An Improved Quarantine Method for Mangoes Against the Mexican Fruit Fly Based on High-Pressure Processing Combined with Heat

High hydrostatic pressure system

Pressure treatments were carried out using a Cold Isostatic Press Model CIP42260 (Avure Autoclave Systems, Columbus, OH), with a high-pressure chamber of 101.6 mm of inner diameter and 584.2 mm of length and operating pressure capacity of 350 MPa. A mixture of 5:1 water:antiabrasive lubricant (Hydrolubric 120-B; EF Houghton & Co., Valley Forge, PA) was used as the high pressurizing fluid (to minimize adiabatic heating), and the temperature of this mixture was adjusted at 50°C before high pressurizing. The isostatic press equipment had no temperature control, and thus temperature changed as affected by the time of pressurization, the pressure level, and the nature of the samples introduced.

Eggs and larvae were pressurized at 25, 50, 75, 100, or 150 MPa for 0, 5, 10, or 20 min using an initial temperature of 50°C. The time required to reach every selected pressure was 8, 17, 44, 60, or 82 sec, respectively. The time period for release of pressure was always less than 30 sec. After pressurizing eggs for 0, 5, 10, or 20 min, the final temperatures were 50°C, 48°C, 45°C, and 40°C, respectively. Final temperatures for larvae samples were 47°C, 45°C, 40°C, and 36–38°C for the same process time. The decreasing of temperature during HPP was considered.

High hydrostatic pressure treatment on eggs

Eggs of 1, 2, 3, or 4 days old were processed independently. An aliquot of an egg/water solution containing 24,200 eggs/mL was taken from a 20 L plastic container to fill up the Eppendorf tubes completely. They were closed immediately to avoid the presence of air bubbles. The isostatic pressurization was in the range of 25–150 MPa for 0–20 min using an initial temperature of 50°C. After treatments, the eggs were extracted from the Eppendorf tubes using a brush and then were arranged in three rows of 100 eggs in Petri dishes using a stereoscope and incubated at 26 ± 1°C during 7 days to record the egg hatch. Three replicates of this bioassay were performed.

High hydrostatic pressure treatment on larvae

At the fourth day after oviposition, 0.1% guar gum (Tic Gums, White Marsh, MD) was added to the liquid containing untreated eggs to prepare a suspension able to maintain a uniform egg distribution. A 2 mL sample of a suspension that contained 600 eggs/mL was dispersed in 300 g of diet and placed in a rectangular plastic container of 1 L. The containers were covered with a cloth mesh, and the eggs were held at 27°C for the first instar (1–3 days old), 26°C for the second instar (4–6 days old), and 25°C for the third instar (7–9 days old). Containers were maintained under complete darkness and 75 ± 5% relative humidity (RH) and used for pressure treatments.

The HPP was applied in the range cited previously to the larvae of first, second, and third instars, which corresponded to the age of 2, 5, and 8 days old, respectively. Approximately 1200 larvae of each instar plus 300 g of diet were introduced into plastic food container of 200 mL. The food containers were hermetically closed just before the high-pressure treatment. After the treatment, they were opened, and the content was returned to a 1 L plastic container, dispersed smoothly, and incubated until the ninth day under the same conditions as before sampling. The living larvae were recorded, and pupation was induced in vermiculite. Third instar larvae were used to determine the percentage of pupation. The survivorship or reproductive capacity of the adults was not registered.

Effect of high hydrostatic pressure on eggs

The effect of the treatments on eggs is shown in Table 1. The hatching ability of eggs from all ages pressurized at 25 MPa for 0 min was minimally affected by the treatments. Hatching decreased drastically by increasing the holding time for eggs in all ages. This finding was similar in each level of pressure. Moreover, the number of hatched eggs decreased by increasing the level of pressure. This effect was more easily appreciated for eggs pressurized at 0 min.

At 25 MPa, eggs of 1-day-old showed to be more resistant to the treatment. The hatching rate was highly decreased when pressure levels of 50–100 MPa were applied for 5–20 min. The hatching ability of eggs pressurized at 150 MPa at 5–20 min was lost completely.

