Enhancing efficacy of Mexican fruit fly SIT programmes by large-scale incorporation of methoprene into pre-release diet

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1 J. Appl. Entomol. ORIGINAL ARTICLE Enhancing efficacy of Mexican fruit fly SIT programmes by large-scale incorporation of methoprene into pre-release diet Y. Gómez 1, P. E. A. Teal 2 and R. Pereira 3 1 Medfly Program DGSV-SAGARPA, Metapa, Chiapas, Mexico 2 Center for Medical, Agricultural and Veterinary Entomology, USDA-ARS, Gainesville, FL, USA 3 Insect Pest Control Section, Joint FAO/IAEA Division of Nuclear Technique in Food and Agriculture, Vienna, Austria Keywords adult diet, Anasptrepha ludens, juvenile hormone, sexual maturation, sexual performance Correspondence R. Pereira (corresponding author), Insect Pest Control Section, Joint FAO/IAEA Division of Nuclear Technique in Food and Agriculture, Vienna, Austria. r.cardosopereira@iaea.org Received: April 7, 2011; accepted: November 14, doi: /j x Abstract The juvenile hormone analogue, methoprene, has been documented to accelerate the development of reproductive competence and sexual signalling of Caribbean (Anastrepha suspensa), the Mexican (Anastrepha ludens), the South American (Anastrepha fraterculus) and West Indian (Anastrepha obliqua) tephritid fruit flies. The incorporation of methoprene into sterile fly release protocols at fly emergence and handling facilities is a key step required for large-scale application of the technology to field release strategies. The goal of our study was to develop a method to supply, in large scale, methoprene to sterile Mexican fruit flies for release in the current Sterile Insect Technique (SIT) programme in Mexico. In field cage tests, the isolation index of sterile males after treatment with methoprene was reduced, increasing the percentage of mating between laboratory sterile males and wild females. In laboratory trials, males fed a diet containing 0.05% or 0.1% methoprene mated 4-day earlier than untreated control males. In a pilot area tested in 3500 ha to measure the impact of the sterile releases when methoprene was supplied, no adult wild flies were detected and no larvae were found after sampling more than 330 kg of fruit both in the treated and untreated blocks. Based on the results obtained in this study, we recommend the incorporation of the methoprene on adult diet to improve the Mexican fruit fly male sexual performance when release in the field, contributing to the increase in the cost-effectiveness of the SIT programme. Introduction The use of the Sterile Insect Technique (SIT), applied as part of an area-wide integrated pest management approach (Hendrichs et al. 2007), provides an environmentally safe and species-specific method to suppress, and in some situations eradicate, tephritid fruit flies of agricultural importance worldwide (Hendrichs and Robinson 2009). In fact, Mexico has been successfully applying the technique for over 30 years at its southern border to exclude the Mediterranean fruit fly (Ceratitis capitata Wiedemann) (Enkerlin 2005). California employs the technique on a routine basis as a preventive measure to avoid the establishment of both Mediterranean fruit fly and Mexican fruit fly (Anastrepha ludens) outbreaks (Dowell et al. 2000; Hendrichs et al. 2005; USDA/ APHIS 2009). Also in Florida, the technique is being used as a prophylactic method to ensure that the Mediterranean fruit fly does not become established (USDA/FDACS 2009). Control is achieved by the mass release of sterile males at overflooding ratios so that they mate with wild females, which then do not produce offspring (Knipling 1955). Continuous vigilance and optimization of the SIT is required to assure that sterile males have the capacity to compete with wild males for mates. Previous studies documented that juvenile hormone is a key component in the regulation of sexual maturity in males of the Caribbean fruit fly, Anastrepha ª 2011 Blackwell Verlag, GmbH 1

2 Large-scale incorporation of methoprene for SIT programmes Y. Gómez, P. E. A. Teal and R. Pereira suspensa (Loew) (Teal and Gómez-Simuta 2002) and that application of juvenile hormone analogues including methoprene and fenoxycarb to either pupae or adults accelerates development of reproductive competence and sexual signalling (Teal et al. 2000, 2007; Pereira et al. 2010a), However, the longevity of males treated with methoprene did not significantly differ from untreated males (Pereira et al. 2010b). Additionally, in the South American (Anastrepha fraterculus [Wiedmann]) and West Indian (Anastrepha obliqua [Mcquart]) tephritid fruit flies, a similar effect of methoprene application has been reported (Aluja et al. 2009; Segura et al. 2009). Our basic research on the mating system of the Mexican fruit fly indicates that application of methoprene successfully accelerates sexual maturation and signalling in the Mexican fruit