Protección vegetal

 

Prediction of surviving larvae of mexican fruit fly after heating in a block system

 

Predicción de la supervivencia de larvas de la mosca mexicana de la fruta después del calentamiento en un sistema de bloques

 

Lorena Caro-Corrales¹, José Caro-Corrales¹*, José López-Valenzuela¹, Ángel Valdez-Ortiz¹, Cuauhtémoc Reyes-Moreno¹, Emilio Hernández-Ortiz²

 

¹ Maestría en Ciencias y Tecnología de Alimentos y Programa Regional del Noroeste para el Doctorado en Biotecnología. Universidad Autónoma de Sinaloa. Culiacán, Sinaloa, México. * Author for correspondence.

² Programa Moscafrut SAGARPA-IICA, Subdirección de Desarrollo de Métodos, Metapa de Domínguez, Chiapas, México.

 

Received: June, 2013.
Approved: February, 2014.

 

Abstract

Modeling thermal death kinetics and heat transfer can allow the prediction of temperature-time combinations for a thermal process, which will facilitate successful development of thermal treatments for pest control. The objectives of this study were: 1) to predict the number of surviving third-instar Anastrepha ludens after heating in a block system using a thermal death kinetic model, 2) to validate predictions of surviving larvae, determining survival of malformed puparia and, 3) to assess heat resistance by gender of eclosed adults. Exposure times for 44, 46, and 48 °C were calculated for 30, 50, 70, 90, and 99 % mortality. Predictions of surviving larvae were successfully validated; for all temperature-time combinations the difference was lower than one larva. Larvae that survived heat treatment and pupariated were scored as surviving larvae. Nevertheless, not all puparia eclosed to adults; some developed as malformed puparia. Adults from treated puparia that did not show malformations emerged at least 24 h later than adults from control puparia. Heat treatment also induced larviform, bottlenose and incompletely-eclosed puparia, but none of them eclosed as adult flies. From treated normal puparia, male larvae were more tolerant to heat in two of the eight temperature-time combinations where there was fly production. The thermal death kinetic model satisfactorily predicted the number of surviving third-instar A. ludens after heating in a block system at the studied sublethal temperature-time combinations.

Key words: Anastrepha ludens, puparia, heating block system.

 

Resumen

El modelado de la cinética de muerte térmica y de la transferencia de calor puede permitir la predicción de combinaciones de temperatura y tiempo para un proceso térmico, lo cual facilitará el desarrollo exitoso de tratamientos térmicos para el control de plagas. Los objetivos de este estudio fueron: 1) predecir el número de sobrevivientes de larvas de Anastrepha ludens en el tercer estadio, después del calentamiento en un sistema de bloques utilizando un modelo cinético de muerte térmica, 2) validar las predicciones de larvas sobrevivientes, determinando la sobrevivencia de pupas deformes, y 3) evaluar la resistencia al calor por género de los adultos eclosionados. Los tiempos de exposición a 44, 46 y 48 °C se calcularon para 30, 50, 70, 90 y 99 % de mortalidad. Las predicciones de larvas sobrevivientes se validaron con éxito; para todas las combinaciones de temperatura y tiempo, la diferencia fue menor a una larva. Las larvas que sobrevivieron el tratamiento térmico y formaron pupas se calificaron como larvas sobrevivientes. No obstante, no todas las pupas eclosionaron a adultos; algunas se desarrollaron como pupas deformes. Los adultos de pupas tratadas que no mostraron deformaciones emergieron al menos 24 h después que los adultos de las pupas control. El tratamiento térmico también indujo pupas larviformes, con nariz de botella y con eclosión incompleta, pero ninguna de ellas eclosionó a moscas adultas. A partir de pupas normales tratadas, las larvas macho fueron más tolerantes al calor en dos de las ocho combinaciones de temperatura y tiempo donde hubo producción de moscas. El modelo cinético de muerte térmica predijo satisfactoriamente el número de sobrevivientes de A. ludens del tercer estadio, después del calentamiento en un sistema de bloques a las combinaciones de temperatura y tiempo subletales estudiadas.

Palabras clave: Anastrepha ludens, pupas, sistema de bloques de calentamiento.

