Alternatives to ginger root oil aromatherapy for improved mating performance of sterile Ceratitis capitata (Diptera: Tephritidae) males

Authors

  • M. Juan-Blasco,

    1.  Unidad Asociada de Entomología IVIA (Instituto Valenciano de Investigaciones Agrarias), CIB (Centro de Investigaciones Biológicas) del Consejo Superior de Investigaciones Científicas (CSIC), UJI (Universitat Jaume I de Castelló), Centro de Protección Vegetal y Biotecnología, IVIA, Moncada, Valencia, Spain
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  • V. San Andrés,

    1.  Unidad Asociada de Entomología IVIA (Instituto Valenciano de Investigaciones Agrarias), CIB (Centro de Investigaciones Biológicas) del Consejo Superior de Investigaciones Científicas (CSIC), UJI (Universitat Jaume I de Castelló), Centro de Protección Vegetal y Biotecnología, IVIA, Moncada, Valencia, Spain
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  • M. A. Martínez-Utrillas,

    1.  Transformaciones Agrarias SA (TRAGSA), Paterna, Valencia, Spain
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  • R. Argilés,

    1.  Transformaciones Agrarias SA (TRAGSA), Paterna, Valencia, Spain
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  • I. Pla,

    1.  Transformaciones Agrarias SA (TRAGSA), Paterna, Valencia, Spain
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  • A. Urbaneja,

    1.  Unidad Asociada de Entomología IVIA (Instituto Valenciano de Investigaciones Agrarias), CIB (Centro de Investigaciones Biológicas) del Consejo Superior de Investigaciones Científicas (CSIC), UJI (Universitat Jaume I de Castelló), Centro de Protección Vegetal y Biotecnología, IVIA, Moncada, Valencia, Spain
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  • B. Sabater-Muñoz

    1.  Unidad Asociada de Entomología IVIA (Instituto Valenciano de Investigaciones Agrarias), CIB (Centro de Investigaciones Biológicas) del Consejo Superior de Investigaciones Científicas (CSIC), UJI (Universitat Jaume I de Castelló), Centro de Protección Vegetal y Biotecnología, IVIA, Moncada, Valencia, Spain
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B. Sabater-Muñoz (corresponding author), Centro de Protección Vegetal y Biotecnología, IVIA, Carretera de Moncada a Náquera km 4,5. Apartado Oficial, 46113 Moncada, Valencia, Spain. E-mails: sabater_bea@gva.es; bsabater@ivia.es

Abstract

Autocidal control of the Mediterranean fruit fly Ceratitis capitata (Wiedemann) is an environmentally friendly and safe practice employed successfully worldwide. A key component in the Sterile Insect Technique (SIT) is to release sterile males that can compete with wild males in nature. Exposure of sterile males to ginger root oil (GRO) in the form of aromatherapy prior to release has been shown to significantly improve male competitiveness and performance. However, alternatives to GRO exposure are desired to (i) reduce personnel costs and handling-time requirements and (ii) find new aromatherapeutic compounds. In this study, alternatives to GRO aromatherapy were investigated, such as incorporating GRO into the post-teneral diet, or employing other molecules (limonene and linalool) occurring naturally in citrus release areas. Our results reveal that both the GRO-supplemented diet and linalool aromatherapy were equal to the GRO aromatherapy control in improving mating performance in sterile males. In contrast, exposure to limonene did not afford a mating advantage to sterile mates when compared to the control. In addition, sterile male survival was not affected by any of the treatments tested. Implications for how both new approaches could be implemented in SIT programmes in citrus production areas are discussed.