fly (Teal et al. 2007; Gómez and Teal 2010a,b; Pereira et al. 2011). Developing techniques that accelerate maturation, reproductive competency and sexual signalling in sterile males will increase male competitiveness and improve SIT efficacy. The high impact of mortality of sterile males in the field before reaching sexual maturation (Hendrichs et al. 1993) can be reduced by the earlier male maturation, and the costs associated with holding adults prior to release can also be reduced. Therefore, techniques for incorporating hormone supplement therapy using juvenile hormone analogues (JH) like methoprene into handling protocols for mass produced sterile males are important steps in the development of cost-effective methods of control of these economic important fruit fly pests. Additionally, the number of sterile flies released to control outbreaks can be reduced because hormone-treated males are more competitive than untreated males (Gómez and Teal 2010, Pereira et al. 2011). However, Pereira et al. (2009) have suggested that accelerated maturity because of hormone manipulation may have serious nutritional consequences as there is less time for adult flies to acquire reserves. Thus, the addition of protein into the adult diet when applying methoprene can be critical to methods employed to accelerate male development by delivering hormone supplements. Although the need for access to protein-rich diets has been demonstrated in adult sterile males and methoprene application results in accelerated development of pheromone calling (Teal et al. 2007) and improved male competitiveness (Pereira et al. 2010a), the technology had not been incorporated into fly emergence and releases facilities. Our study focused on assessing the: (i) mating competitiveness of A. ludens males treated with methoprene by topical application; (ii) quality and mating competitiveness of sterile males fed on a diet incorporating methoprene prior to release; and (iii) the effect of releasing sterile Mexican fruit flies treated with juvenile hormone on a wild population and the related level of fruit infestation in a pilot area at Montemorelos, Nuevo Leon, Mexico. Materials and methods The laboratory and field cage tests were carried out in the Mediterranean fruit fly Programme Methods Development Department located in Metapa de Dominguez Chiapas, Mexico. Methoprene application We selected methoprene as the analogue of juvenile hormone (JH) for all our studies. For the laboratory experiments, as well as the evaluation of the largescale handling of sterile flies emerged in towers, methoprene was provided as part of the adult diet. For the incorporation of methoprene into the sterile fly adult diet, we selected Precor 1.2% MR (imported by Better World Manufacturing, D.F. Mexico); it was locally available, being marketed as an insect growth regulator. The formulation contained 1.2% methoprene in a water soluble formulation. Concentrations of 0.025%, 0.5% and 0.1% of methoprene were prepared by dilution in water and used to prepare the adult diets. For the field cage experiment, methoprene was applied topically to sterile flies with 5 ug of methoprene in 1 ul acetone solution following the protocol described by Pereira et al. (2009). Experiment 1: Mating performance of sterile males treated with methoprene incorporated into the adult diet in laboratory Forty-eight hours before fly emergence, the pupae of A. ludens were irradiated in a cobalt 60 source (Panoramic wet source Irradiator model JS-7400 Nordion International, Kanata Ontario, Canada) with a dose of 80 Gy. This is the recommended dose to guarantee the sterilization of males (FAO/IAEA/ USDA 2003). Within a few hours of adult emergence, the sterile flies were sorted by sex and placed in separate 30 by 30 by 30 cm Plexiglass cages. Flies were fed with adult food (yeast hydrolysate/sucrose; 1 : 3 ratio) mixed with water containing 0.025% of methoprene (treatment 1), mixed with water containing 0.05% of methoprene (treatment 2), mixed 2 ª 2011 Blackwell Verlag, GmbH

3 Y. Gómez, P. E. A. Teal and R. Pereira Large-scale incorporation of methoprene for SIT programmes with water containing 0.1% of methoprene (treatment 3), and without methoprene treatment (control). Wild flies were collected as larvae from host fruit available, mainly Sour Orange Citrus aurantium and white zapote Casimiroa edulis in the region. The fruits were placed on a fruit sampling container with moistened vermiculite at the bottom, where the wild pupae were collected. Pupae were then placed in flight ability tubes to select only the fliers to conduct the experiment. The Plexiglass cages were held indoors at 25 C, 75% RH with a 14 : 10 L/D cycle. Ten sterile males and 10 wild mature females (since the flies were 1 day old) were released into the 30 by 30 by 30 cm Plexiglas cages, and the numbers of matings per day were recorded. Ten replications were conducted