 

INTRODUCTION

Fruit flies of the genus Anastrepha are one of the three main problems of fruit production in México (Ibañez-Palacios et al., 2010). Temperature is one of the main factors delimitating survival and natural mortality in pest populations and offers a rich potential that can be exploited for developing environmentally safe pest management strategies (Hallman and Denlinger, 1998). Heat treatments are relatively short, and the heating rate is affected by many factors; for example, heterogeneity in mangoes' (Mangifera indica L.) shape and size produces variations in heat penetration, and different temperature-time combinations are needed to disinfest them (Matias-Hernandez et al., 1998). Hence, developing a heat treatment strategy for a commodity has typically been specific to pest and commodity variety, and even to the shape, size and weight of the commodity (Hallman et al., 2005).

Research to develop treatments for pest control has used millions of immature flies (Hernández et al., 2007; NAPPO, 2010). Anastrepha ludens (Loew) (Diptera: Tephritidae) is a quarantine pest and among its hosts there are important fruits such as citrus (Citrus sinensis L. (Osbeck), C. aurantium L.) and mangoes (Aluja et al., 1987); it is more attracted to green orange and mango fruits than to yellow ones (Garcia-Ramirez et al., 2004). Importing mangoes to the USA from México where this fly is indigenous, either requires disinfestation treatments with chemicals such as methyl bromide or non-chemical treatments such as irradiation, vapor heat, steam, cold, and hot water immersion. In hot water immersion treatments, the dip time at 46.1 °C depends on the shape and size of the fruit. For instance, rounded varieties of mangoes in weight bands up to 500 g, 501 to 700 g, and 701 to 900 g are immersed 75, 90, and 110 min, in water at 46.1 °C (NOM-075-FITO-1997, USDA-APHIS 2011) to disinfest the fruit. However, if hydro-cooling starts immediately after the hot water immersion treatment, the dip time must be extended for 10 min. To disinfest the fruit of tephritid immatures, USDA-APHIS (2011) quarantine restrictions mandate a treatment efficacy at the Probit 9 level, equivalent to 99.9968 % mortality (Follet and Neven, 2006).

One promising route to reduce the time and cost needed to develop phytosanitary heat treatments is by modeling thermal death kinetics for quarantine pests coupled with heat transfer modeling inside commodities (Jang, 1996). The first step is having an adequate thermal death kinetic model for sublethal temperature-time combinations to be validated using in vitro experiments. A quarantine treatment should be effective against the most tolerant stage. Mangan et al. (1998) reviewed several studies and concluded that third-instar A. ludens was the most heat tolerant stage based on tests with eggs and larvae inside mangoes and in vitro hot water immersion. A fractional order thermal death kinetic model fits heat mortality of A. ludens (Aispuro-Coronel, 2007; personal communication); thermal constants at 44, 46, 48, and 50 °C are shown in Table 1.

The objectives of this study were: 1) to predict the number of surviving third-instar A. ludens after heating in a block system using a thermal death kinetic model; 2) to validate predictions through surviving larvae, determining survival of malformed puparia; and, 3) to assess heat resistance by gender.

 

MATERIALS AND METHODS

Insects

Larvae of A. ludens were obtained from the Moscafrut Facility at Metapa de Domínguez, Chiapas, Mexico, and grown on an artificial diet according to the methodology described by Stevens (1991) and Domínguez et al. (2010). Rearing conditions for larval development were 26 °C, 70±10 % RH, and photoperiod of 14:10 h (L:D). When the larvae were at third instar, they were separated from the diet using steel tweezers. Larval stages were observed with a stereomicroscope (Motic, SMZ-140-N2GG, USA) with software (Motic Images 2000, USA) to capture and record images in a computer. Analysis of the images confirmed the third instar when the mouth hook was black, the larva and anterior spiracle were well developed and showed high mobility.

 

Prediction of surviving larvae

Prediction of surviving third-instar larvae of A. ludens was made using the thermal death kinetic model described by Gazit et al. (2004) based on the following equation:

where N₀ and N are the initial and surviving numbers of larvae, t is the exposure time (min), k is the thermal death rate constant (min^—1) when temperature is constant, and n is the kinetic order of death. The integration form of this equation for a fractional order is:

where c' is the ordinate at origin and k' is the slope in a (N/N₀)^1-n versus t graph.