Introduction

In Mediterranean fruit fly (Ceratitis capitata) Sterile Insect Technique (SIT) programmes, millions of male flies are mass-reared, sterilized, marked and released into the field. The success of an SIT programme in controlling Mediterranean fruit fly depends greatly upon the mating performance of the released sterilized males. The economic effectiveness of SIT programmes requires mass rearing and continuous and timely releases (Dyck et al. 2005); thus the millions of flies produced daily should achieve sexually maturity quickly, be sexual competitive and survive long enough to induce sterility on wild populations. The mass-rearing process with its inherent overcrowding conditions can negatively affect the quality of produced males (Shelly et al. 1994; Briceño and Eberhard 1998; Cayol 2000; Gaskin et al. 2002; Franz 2005). Therefore, much research has addressed sterile male mating competiveness during recent years to implement cost-effective SIT programmes (Barry et al. 2003; Shelly and Villalobos 2004; Liedo et al. 2007; Papadopoulos et al. 2007; Shelly et al. 2007c; San Andrés et al. 2009).

Several natural attractants have been shown to increase the mating success of males through aromatherapy (Shelly and McInnis 2001; Shelly et al. 2005, 2007d), one of which is ginger root oil (GRO, Zingiber officinale Roscoe). This oil contains in low proportion a natural attractant of C. capitata males, α-copaene, capable of stimulating mating behaviour (Nishida et al. 2000; Shelly 2001; Shelly et al. 2004a; Briceño et al. 2007). Aromatherapy with GRO is currently being used to improve mating competitiveness of released males in many SIT programmes, including USA and Western Australia (Shelly et al. 2006) and Spain (R. Argilés, TRAGSA, S.A., Spain, unpublished results). Nevertheless, its use has increased programme costs in terms of infrastructure, personnel and handling time requirements. For exposure, 24 h prior to release, at least three independent holding rooms are needed for each batch of C. capitata (pre-exposure, exposure, post-exposure/chilling room). The first objective of this work was to explore other options with a view to reducing costs at fly emergence and release facilities, such as the addition of essential oils directly to the post-teneral diet, which would reduce the need for independent holding rooms and personnel required to allocate the flies to each treatment room.

Despite the advantages of using GRO, other chemicals, also involving the α-copaene, produced by host plants or fruit exudates (such as citrus, guava, mango, grapefruit, or fig) could trigger host-plant searching or feeding behaviours that detract from the main objective of releasing these males, that is, to track and mate with wild females (Warthen and McInnis 1989; Shelly and Villalobos 2004; Papadopoulos et al. 2007; Shelly et al. 2007c). Katsoyannos et al. (1997) reported the effect of chemicals from citrus fruit juice and peels on C. capitata male and female mating behaviours. Other authors (Papadopoulos et al. 2001, 2006; Jang 2002; Shelly 2004, 2009; Shelly et al. 2004b, 2008a) have reported similar enhancement of sterile males’ mating competitiveness by other essential oils, plant or fruit exudates. In all cases, the sesquiterpene α-copaene is present in these plant extracts and is considered the component responsible for arousing mating behaviour. However, other compounds present in those essential oils are also likely to have an effect on the mating behaviour of the Mediterranean fruit fly, and deserve further research (Jang et al. 1989; Warthen and McInnis 1989), which is one of the objectives of this work.

The monoterpenes limonene and linalool are major components of orange oils. These compounds also cited as common in other citrus species, have been found in the blend of the male Mediterranean fruit fly sex pheromone (Papadopoulos et al. 2001; and references therein). More recent studies have shown that females prefer males with a specific scent from host plants, such as Citrus species (Shelly 2004; Papadopoulos et al. 2006; Shelly et al. 2007c). Thus, the second objective of this study was to identify other natural chemicals, which could replace GRO as a mating competitiveness enhancer in sterile males.

In summary, the aims of this work were (i) to provide a feasible alternative to GRO aromatherapy by incorporating GRO into the post-teneral diet, and (ii) to identify other molecules to improve the mating performance of the released sterile males.