Effect of high hydrostatic pressure on larvae

Larvae showed very low resistance to HPP even at 25 MPa (Table 2). The surviving of larvae decreased drastically as affected by the increasing time and pressurizing level. At 25 and 50 MPa, second instars showed the lowest resistance when time was increased from 5 to 20 min.

Larvae of all instars did not survive when pressurized at 75 and 100 MPa for 5–20 min. Similar behavior was observed by pressurizing at 150 MPa for 10–20 min. However, some larvae from first and third instars survived after 5 min at 150 MPa.

Several larvae survived to the treatments and preserved their ability to pupate (Table 3), especially larvae treated at 25 MPa for 0 min. Most of the larvae that survived pressurizing treatment above 50 MPa lost their ability to pupate. Some of them resisted 50 and 75 MPa for 0 min, and surprisingly some larvae were able to pupate even after treatments of 150 MPa for 5 min.

Effect on eggs hatching

The hatching of the Mexican fruit fly eggs was inhibited by HPP at initial 50°C, affected by both, time and level of pressure. The eggs were affected even by HPP of 25 MPa. This level of pressure did not inhibit the hatching of the eggs when applied at 25°C or 0°C (Candelario-Rodríguez et al., 2009; Velazquez et al., 2009). In this study, the effect of pressurizing time was highly influenced by the temperature. The quarantine methods for mangoes include a water immersion at 46.1°C for 65–90 min depending on the size of fruit based on the fruit weight, to destroy eggs or larvae present in the fruit before exportation (Armstrong and Mangan, 2007). The resistance of fruit fly eggs to heat varies between species, but the resistance decreases strongly above 40°C. The minimal time required to obtain 100% mortality at 44°C varied from 50 to 80 min depending on the species, but at 50°C only 0.5–3 min is required (Armstrong et al., 2009).

The importance of temperature on the effect of HPP inhibiting the hatching of eggs can be observed in those samples that were just pressurized and released (0 min). Under these conditions, egg hatching was considerably inhibited only when 100 MPa was used, indicating that this pressure affects the eggs. The time required to reach a determined pressure increased linearly. It is important to consider the combined effect of time and the level of pressure and the decrease of temperature during the time of pressurizing because temperature was not constant in this study. Samples pressurized at 25 MPa required 8 sec, whereas samples pressurized at 150 MPa required 82 sec, and the final temperature of pressurizing vessel after reaching 150 MPa was 49°C. The hatching inhibition did not show a linear effect, as did the time required to reach pressure and the temperature decreasing by the effect of time, indicating that HPP above 100 MPa affected the eggs strongly.

Pressurizing time at initial 50°C was important to inhibit hatching. Hatching was highly inhibited even by pressurizing 5 min above 50 MPa. Although increasing the time caused an important decrease in the number of eggs hatching, some eggs were able to hatch even after 20 min at 100 MPa. It is important to consider the decrease in temperature during the HPP.

Treatments at 150 MPa for 5–20 min were able to inhibit the hatching of eggs, indicating that this treatment could be a useful quarantine treatment for Mexican fruit fly. However, preliminary studies showed that mangoes might suffer some physical damage when pressurized at 150 MPa, especially in ripe fruits. Green mangoes showed more resistance to this pressurization level (Candelario-Rodríguez et al., 2009).

These results indicate that HPP seems to be appropriated as a quarantine method to control insects which induces damage to fruits, by destroying the laid eggs. Note that the resistance of insect eggs to HPP varies among species. The eggs of the Mediterranean fruit fly were destroyed after 5–20 min at 50–100 MPa at 25°C, but lower levels of pressure were required when HPP was conducted at 40°C or 0°C (Butz and Tauscher, 1995). The eggs of the western cherry fruit fly required 172.4 MPa for 1 min at 16–18°C and codling moth eggs lost their ability to hatch under HPP above 200 MPa (Neven et al., 2007). There is no information about the causes associated with differences on the resistance of eggs to HPP.