Since the purpose of this study was to predict the number of surviving larvae, at each temperature for a given mortality percentage [(N₀—N)/N₀]100, the exposure time was calculated using thermal death constants (Table 1) and the number of surviving larvae for that exposure time was predicted using the initial number of larvae. Therefore, every temperature-time combination is associated with a given mortality. For instance, when the aim is to achieve a 30 % mortality, then [(N₀—N)/ N₀]100 = 30, from here the survival ratio was N/N₀=0.70. After using the thermal death kinetic model, the calculated exposure time at 46 °C (n=0.55, k'=0.0339, and c'=1.0200, Table 1) was 4.96 lethal min. From the survival ratio, since the initial number of larvae was N₀=80, the predicted number of surviving larvae for that exposure time was N=56 larvae. After all predictions were made, they were experimentally validated at each temperature by applying the calculated exposure time for a given mortality and registering the number of surviving larvae.

 

Experimental design and treatment application

A completely random design was used; factors were temperature (44, 46, and 48 °C) and time associated to 30, 50, 70, 90, and 99 % mortality. For these mortality levels, the calculated exposure times at 44 °C were 17.35, 31.52, 48.59, 72.46, and 93.11 min; at 46 °C they were 4.96, 8.49, 12.93, 19.62, and 26.37 min; at 48 °C calculated exposure times were 0.24, 1.33, 2.70, 4.76, and 6.85 min, respectively. The response variables were the number of surviving larvae (N), the ratio of number of surviving larvae to the initial number of larvae raised to the (1—n) power, i.e. (N/N₀)^1—n, the number of eclosed males and females, and the type of puparia (normal, larviform, bottlenose, and incompletely-eclosed puparia). Thomas and Mangan (1995) found that in the bottlenose malformation the anterior-most segment of the puparium is abnormally constricted, otherwise morphologically asymptomatic. Larviform malformation was characterized by a failure to constrict into a typical barrel shape (Thomas, 1985). Incompletely-eclosed pupariation is the phenomenon where larvae form normal puparia and develop to adult, but these fail to emerge from puparium.

Five replicates for the 15 combinations were made with 80 third instars per treatment giving a total of 6000 larvae. For every response, an analysis of variance was accomplished and the Tukey's test (p≤0.05) was used to obtain differences between means of the responses.

A heating block system built as described by Ikediala et al. (2000) was used to heat third instar larvae. Two aluminum blocks (25×25×2 cm) and an intermediate thin aluminum slab of 25×25×0.3 cm with four square holes of 5×5 cm located symmetrically near the center could fit together. This achieved four closed square chambers where the larvae could be placed. One T type thermocouple was inserted in each holding chamber and connected to a data acquisition module (Omega, OMB-DAQ-55, Stamford, CT, USA) to record temperature histories. Heat was supplied by electric heating pads attached to the upper and lower surfaces of each plate, connected to a voltage regulator (ISB Sola Basic, microvolt inet, Mexico) and controlled by a standard rheostat through a T type thermocouple.

The bottom block and intermediate slab were joined together and 80 third instar larvae (20 per chamber) along with 7 mL of rearing medium were placed on them and closed with the upper block. Initial temperature was set at 25 °C and temperature was raised at 1 °C min^—1. Temperature was recorded every 3 s and treatment time initiated when temperature reached the target temperature (44, 46, and 48 °C), which was held for the selected exposure times. Immediately after the exposure time, the upper aluminum block was removed and larvae along with new rearing medium were transferred to plastic containers by gentle brushing. These were then sealed with a mesh to allow air to pass through, sprayed every day with water and placed in a room at 26 °C, 80 % RH, and a photoperiod of 14:10 h (L:D). Treated larvae were observed for 5 d, because dead larvae are not always obvious (Hallman et al., 2005). If the larva had turned dark, had not initiated pupariation, or did not respond to gentle stimulation, it was considered dead. Control insects were treated the same way but without heat at 25 °C. Abbott's equation for calculating control mortality (Abbott, 1925) was applied.

Five days after treatments, surviving larvae and puparia were transferred to different plastic containers with pupariation medium (slightly moistened sterile vermiculite, Strong-lite fine, Sun Gro Horticulture, Seneca, IL, USA) using steel tweezers at the same room conditions for 14 d.