Experimental Procedures

Strains and rearing conditions

The wild-type strain (wt) adults were obtained from a laboratory colony (generation XI and XII) housed at the Generalitat Valenciana-IVIA emergence facility (Moncada, Spain), which is refreshed twice annually with wild individuals from field-infested fruits. The new individuals are introduced in a new setup colony cage in the same number as those from the colony strain, producing a small bottleneck in the population, and allowing random mating with the resulting introduction of the new individuals’ genes into the colony. Every 2 years, this colony is completely replaced by wild individuals. Sterile males of the Vienna-8 temperature sensitive lethal (tsl) Genetic Sexing Strain mix 2002 strain [also named GS1/D53 or T(Y; 5–30C) (Franz 2002)], currently under production at the mass-rearing facility in Caudete de las Fuentes, Valencia Province, Spain, were obtained 2 days prior to fly emergence, as marked and irradiated pupae.

Soon after emergence, about 1000 adult wt females, wt males (<24 h old) or Vienna-8 males were separated by sex and strain into perspex [poly(methyl methacrylate) or Plexiglas®] cages (20 × 20 × 20 cm) with 250 individuals per cage. Adults were maintained at 25 ± 4°C, 75 ± 5% RH and 14:10 h (L: D) photoperiod (photophase started at 06:00 h including a 1 h dawn and dusk period) in an environmental chamber. Female wt adults were fed a mixture of sugar and hydrolyzed yeast (4 : 1; w : w; Biokar Diagnostics Co., Pantin, France), whereas males (wt or Vienna-8) were fed only sugar. Water and diet were provided ad libitum.

Effect of GRO-supplemented post-teneral diet

Mating experiment

Four treatments of sterile males were compared: (i) GRO-supplemented diet (GROd); (ii) GRO-supplemented diet plus aromatherapy (GROda); (iii) control diet plus aromatherapy (GROa); and (iv) control diet without aromatherapy (control). The control diet consisted of 10 g of sugar mixed with 1.5 ml of 20% sugar (w/v) to obtain a similar texture as the GROd. The GROd consisted of 10 g of sugar complemented with 1.5 ml of 16.7% (v/v) GRO in 20% sugar (w/v) (GUINAMA, Valencia, Spain) to resemble a sugar block. The GRO dose in the diet was determined by previous assays in which mortality was not affected (data not shown). Periodic surveillance of the experimental treatment cages showed that males were in contact with, and fed on, the GROd. For all the aromatherapy treatments (GROa and GROda), 1 day before testing, sterile Vienna-8 males were exposed to GRO for 3 h (dose of 310 μl/m3) in the forced-ventilation exposure room of the fly emergence and release facility. Virgin wt females and males, and sterile males were kept apart in different rooms to prevent any pheromone and/or aromatherapy effects.

For all the mating experiments, wt females were 10-day-old, wt males 7-day-old and sterile Vienna-8 males 3-day-old (Cáceres et al. 2007; Shelly et al. 2007b; Shelly 2012). The mating arena consisted of perspex cages (30 × 40 × 30 cm) with four lateral (15 × 15 cm) and one upper (20 × 35 cm) ventilation openings. Fifty males wt and 50 Vienna-8 (1 : 1) were placed in the mating arena first (at 8:00 h) and left to settle for 15 min, then 50 females were introduced, comprising the final ratio (1 : 1 : 1) tested. In each arena, observations for mating pairs were carried out continuously for 3 h removing each couple as formed by gentle soaking into 50 ml vials; after this time, the arena were supervised discontinuously (each 15–20 min) for additional 3 h after which any remaining uncoupled female was discarded. After copula completion (Taylor et al. 2000), the male type in each pair was determined by the presence (sterile) or absence (wt) of fluorescent dye (Dyck et al. 2005). Mating experiments were done with four replicates and repeated twice (eight replicates in total for each treatment).

Longevity

Longevity experiments were carried out only with sterile males subjected to the same four treatments. The experimental arena consisted of ventilated plastic cylinders (16 cm high × 13 cm in diameter) that were placed in an environmental chamber (25 ± 4°C, 75 ± 5% RH, and 16:8 h (L:D) photoperiod including a 1 h dawn and dusk period). Forty 3–day-old sterile males were confined per container and a total of four containers per treatment were tested. Longevity was assessed under no stress feeding scenario (sugar and water ad libitum). For all the experiments, the arena was checked daily at the same time (13:00 h), until post-treatment day 15 (18 days of adult male), and dead males were recorded and removed.