for each sterile male age (1 8 days of age), both in treatment and the control. Experiment 2: Mating performance of JH topically treated sterile males in field cages In the mating performance test in field cages (2 m high and 3 m diameter) with two potted plant (mango, Manguifera indica and sour orange C. auratium tree) about 1.8 m high, the experiment was carried out following the standard protocol (FAO/IAEA/ USDA 2003). The following treatments were tested: (i) 6 days old sterile males and females without treatment; (ii) 6 days old sterile males and females treated; (iii) 12 days old sterile sexually mature males and females without treatment. In all cases, sterile flies were competing with 18 days old sexually mature wild male and female flies without methoprene treatment. Within a few hours after adult emergence, sterile flies were sorted by sex and placed in separate 30 by 30 by 30 cm Plexiglas cages. Flies were provided with adult food (hydrolysate yeast/sucrose; 1 : 3 ratio). On the day of fly emergence, males (treatment 2) were treated topically with 5 lg of methoprene (Teal et al. 2000). Flies of treatments one and three were not treated with methoprene. Wild flies were obtained as described for the field cage mating performance test. Wild flies were handled and fed exactly the same way as sterile flies. At the day of emergence, the wild flies were sorted by sex and placed in separate 30 by 30 by 30 cm cages 18 days before the test (according the treatment). All flies were held indoors at 25 C and 75% RH with a 14 : 10 L/D cycle. To separate among treatments, flies were marked with a dot of water-based paint on the thorax (FAO/IAEA/USDA 2003). On the day of the test, 20 males and 20 females from each treatment per cage competing with 20 wild males and 20 wild females were introduced into a field cage at 17:00 h. Mating was monitored until 19:00 h, and the mating performance was estimated based on the type of mating found using the indices proposed by Cayol et al. (1999): ISI (index of sexual isolation), FRPI (female relative performance index), MRPI (male relative performance index), as well as the RSI (relative sterility index) proposed by McInnis et al. (1996). For each treatment, four replications were conducted. Experiment 3: Sterile fly quality after incorporation of methoprene into the adult diet and handling in two types of fly emergence towers This evaluation was conducted at the Mexican fruit fly emergence and release facility in Montemorelos, Nuevo Leon, Mexico. Sterile Mexican fruit flies pupae are received and packed daily using the standard protocol authorized by the Mexican National Fruit Fly Campaign (DGSV/CESAVESIN 2006). The flies are produced, sterilized and shipped (over 16 h average) to the sterile fly emergence facility as pupae from the mass-rearing facility in Metapa de Domínguez, Chiapas, Mexico. Two types of fly emergence towers were evaluated. The Worley tower holds pupae of Mexican fruit fly per aluminium rack (with 75 racks per tower) (FAO 2007), and the Mexico tower has higher aluminium rack with screens on the four sides, holding pupae per rack (18 racks per tower). Both types of towers are under use at the Mexican National Fruit Fly Campaign and were prepared following the same procedures (Fig. 1). The water was provided in a special impregnated cotton device with 5 by 20 cm. Food used was the Mubarqui adult diet composed of Amaranto powder, glase sugar, peanuts and egg (DGSV/CESAVESIN 2006). The total capacity of both towers was pupae and pupae, respectively, in the Worley and Mexico fly emergence towers, and pupae were loaded onto towers on the day of the arrival of shipments. A representative sample of irradiated pupae of Mexican fruit fly per shipment was randomly selected, and the following four treatments were evaluated: 1 Flies fed on Mubarqui adult diet plus methoprene held in Worley fly emergence towers; 2 Flies fed on Mubarqui adult diet without methoprene held in Worley fly emergence towers; ª 2011 Blackwell Verlag, GmbH 3

4 Large-scale incorporation of methoprene for SIT programmes Y. Gómez, P. E. A. Teal and R. Pereira Worley tower Mexico tower Fig. 1 Fly emergence towers used (Worley tower [left] and Mexico tower [right]) for the evaluation of methoprene incorporation into the Anastrepha ludens sterile fly Mubarqui adult diet. 3 Flies fed on Mubarqui adult diet plus methoprene held in Mexico fly emergence towers; 4 Flies fed on Mubarqui adult diet without methoprene held in Mexico fly emergence towers. The incorporation of methoprene was performed by diluting 100 ml of the Precor 1.2% MR (formulation containing 1.2% methoprene) in 900 ml of water. This solution was mixed with 1 kg of Mubarqui adult diet in use for the Mexican National Fruit Fly Campaign. Once the methoprene was incorporated into the adult diet, it was then coated on a cm piece of paper. Each piece of paper contains approximately 