Introduction

Huanglongbing (HLB) is considered the most destructive citrus disease. Its capacity of dissemination and pathogenicity represents a high risk for citrus production in Mexico and other parts of the world (^{SENASICA, 2014}). Diaphorina citri Kuwayama (Hemiptera: Liviidae), also known as the Asian Citrus Psyllid (ACP), is the primary vector of HLB, thus its control is vital for the adequate management of the disease (^{Halbert and Manjunath, 2004}; ^{SENASICA, 2014}). The association of ACP and HLB has left defenseless more than 67 000 citrus producers in Mexico, who concentrate a production of more than 7 million Mg, with an approximate value of MX$10.2 billion (^{SENASICA, 2014}). To halt the expansion of the disease, the Mexican government, as in other countries, has initiated a control program based on three strategies: chemical control of the vector (ACP) in Regional Control Areas (RCA), the production and use of certified plants and the elimination of infected trees at the moment of their detection (^{SENASICA, 2014}; ^{Lewis-Rosenblum et al, 2015}). However, the common practice among the producers is the indiscriminate application of various active insecticide materials each season (^{Boina et al, 2009}). In the present situation, it is foreseen that the development of resistance to different active ingredients will increase considerably in the coming years. ^{Tiwari et al. (2012}) documented that ACP can survive the application of various insecticides; mainly those that share at least one resistance mechanism and that were previously used intensively to control other citrus pests. This meant that to control D. citri in orchards of Florida, U.S., the initial dose of imidacloprid would be increased 35 times and 18 that of chlorpyrifos (^{Tiwari et al, 2011}).

In integrated pest management it is important to detect early changes in susceptibility to insecticides in use in field populations and thus avoid unnecessary applications to prolong the useful lifespan of the available insecticides (^{Georghiou and Taylor, 1986}; ^{Diaz-Zorilla etal., 2011} ). This early detection facilitates the application of optional control measures and is the basis of the chemical control programs of a determined pest or vector. The objective of the present study was to determine the susceptibility of a population of D. citri, from Martínez de la Torre, to different insecticide materials of common use in the citrus regions of Veracruz, Mexico. The susceptibility of this population was compared with that of a laboratory population to determine the relative response of activity and to infer the development of resistance.

Materials and Methods

The study was carried out from May of 2012 to April of 2013 in the Vectors Laboratory of the Graduate Program of Plant Health-Entomology and Acarology, Colegio de Postgraduados, Campus Montecillo, Estado de Mexico, and in the Local Board of Plant Health of the ejido La Palma in Martínez de la Torre, Veracruz.

Results and Discussion

The adults of D. citri exhibited different susceptibility to the seven insecticides evaluated. The Cazones colony was more susceptible than the Martinez colony; the former was maintained isolated from the selection pressure of insecticides for more than five years, which allowed it to be considered as the reference colony in this study. Resistance of pests to insecticides is one of the most serious problems that prevents the advancement of vector management programs. Switching insecticides when the one previously used is no longer effective is not a viable solution (^{Ortega et al, 1998}). Knowing the susceptibility of field populations to the insecticides can help to reduce the use of non-effective products, which increase production costs and contaminate the environment.

Lambda-cyhalothrin showed highest toxicity in the Cazones colony, according to the LD₅₀; in decreasing order, it was followed by abamectin, dimethoate, methomyl, malathion, imidacloprid and endosulfán. The group of pyrethroids (PIRT) presented the highest toxicity, followed by the microbials (I-MICR), aliphatic phosphates (FA-SM), carbamates (CA-MM) and carboxylated phosphates (F-Cx). The group of organochlorine cyclodienes (ОС-Cd) was the least toxic (Table 1). Slopes registered for all of the insecticides were higher than 0.84±0.13, which indicated certain uniformity of the colony for responding to the application of the products evaluated.

Table 1 Toxicity of insecticides in adults of D. citri, from Cazones de Herrera (Cazones colony) and Martínez de la Torre (Martínez colony), Veracruz, Mexico. 