Effect of aromatherapy with essential oils

Mating experiment

The aroma-deprived control was compared with three aromatherapy treatments: (i) GRO (as described above); (ii) Limonene (Limonene 145, ≥99.0%, sum of enantiomers; FLUKA, Seelze, Germany); and (iii) Linalool [±Linalool, ≥95.0% (GC); FLUKA]. One day before initiating a mating assay, males were exposed to essences of the above compounds for 3 h at a dose of 310 μl/m3. The GRO treatment was conducted in the forced-ventilation exposition room (143 m3) of the fly emergence and release facility as described earlier. The Limonene and Linalool treatments were conducted in small rooms (of 24 and 33.8 m3 respectively) without forced ventilation. To assure aroma scenting by the flies, 2 μl of each essential oil at a dose of 310 μl/m3 was deposited in a piece of filter paper and introduced inside of a gauze-covered cup in each cage (room size was also taken into account to achieve the dose of 310 μl/m3). Exposure was conducted simultaneously in isolated rooms thus avoiding inadvertent exposure of the control and aroma-deprived males to essential oils. The four treatments, with four replicates per treatment, were compared for mating success as previously described.

Longevity

The longevity assay was carried out with sterile males subjected to the same treatments including the aroma-deprived control. The experimental arena was set up as described above under the same feeding scenario. In the same manner, mortality of sterile males was counted daily at the same hour (13:00 h) until post-treatment day 15 (18 days of adult male age). Dead males were recorded and removed from the arena.

Statistical analysis

In all mating experiments, the percentage of sterile matings was calculated based on the total number of matings observed (matings sterile + wild) in each cage. Data were submitted to one-way anova followed by Tuckey’s HSD test for multiple comparisons (P < 0.05). The Kaplan–Meier survival analysis followed by Breslow tests were used to compare cumulative survival rates. The Bonferroni method was used to determine statistically significant differences among treatments in pair-wise comparisons after the Breslow tests. SPSS 10.05 was used for all statistical analyses (SPSS, Chicago, IL).

Results

Effect of GRO-supplemented post-teneral diet

Mating test

Mating competitiveness of 3-day-old sterile males exposed to any of the three treatments was significantly higher than the control (F = 12.64; d.f. = 3, 30; P < 0.0001) (fig. 1). The number of sterile males mating when GRO was added to the post-teneral diet (GROd), did not differ from those exposed only to GRO aromatherapy (GROa). However, when GRO aromatherapy was combined with a GRO-supplemented diet (GROda), a significant higher number of sterile matings were observed, compared with the treatment where males were only exposed GROa and with the control without GRO exposure.

Figure 1.

 Percentage of matings (mean ± SE) achieved by 3-day-old sterile Vienna 8 tsl Mediterranean fruit flies exposed to ginger root oil aromatherapy (GROa), GRO-supplemented diet (GROd), combined treatment of GRO aromatherapy and GRO-supplemented diet (GROda) and untreated control; in competition with laboratory wild-type males at a 1 : 1 ratio.

Longevity

Sterile male survival was not affected by the addition of GRO to the post-teneral diet (GROd and GROda) or presented as aromatherapy (GROa) when compared to control males (χ= 1.942; d.f. = 3; P = 0.585) (fig. 2). Survival rates for all treatments throughout the 15 days period were higher than 80%.

Figure 2.

 Survival (mean percentage) of 3-day-old sterile Ceratitis capitata Vienna 8 tsl males subjected to aromatherapy with ginger root oil (GROa), GRO-supplemented diet (GROd), combined GRO aromatherapy and GRO-supplemented diet (GROda), and a control (no GRO exposure) in a post-treatment feeding regime of no-stress (with ad libitum access to water and sugar).