25 g of dry adult diet. One piece of paper with the adult diet was added to each rack containing and pupae in Worley and Mexico towers, respectively, for towers of treatments A and C. The diet for treatments B and D was prepared the same way but without the incorporation of methoprene. The towers were moved to the fly emergence rooms, following the standard protocol (DGSV/CESAVESIN 2006). Six days after loading pupae into towers (flies approximately 4 days old), the towers were moved to the chilling room (3 C for 30 min) to knockdown flies to immobilize and transfer them to the chilled adult box prior to aerial release. The quality control parameters evaluated were the percentage of emergence and fliers (FAO/IAEA/ USDA 2003), absolute fliers index (described later) and the sexual activity of the males (pheromone calling). The absolute fliers index was obtained using the same procedure as for the emergence and flight ability tests (FAO/IAEA/USDA 2003), but with a sample of 100 chilled adults. The percentage of absolute fliers was obtained by determining the amount of flies that left the tube. To determine the effect of the methoprene on pheromone calling behaviour, 20 males per treatment were transferred from the aerial release machine to a 30 by 30 by 30 cm Plexiglas cage. Water and food (Mubarqui diet without methoprene) were provided to all flies. The pheromone calling behaviour of males was recorded 1 day later (when flies were about 5 days old, i.e. 1 day after the age when flies are released). The number of males calling was registered every 15 min from 16:00 to 18:00 h (peak of sexual activity of A. ludens). Twelve replications per treatment were conducted. Experiment 4: Comparison of the impact of sterile flies treated with and without methoprene and aerially released in a pilot area To evaluate the effect of the methoprene on the released flies, we selected two areas with similar environmental conditions and with the same level of fertile fly captures (lower than 0.01 fly per trap per day [FTD]) at Montemorelos, Nuevo Leon, Mexico. One block was selected for releasing flies treated with methoprene (from both treatments Worley and Mexico towers), and other block (two blocks of 4000 ha away) was selected to release the control flies (without treatment). Flies were released at the adult age of 4 days (6 days after reception and packing of pupae into towers). Both release blocks contained approximately 3500 ha of citrus. Once a week, for 16 weeks, 2500 flies per ha were released in both blocks and monitored using adult trapping (multilure traps with hydrolizate protein as attractant per each 100 ha) and fruit sampling taking a sample of host fruit approximately each 15 ha, depending on the abundance of fruits. Statistical analysis In experiment 1, the percentage of mated males at day 6 was compared among treatments by means of a one-way anova. In experiment 2, the mean indexes registered during the field cage tests were compared by a one-way anova. In experiment 3, 4 ª 2011 Blackwell Verlag, GmbH

5 Y. Gómez, P. E. A. Teal and R. Pereira Large-scale incorporation of methoprene for SIT programmes adult emergence, absolute fliers and per cent of calling males were compared among treatments by means of a two-way anova, with type of tower and methoprene treatment as factors. All the data were ArcsenÖx transformed for the statistical analysis. In the case of anova-detected significant differences, multiple comparisons were carried out, and JMP 5.01 Statistical Software was used to perform the analysis. Results Experiment 1: Mating performance of sterile males treated with methoprene incorporated into the adult diet in laboratory 2,4 P = ). Multiple comparison showed that 6-day-old sterile males treated with methoprene exhibited a significantly lower ISI ( ) than the recorded ISI for the non-treated males of the same age ( ), but not significantly different from the mature 12-day-old sterile males ( ). The FRPI presented significant differences among treatments (F = , d.f. 2,4 P = ). FRPI recorded for both treated () ) and mature females () ) showed higher values than untreated 6 days old females () ) (Fig. 3). The MRPI showed significant differences among the three treatments (F = , d.f. 2,4 P < 0.001). MRPI multiple comparisons reflect a significantly The mating percentages with wild females in laboratory cages of A. ludens sterile males at different ages, and fed with or without methoprene, are shown in Fig. 2. In all the treatments with methoprene, approximately 50% of males mate when they reach 5 days of adult age, while untreated males reach that percentage of mating only on day 8. The differences in sexual maturation were statistically significant (F = 6.577, d.f. 3,5, P = ). Multiple comparison showed that the concentration of 0.05% and 0.1% was significantly more efficient to accelerate sexual maturation than the other tested