Normal and damaged puparia were separated and held for emergence. Puparia were placed into 2 L plastic containers sealed with a white nylon mesh in the cover to facilitate access to the flies after they emerged. Three days after treated normal puparia eclosed, larviform and bottlenose puparia were discarded. Adults were continuously fed with a 3:1 mixture of sugar and hydrolyzed enzymatic yeast ICN* (Biomedicals, Costa Mesa, CA) (Message and Zucoloto, 1989). Water was supplied into 50 mL bottles with a filter paper which was impregnated by capillarity at a maximum of 10 d.

 

RESULTS AND DISUSSION

Control mortality for the third larval instars of A. ludens was less than 2 %. Predicted and experimental survival of third-instars of Mexican fruit fly is shown in Table 2. Experimental values are the mean of five replicates (each with N₀=80 larvae) and were rounded to one decimal. At each temperature, from regression analyses between predicted and experimental numbers of surviving larvae, the coefficients of determination were R²>0.99 (p≤0.01). At 44 °C, the largest difference between predicted and experimental number of larvae corresponded to 30, 90, and 99 % mortality and this difference was 0.4 larvae. However, for 50 and 70 % mortality, the difference was less than or equal to 0.2 larvae. At 46 °C, the model generates a better estimation, except for 30 % mortality where the difference was 0.4 larvae. At 48 °C, the largest difference between predicted and experimental numbers is 0.4 larvae corresponding to 30, 50, 90, and 99 % mortality. For all the temperature-time combinations the difference was lower than one larva. The thermal death kinetic model predicted satisfactorily the number of surviving larvae for the studied sublethal temperature-time combinations. As exposure time is increased at a constant temperature, the number of surviving larvae decreased. Johnson et al. (2004) found that red flour beetle larvae from dried fruit and nuts were very tolerant at 48 °C (77 min), but at 50 and 52 °C the exposure times (9 and 1.6 min) were more acceptable. At a constant temperature, all ratios of surviving number of larvae to the initial number of the population raised to the (1-n) were different. The slope increased sharply as temperature increased, resulting in lower times to achieve 100 % kill (Figure 1). This result is in agreement with that reported by Wang et al. (2002). At each temperature, from regression analyses between predicted and experimental (N/N₀)^1—n values, the coefficients of determination were R²>0.99 (p≤0.01). According to the thermal death model, with a kinetic order of death n=0.55, the exposure time to reach Probit 9 (99.9968 % mortality) is 100.2, 29.8, 7.9, and 2.5 min for 44, 46, 48, and 50 °C, respectively. For every 2 °C of temperature increase, the lethal (exposure) time to accomplish this mortality decreased by about 70 %. A regression analysis between the log of lethal time (LT) and temperature (T) led to a temperature increase of z=3.7 °C for a 10-fold decrease in lethal time required to achieve the same level of mortality (99.9968 %). The z value is practically the same independently of any fixed kinetic order of death taken from Table 1. The lowest and highest values are z=3.702 °C and z=3.704 °C. This result is similar to the value of 3.5 °C reported by Hallman et al. (2005) for third instars of Mexican fruit fly. The regression equation was log (LT_99.9968) = — 0.27 T + 13.9, where z is the negative reciprocal of the slope of the thermal death curve. The coefficient of determination was R²=0.9994 (p≤0.01), which indicates that lethal time decreases exponentially with temperature in the studied range.

The heating block is a homogenous heating system. However, in practice the variability in heat transfer inside the fruit should be taken into account as commodity variety, shape, thickness, and size of the fruit have an effect on heat transfer. At a selected temperature, the thermal death kinetic model can be used to predict the lethal time required to achieve a given mortality. From temperature histories inside the fruit, the process (dipping) time to get that given lethal time (or mortality) can be calculated from a heat penetration analysis.

Thomas and Mangan (1995) showed that larval mortality rate increased sharply for 1 h of immersion in hot water at 42 °C for A. ludens. They also reported that 2.5 % of larvae survived after 1 h of immersion in hot water at 44 °C. We found that after 72.46 min at 44 °C, using a heating block system, 9.5 % of larvae survived while the thermal death kinetic model estimated a value of 10 %. Most surviving larvae recovered their mobility within 24 h and some without movement pupariated 5 or 6 d after treatment. Hansen and Sharp (1994) used the terms acute and chronic deaths. Acute death is characterized by the death of the larvae immediately after the treatment and chronic death is based on the number of larvae unable to survive to the adult stage. The temperature at which acute death is present is called temperature-mortality threshold.