Effect of aromatherapy with essential oils

Mating test

The number of 3-day-old sterile males that mated was significantly higher in the GRO and linalool aroma treatments when compared to the limonene and control treatments (F = 18.79; d.f. = 3, 15; P < 0.0001) (fig. 3).

Figure 3.

 Percentage of matings (mean ± SE) achieved by 3-day-old sterile Vienna 8 tsl Mediterranean fruit flies exposed to aromatherapy with ginger root oil (GRO), limonene, linalool and control; in competition with laboratory wild-type males at a 1 : 1 ratio.

Longevity

Breslow tests within the Kaplan–Meier survival analysis for 3-day-old sterile males did not reveal significant differences in survival among treatments when adults had access to water and sugar (no stress χ= 8.073; d.f. = 3; P = 0.045) (fig. 4). Survival rates for all treatments throughout the 15 days period were higher than 80%.

Figure 4.

 Survival (mean percentage) of 3-day-old sterile Ceratitis capitata Vienna 8 tsl males subjected to aromatherapy with ginger root oil (GRO), limonene, linalool and a control (no aromatherapy exposure) in a post-treatment feeding regime of no-stress (with ad libitum access to water and sugar).

Discussion

In this laboratory trial, we found that adding GRO to the post-teneral diet combined with GRO aromatherapy enhanced mating success of treated sterile males (56.9% vs. 37.7% of sterile male matings in the control). Ginger root oil in the post-teneral diet alone led to an increase in matings similar to GRO aromatherapy alone (51.7% and 46.8%, respectively). This contrasts with previous studies conducted in field cages that showed no positive effect on mating success either by GRO-supplemented larval diet or by allowing the pre-release males to be in contact (not feeding) with GRO (Shelly 2001; Shelly et al. 2008b). Nevertheless, our results showed an additive positive effect of GRO-supplemented diet when males were also subjected to GRO aromatherapy. None of the GRO treatments tested in this study produced a detrimental effect on longevity of the treated males, in accordance with other studies (Shelly et al. 2007a; San Andrés et al. 2009).

Despite its positive effects on mating performance in the laboratory, the GRO-supplemented pre-release diet increases costs as more GRO (approximately 3×) is needed than in the aromatherapy alone. On the other hand, when compared to aromatherapy, this cost is offset by the reduction in handling time and associated personnel costs, and costly upgrading and management of compartmentalized release facilities. Further research must be performed to evaluate the effect on the male sexual performance of the studied treatments when tested under semi-natural conditions (field cages) and also to define what GRO concentration is needed in the adult diet to improve sterile male mating rates.

In addition, it is necessary to consider that host-plant volatiles in release areas could interfere with the interactions between α-copaene (from the GRO) and enhanced mating competitiveness in sterile males. As stated by several authors (Nishida et al. 2000; and references therein), a rendezvous role of tephritidae host-plant volatiles is evident, increasing local female traffic and thus influencing lek formation. This attraction has mainly been attributed to the sesquiterpene α-copaene; however, other trace chemicals (sesquiterpenes, monoterpenes, etc.) in these host plants also seem to be of importance (Jang 2002; Shelly 2004, 2009; Shelly et al. 2008a). As demonstrated in previous works, wild females are more likely to visit leks or host plants harbouring males, when these males have acquired scent from citrus, guava or papaya (Nishida et al. 2000; Jang 2002; Shelly and Villalobos 2004; Papadopoulos et al. 2007).