treatments. Experiment 2: Mating performance of JH topically treated sterile males in field cages The results of A. ludens sterile male performance when competing in field cages with mature wild males are presented in Fig. 3. The ISI showed significant differences among treatments (F = 6.792, d.f. Mating (%) Control 0.02% 0.05% 0.10% Age (days) Fig. 2 Laboratory mating percentage by Anastrepa ludens sterile males at increasing ages, mating with wild mature females. Sterile Males were fed with adult diet (sugar/yeast hydrolysate; 3 : 1), mixed with a solution of water with or without (untreated control) different concentrations of methoprene. Fig. 3 Sexual performance indices (SD) of Anastrepha ludens sterile flies when competing in field cage tests with 18 days old wild males and females as (a) 6 days old sterile males and females without treatment (untreated control); (b) 6 days old sterile males and females treated with methoprene (treated); and (c) twelve days old sterile males and females (sexually mature). Indices in the figure are: Index of Sexual Isolation; Male Relative Performance Index; Female Relative Performance Index; and Relative Sterility Index. ª 2011 Blackwell Verlag, GmbH 5

6 Large-scale incorporation of methoprene for SIT programmes Y. Gómez, P. E. A. Teal and R. Pereira better mating performance for 6-day-old treated males () ) and also mature males () ) than for untreated 6-day-old males () ). Thus, methoprene-treated sterile males obtained more mating than the control group of mass-reared laboratory males. The RSI statistically differed among treatments (F = 5.003, d.f. 2,4 P = 0.021). RSI recorded for methoprene-treated males ( ) was significantly different than the data reported for untreated males ( ) and shows no significant differences when compared with the fully mature males ( ). Experiment 3: Sterile fly quality after incorporation of methoprene into the adult diet and handling in two types of fly emergence towers The average of adult emergence from the pupal batches used during the test was 91 5%. Regardless of methoprene application and tower used, there were no significant effects (F = 0.092, d.f. 3,9, P = 0.932, for methoprene application and F = 0.091, d.f. 3,9 P = for tower type) or interactions between methoprene and tower (F = 0.093, d.f. 3,9 P = 0.910) on absolute fliers. The results obtained clearly indicated that the incorporation of methoprene into the adult diet had a positive effect on the pheromone calling behaviour of A. ludens males (Fig. 4). There was no influence of both tower systems on male pheromone calling behaviour. In Mexico towers, the males fed with adult diet containing methoprene showed significantly earlier calling behaviour than males fed on a diet without methoprene (F = 106.5, d.f. 1,11, P < 0.001). In Worley towers, there was a similar response, with males feeding on a diet containing Males calling (%) 80 a Mexico methoprene b Mexico control Treatments a Worley methoprene b Worley control Fig. 4 Percentage (SD) of Anastrepha ludens sterile males showing pheromone calling behaviour 1 day after release (about 5 days of age), after being packed in Mexico or Worley fly emergence towers with and without methoprene incorporated into the adult diet. methoprene showing earlier calling behaviour than males fed without methoprene (F = 19.74, d.f. 1,11, P = 0.001). Experiment 4: Comparison of the impact of sterile flies treated with and without methoprene and aerially released in a pilot area Weekly trapping data from treatment and control blocks showed a sterile male recapture rate, respectively, of FTD and FTD, whereas for wild males, the FTD for both treatments was zero. In terms of fruit infestation, we collected and dissected a total of 268 samples of citrus fruit from the treated block receiving sterile flies treated with methoprene (158 samples were collected from trees and 109 samples from the soil), and in the control block receiving untreated sterile flies, we collected a total of 279 fruit samples (156 samples were collected from trees and 123 samples from the soil); however, no larvae were found in both release blocks. Discussion The overall results of these experiments showed that incorporation of methoprene into the diet enhanced the sexual behaviour of sterile flies. This was reflected by acceleration in reproductive development time, an earlier pheromone calling behaviour, and a significantly higher mating performance in methoprene-treated males. Therefore, the incorporation of the methoprene into the adult pre-release diet makes young sterile males sexually more competitive when compared with young untreated males. The quality control procedures described in the FAO/IAEA/USDA (2003) international manual establish that the ISI for laboratory and wild populations of C. capitata should normally range between 0.15 and 0.4. Our results from field cage studies with A. ludens showed an ISI value of 0.4 (Fig. 3), indicating that the reproductive behaviour of young sterile males that become sexually mature early by feeding as adults on methoprene is close to the internationally acceptable standards for the Mediterranean fruit fly. The high value of the ISI for untreated sterile males indicates that the common practice of releasing immature 6-day-old males results in few matings and a high mortality until these males reach their sexual maturity (Gómez and Teal 2010). On the other hand, sterile males treated with methoprene have not only a higher probability of surviving 6 ª 2011 Blackwell Verlag, GmbH