^†LD or LC₅₀: Lethal dose or concentration 50 in ng of active ingredient per insect (ng AI insect^-1); ^¶LC: Confidence limits at 95 %; ^§b±EE: slope with standard error; ^ϸϰ²: estimation of the straight line in the regression; ^¤FR₅₀: resistance factor 50 (LD₅₀or LC₅₀ of Martinez/ LD₅₀ or LC₅₀ of Cazones). The value of imidacloprid is expressed in mg mL^-1 to represent a Lethal Concentration 50 (LC₅₀).

Lambda-cyhalothrin was also the most toxic product for the Martinez population, according to the DL . Methomyl showed the lowest toxicity. Between these two products in order of effectiveness are abamectin, endosulfán, malathion and dimethoate. The PIRT group was also effective, in contrast to the Cazones colony, the CA-MM were least toxic. Registered slopes varied from 0.51 ± 0.03 (dimethoate) to 1.29±0.087 (abamectin), which indicated that population response was uniform to selection with abamectin, lambda-cyhalothrin and endosulfán, and heterogeneous for malathion, dimethoate and methomyl. The latter two presented a high resistance factor (RF₅₀) (Table 1).

The RF₅₀ of each insecticide evaluated in the Martinez colony (Table 1 ), with respect to the Cazones colony, indicated susceptibility to the insecticides abamectin, endosulfán, lambda-cyhalothrin, imidacloprid and malathion, considering that values were similar to those of the reference population and to the overlap of the confidence limits. Estimated methomyl and dimethoate RF₅₀ values indicated that genes that supplying resistance to these insecticides were expressed in adults of D. citri from Martinez de la Torre. Separation of the confidence limits of log dose-Probit lines allowed the confirmation of the lack of susceptibility of the Martinez colony according to the LD₅₀ of the evaluated toxic. This showed the capacity of field population adults to survive, independently of the presence of these products (Table 1).

Cazones colony has been maintained free of the pathogen for more than five years and from the application of pesticides. Adults of this colony were susceptible to the seven insecticides evaluated, thus the log dose-Probit lines of each insecticide can be used as reference for managing the resistance of the psyllid in Veracruz, Mexico (^{Prabhaker et al, 2006}). According to ^{Tiwari et al. (2010}), the use of insects free of HLB gives certainty to the bioassays, since the susceptibility of insects to insecticides is reduced in populations of ACP infected with Candidatus Liberibacter asiaticus.

Martinez colony contains resistance genes in detectable frequency for the insecticides methomyl and dimethoate. In Mexico and in particular in the Persa lime producing region of Martínez de la Torre, insecticides of the principal chemical groups (organochlorines, organophosphates, carbamates, neonicotinoids and pyrethroids, among others) have been used against D. citri, since they first were sold for this purpose (^{Córtez-Mondaca et al, 2010}; ^{Díaz-Zorrilla et al, 2011}). It is probable that the resistance levels registered in our study are related to the previous history of exposure. This would concur with the observations of some farmers of the region, with respect to the lack of control of D. citri with applications of conventional insecticides, and with ^{Tiwari et al. (2011}); they indicated that the resistance of D. citri to organochlorines and neonicotinoids insecticides was higher in areas of Florida, where they were used with more frequency.

In contrast, the relative susceptibility of the Martinez colony to abamectin, lambda-cyhalothrin, endosulfán, malathion and imidacloprid could be explained by the scant selection of the resistance mechanisms associated to these molecules. Therefore, the frequency of resistance genes is not yet found at detectable levels. High levels of resistance sometimes have shown opposite effects. In populations of D. citri of Florida exposed to sub-lethal doses of imidacloprid, increments of detoxifying enzymes (glutathione transferase (GTS) and cytochrome P450 oxidases) were detected, allowing them to survive the exposure to insecticides (^{Tiwari etal, 2011}; 2012). The resistance to chlorpyrifos and fenpropathrin continues to increase in Florida, and these products are used intensively for the control of this pest (^{Tiwari et al, 2013}). Imidacloprid is a costly product, but it is effective against D. citri (^{Hernández-Fuentes et al., 2012}). The product exerts an agonist action in the postsynaptic acetylcholine receptors (^{IRAC, 2014}), which is the specific site of the insects that confers them low toxicity to mammals (^{Liu etal, 2005}; ^{Phua et al, 2009}). Furthermore, it requires an adequate and opportune application to be translocated and to take down the populations (^{Tomizawa and Casida, 2003}).

