes are involved in the resistance to organophosphate insecticides (Ortelli et al., 2003); however, the elevated production of esterases in an enzyme more related to this toxicological group (Bisset et al., 2001).

Conclusions

In the production zone of the state of Guanajuato, Mexico, esterases and oxidases are the enzymes with the highest presence, responsible for the resistance in P. xylostella. As for GST and AChE, they do not present relevance as a detoxification mechanism, therefore reducing the application of organophosphate and carbamate products is proposed.


fn1Cite this paper: Cerna Chávez, E., Rodríguez Rodríguez, J. F., Hernández Juárez, A., Aguirre Uribe, L. A., Landeros Flores, J., Cervantes Ortiz, F., Guevara Acevedo, L. P., Ochoa Funetes, Y. M. (2018). Quantification of enzymes associated to insecticide resistance in different populations of Plutella xylostella L. (Lepidoptera: Plutelliidae) from the State of Guanajuato, Mexico. Revista Bio Ciencias 5(2), e424. doi: https://doi.org/10.15741/revbio.05.nesp.e424

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