Effect on surviving larvae

Larvae were less resistant to HPP than eggs. These results agree with those previously reported for HPP of the Mexican fruit fly at 25°C or 0°C (Candelario-Rodríguez et al., 2009; Velazquez et al., 2009). In this study, HPP at initial 50°C induced higher levels of mortality even at 25 MPa. Larvae of the Mexican fruit fly are very sensible to heat treatments. In the range of 44°C to 50°C, each 2°C increase resulted in a reduction of time (75%) required to achieve 100% mortality of third instars. Larvae die after 2 min at 50°C, after 6 min at 48°C, and after 100 min at 44°C (Hallman et al., 2005). Recently, it has been reported that the third instar larvae from different fruit flies require as minimal 1–4 min at 50°C to die (Armstrong et al., 2009).

The rates of larval mortality reached at pressures higher than 75 MPa were 100% by holding the pressure for 10 or 20 min. Although 100% mortality was observed at 75 MPa for 5 min, some larvae were able to survive after 5 min at 100 MPa. This behavior might be associated with the resistance to HPP observed for some organisms. As discussed previously, it is important to consider the decreasing in temperature during the time of pressurizing.

The lower resistance to HPP for larvae as compared with eggs indicates that further studies in the resistance of the Mexican fruit fly to HPP might be addressed to find the conditions required to destroy eggs. However, in practical studies dealing with the efficiency of treatments to kill eggs and larvae inside the fruit, it must be considered that although the egg is a very thermotolerant stage, the larvae that tunnel deep into the fruit may be more difficult to kill because the surrounding fruit pulp acts as a heat sink and insulates the larvae (Denlinger and Yocum, 1998; Armstrong and Mangan, 2007). This is an important factor to consider in the HPP proposed as quarantine treatments because although pressure is reached simultaneously in all the fruits inside the container, the heat might not reach some parts of the fruit because of the very short processing time (0–20 min).

HPP induces a strong physical damage on most of the larvae, killing almost all of them. The physical damage to larvae by HPP has been described recently (Candelario-Rodríguez et al., 2009; Velazquez et al., 2009), and it was continued in this study and illustrated in Figure 1. Surviving larvae did not show the dark regions, air bubbles, separation from external membrane, and liquid accumulation present in larvae which did not resist HPP.

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FIG. 1.

Anastrepha ludens third instar showing visual differences between dead larvae during pressurizing treatment (150 MPa for 20 min) and a survival-resistant larva with less visual damage.

Several larvae that survived the treatments were able to pupate. Although no efforts were conducted to determine if puparium was able to form viable adults, it has been reported that adults may emerge even from puparium which did not look normal after heat treatments (Thomas and Mangan, 1995; Hallman, 2000).

The effectiveness of HPP in larvae from other insect species has been reported previously. The larvae of the western cherry fruit fly died after pressurizing at 172.4 MPa, but the resistance to HPP depended on the stage, decreasing in the next order: third, first, and second instar. Larvae of codling moth resisted a pressure of 209 MPa before dying (Neven et al., 2007). The different resistance to HPP among species might be associated with a different composition and changes in the structure of proteins in each organism. HPP and heat treatments are associated with protein denaturation and aggregation that affect the biological functions of proteins in the organisms inducing the death (Téllez-Luis et al., 2001).

HPP as a quarantine treatment

Quarantine treatments are designed to kill insects present in the commodities even at a cost of reducing the quality of the product (Hallman, 2000). Using HPP as a quarantine method must consider the effect on fruits. As mentioned above, ripe mangoes do not resist HPP treatment because even a low level of pressurizing (50 MPa) causes immediate physical and physiological changes in the structure and appearance of skin.

In Mexico, recently the irradiation treatment has been approved for guava fruit and mangoes as a quarantine method for fruit flies. Preliminary studies indicate that irradiation might affect some varieties of mangoes more than others (Mercado-Silva et al., 2009). Other emergent technologies such as pulsed electric field, radiofrequency, and microwave-vapor heat are still under experimentation (Hallman and Zhang, 1997; Monzon et al., 2007; Varith et al., 2007; Wang et al., 2007; Tiwari et al., 2008).

However, studies maintaining a constant temperature of the pressurizing vessel are required to reduce the level of pressurization and to allow the HPP treatment become a useful quarantine method. The effect of HPP in the shelf life of mango at different stage of ripeness should be also studied.