Dimethoate is a product used in Mexico for the control of sucking insects for various decades (^{Hernández-Fuentes et al, 2012}). It inhibits the action of the acetylcholinesterase (^{IRAC, 2014}), and it is used in the control of D. citri in orchards with a low infestation rate. Dimethoate is widely accepted by the growers because apparently it is effective against diverse pests. This product is recommended in technical brochures distributed by the Federal Government (^{Córtez-Mondaca et al, 2010}; ^{Díaz-Zorrilla et al, 2011}; ^{Robles-González etal, 2011}). However, in those texts it is emphasized that the product should only be applied once a year and at the recommended dose to avoid the selection of resistant insects (^{Córtez-Mondaca et al,}2010). Dimethoate can be degraded by more than four types of detoxifying enzymes, thus resistance crossed with various insecticides that share at least one resistance mechanism could be created (^{Lagunes-Tejeda and Villaueva-Jiménez, 1994}). This phenomenon is common among organophosphate compounds and carbamates (^{Tiwari et al, 2012}) and it can explain partially the tolerance of the field population to methomyl (83-58 X) and the separation of the response lines between the colonies studied. Acetylcholinesterase (АСҺЕ) and GTS (^{Zhu and Gao, 1999}; ^{Gao and Zhu, 2000}; ^{Tiwari et al, 2012}) are the enzymes present in common mechanisms that were identified in the tolerance of various insects to dimethoate and methomyl. Methomyl is not authorized for the control of D. citri in Mexico and apparently was not used in recent years to combat ACP in Martínez de la Torre. This confirms the importance of having the susceptibility reference for most of the possible toxicological groups, even products that could be authorized soon for the control of ACP, and prevent control problems (^{Prabhaker et al, 2006}). The continuous and anarchic application of insecticides in the citrus regions of Veracruz will lead to reducing the useful life of the toxicological groups. This may be the case of dimethoate with chlorpyrifos, which share resistance mechanisms by action of GTS and cytochrome P450 oxidases (^{Ortega, 1998}; ^{Ortega et al, 1998}; ^{Tiwari et al, 2011}). The use of dimethoate and methomyl should be limited in Martínez de la Torre, due to the resistance levels found in D. citri, in order not to continue incrementing resistance in these and other products. According to ^{Georghiou and Taylor (1986}), the capacity of controlling a species could be lost with presence of 1 % of resistant individuals in one to seven generations.

The development and establishment of resistance is possible in scenarios with high selection pressure, without adequate rotation of insecticides or with the use of the same toxicological group (^{Ortega et al, 1998}; ^{Lagunes-Tejeda et al, 2009}). Resistance is a dynamic process (^{Georghiou and Mellon, 1983}), thus it appears necessary to carry out a constant monitoring in each agricultural area. The base lines obtained in this study are a reference for monitoring resistance to these insecticides applied against D. citri. According to ^{Tiwari et al. (2011}), in 1998 the presence of D. citri was reported in citrus orchards of Florida; after that, the regional management of the pest has been carried out continuously, based on the rational and selective use of insecticides. Management of D. citri is feasible based on toxicological groups; therefore, the frequency of application is reduced, the use of mixtures and of insecticides of prolonged environmental persistence is avoided, the most opportune moments are defined for carrying out chemical control actions per region and methods of lower environmental impact are incorporated.

Conclusions

The Cazones colony of D. citri originally from Cazones de Herrera, Veracruz, was susceptible to the insecticides endosulfán, dimethoate, imidacloprid, malathion, methomyl, abamectin and lambdacyhalothrin, The Martinez colony, Martínez de la Torre, Veracruz, showed susceptibility to endosulfán, imidacloprid, abamectin, malathion and lambda-cyhalothrin and resistance to dimethoate and methomyl.

The log dose-Probit lines of each insecticide of the Cazones colony can be a reference for decision making in the management of resistance of D. citri.