7 Y. Gómez, P. E. A. Teal and R. Pereira Large-scale incorporation of methoprene for SIT programmes towards sexual maturity in the field, but also of mating immediately after release with wild females, which increases the overall SIT efficacy. The per cent of mating between wild males and wild females decreased when methoprene-treated sterile males were present in the field cages, this is reflected in the value of RSI obtained. This is a highly desirable feature for SIT application because more sexually competent sterile males are available to compete with wild males (Knipling 1955). Our results also showed that methoprene had no effect on sterile females because the RSI between sterile males and sterile females was reduced in treated flies. Therefore, as reported by Segura et al. (2009) for A. fratreculus flies, methoprene-treated A. ludens sterile females, unlike treated sterile males, did not engage in mating earlier in life, but the anticipation is parallel to the male maturation (Pereira et al. 2011). Normally, sterile Mexican fruit flies are released when they are 6 days old and they need to survive in the field looking for food and water before reaching sexual maturity and mating at about 12 days of age (USDA/APHIS 2009). During that period, the sterile flies are exposed to adverse environmental conditions and predators. Recapture data show that high number of flies in the field decreases rapidly before they complete their reproductive development and are able to transfer sterile sperm to the wild population. Our data show that both tower systems used are viable for large scale emergence and handling of sterile flies. Both towers are similar in cost, but the Mexico-type tower has better air circulation for the screen side open in that way that is not necessary to use the fan extractor used for Worley-type tower. This characteristic reduces the holding, and the flies showed better calling behaviour as was recorded in our experiment. In both systems, there was a significant increase in the numbers of males pheromone calling at 5 days after feeding methoprene incorporated into a yeast hydrolysate/sugar pre-release diet. The comparison of the release of treated and untreated sterile insect in the pilot area did not reflect a high impact in reducing the wild population because the density of wild flies was very low and no wild flies were captured in the traps or infested fruits were found in our treatment and control blocks. Nevertheless, sterile male recapture data indicate that the incorporation of methoprene into the action release protocols increased the recapture rate of sterile insect released, and these results reflect that more sterile males are on the field after release. Similar results were obtained when methoprenetreated sterile males were released in Sinaloa Mexico (personal communication by Comite Estatal de Sanidad Vegetal de Sinaloa), the recapture data reported a FTD of 1.4 for methoprene-treated sterile flies and a FTD of 0.67 for non-treated sterile flies. In conclusion, our studies on sexual behaviour of methoprene-treated sterile A. ludens males indicated that incorporation of methoprene into the adult diet effectively accelerated the reproductive development. Under normal circumstances, releasing 5 6 days old untreated flies into the field, we estimate that most of the released flies never reach sexual maturity. The method developed of incorporating of the methoprene into the adult diet was shown to be viable in large-scale operations, accelerating significantly male maturation without increasing much the handling time at the fly emergence facility or the total cost of the adult food. This methodology has been transferred and will contribute to the efficiency of the Mexican fruit Fly SIT programme, since it will result in the release of sexually mature males ready to mate with wild females after release. Acknowledgements This project was funded in part by the International Atomic Energy Agency (Research Contract OIEA/ MEXICO/12861), acknowledgments also to Javier Coutiño Ibarias for the technician support; Salvador Meza for the analysis and the field cage test and the Comite Estatal de Sanidad Vegetal de Nuevo Leon and Martin de los Santos fort their collaboration. 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