Development of puparia from surviving larvae of A. ludens after heat treatment at 44, 46, and 48 °C is shown in Table 3. Number of dead larvae represents acute death, and chronic death corresponded to malformed puparia (larviform and bottlenose) and incompletely-eclosed puparia.

Treated normal puparia successfully emerged as adults. At each temperature, except for 0 vs. 0 and 0 vs. 0.2 comparisons, all numbers of treated normal puparia were different. Larvae of A. ludens treated using a heating block system showed acute and chronic death at 44, 46, and 48 °C. The percentage of incompletely-eclosed puparia in the control was very low (less than 1 %) compared to that from treatments.

The number of treated normal puparia decreased continuously while the number of malformed puparia (larviform and bottlenose), at each temperature, increased from 30 to 50 % mortality and then decreased as exposure time increased. Bottlenose puparia frequency was higher than larviform or incompletely-eclosed puparia frequency. This could be due to heat damage preventing larvae from completing metamorphosis. After heat treatment, surviving larvae pupariate; however, not all of them produce adults, some will develop as malformed or incompletely-eclosed puparia. In our study none of the malformed puparia produced adults three days after the first fly emerged from treated puparia. At 46 °C and 70 % mortality, from 400 initial larvae 121 (24.2×5) puparia were produced and 23 (4.6×5) developed to adults, meaning only 5.75 % emerged as adults and finally 377 larvae did not arise to adults, and this is scored as 279 dead larvae. At conditions near 100 % mortality (90 and 99 % mortality) less than 10 % of puparia were produced; they were malformed or incompletely-eclosed puparia and were unable to develop to adults. Therefore, at these conditions the percentage of dead larvae and puparia was 100 % as they did not develop to adults, and it is scored as 90 or 99 % of larval mortality.

The number of bottlenose puparia decreased as mortality percentage increased from 50 to 99 % at a constant temperature. Larviform malformation did not show the same tendency, except from 90 to 99 % mortality. There were not incompletely-eclosed puparia in controls. At 90 and 99 % mortality, we did not observe incompletely-eclosed puparia, except at 44 °C and 90 % mortality, where only one incompletely-eclosed puparium was found.

During heating in the block system, layers that form the puparium dried up and were abnormally constricted forming smaller layers leading to bottlenose puparia. When larvae survived the heat treatment, but did not reach entire metamorphosis, a hard outer layer was formed which characterizes the puparium stage producing larviform puparia. When metamorphosis of the larva is achieved and a part of its body remains inside the puparium, the fly is unable to break the layer of the puparium giving rise to incompletely-eclosed puparia. This could be attributed to heat affecting some morphological structures related to the locomotion efficiency of flies and the muscle contractions were insufficient for successful eclosion.

Eclosed adults by gender are shown in Figure 2. Adults from treated normal puparia emerged at least 24 h later than adults from control puparia and showed a normal physiological maturation. The amount of eclosed females was higher in the control at 44 and 46 °C. (Figure 2). Folk et al. (2006) found that females had higher survivorship, as they had more heat-shock proteins than males. There was no adult fly production when total puparia were less than 10 % of initial larvae. At temperature-time combinations where there was fly production, both males and females eclosed and means of adult males were higher (p≤0.05) for combinations of 44 °C, 70 % and 46 °C, 30 % mortality. The number of adult females and males was not different for all other treatments.

 

CONCLUSIONS

The thermal death kinetic model satisfactorily estimated the number of surviving third-instar A. ludens after heating in a block system at the studied sublethal temperature-time combinations. Therefore, at a selected temperature, the lethal time can be estimated from the thermal death kinetic model (and its appropriate parameter values) for a target sublethal mortality level.

The thermal susceptibility of larvae to temperature change is characterized by the z value and a significant advantage of this value is the ability to estimate the efficacy of a thermal treatment based on temperature histories in host fruits. Therefore, the lethality of a thermal treatment can be estimated from the obtained z value and temperature histories inside host commodities. This will facilitate successful development of thermal treatments for pest control.

Larviform, bottlenose, and incompletely-eclosed puparia were unable to produce adult flies. From treated normal puparia, male larvae were more tolerant to heat in two of the eight temperature-time combinations where there was fly production.

 

ACKNOWLEDGMENTS

This work was supported by Programa de Fomento y Apoyo a Proyectos de Investigación [PROFAPI 2010/027]: Universidad Autónoma de Sinaloa.

 

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