Bearing in mind these plant chemical cues, alternatives to GRO were tested as aroma-therapeutic agents in the present study to evaluate their possible effects on the mating success of sterile males. Limonene and linalool were chosen due to their influence on male mating behaviour [found in pheromones produced by calling males, by Jang et al. (1989)] and tested with a view to understanding which component(s) of the citrus oils is/are responsible for enhanced mating (Papadopoulos et al. 2001; Shelly et al. 2004b, 2008a; Kouloussis et al. 2013). In response to this second research question, we obtained different results in mating performance with the tested components of orange oil. Sweet orange oil (GUINAMA, obtained from cold pressure of fruits) was included in the pre-test to determine effect of one natural mixture of limonene and linalool, and the results in sterile mating enhancement were resembling those obtained with GRO aromatherapy (in the same dose and application procedure; data not shown). The monoterpene limonene, the primary volatile emitted from citrus oils; did not seem to affect the mating performance of treated males (45.3% vs. 43.3% of sterile matings in the aroma-deprived control), whereas surprisingly, linalool enhanced mating performance similar to those obtained with GRO exposure (approximately 52.6% vs. 58.6%, fig. 3), which coincided with results from other studies (e.g. Shelly et al. 2004a). Previous investigations (Salvatore et al. 2004; Chang et al. 2009) have reported toxic effects of linalool application on adult survival or female oviposition preference; however, in our study, treatment with this component was not linked to any detrimental effect on longevity. To our knowledge, this is the first time that this component has been used successfully to boost male competitiveness with promising results, suggesting that it could be implemented in SIT programmes. Despite this result, in one recent work (Kouloussis et al. 2013) only a small advantage to increase matings was observed in linalool-treated males. Further work is needed to establish the appropriate application dose and test the effect under semi-natural conditions.

As stated before, the use of an essential oil that is already present in the programme area (i.e. linalool) may mean that exposed sterile males do not need to locate these chemical sources in the environment, thus reducing the time and energy costs associated with searching. It follows that these males might devote more time/energy to tracking wild females, which is the main objective of SIT programmes, thereby representing an advantage over GRO. However, all these associated behaviours (chemical and protein sources, and female search) deserve further research. Furthermore, the application of aromatherapy with linalool from locally available sources is advantageous to the citrus industry as it utilizes a by-product. Recently Kouloussis et al. (2013) demonstrated that males exposed to different citrus fruits (commercial oils or wounded fruits) and to different doses of sweet orange oil, acquired a significant mating advantage. Even more, some males were often observed attempting to feed on these oils, thus assessing the advantage of citrus oils or mixture of compounds over GRO. These results agree with ours, despite the fact that the applied doses were lower and oil sources and exposure methods were different. Therefore, further work should be invested to establish an application dose, to select the more convenient and economically affordable linalool source (citrus oil from local industry) and its effect under natural conditions. Concerning cost reduction, the cost of aromatherapy in the Comunitat Valenciana SIT programme represents only 0.001% of its total cost [calculated for current GRO aromatherapy, R. Argilés (TRAGSA, Spain)]. In the Comunitat Valenciana SIT programme, GRO and the tested essential oils are currently supplied by foreign manufacturers, consequently, the actual availability of local producers of these oils, mainly the proposed linalool would represent a limited cost reduction.

In conclusion, our study demonstrates that (i) a GRO-supplemented diet increase male mating performance in levels comparable to GRO aromatherapy, and could therefore result in a net reduction in programme costs; and (ii) linalool aromatherapy improved mating performance in sterile males, and could serve as viable alternative to GRO aromatherapy. With respect to the SIT programme success by boosting sterile male performance through aromatherapy, further studies are needed to find the best and most economically viable variety of citrus oils that contain higher percentages of linalool [including also studies of dose and exposure methodology optimization as in Kouloussis et al. (2013) and Paranhos et al. (2013)]. In particular, field cage studies under semi-natural conditions and further field studies should be conducted to compare the induced sterility in nature.

Acknowledgements

We thank the TRAGSA Emergence and Release facility personnel for their help in the mating experiments and to Don McInnis (USDA-ARS Hawaii) and Rui Pereira (IAEA/FAO Entomology section) for their helpful comments on earlier versions of this manuscript. This work was partially supported by IAEA Contract no. 12864, the Conselleria d’Agricultura, Pesca i Alimentació de la Generalitat Valenciana and FEOGA COOPERACIÓN. M. A. Juan-Blasco was supported by an IVIA PhD fellowship. B. Sabater-Muñoz is a recipient of a cofunded EC-SF fellowship- CCAA-INIA.

This article was published online on 13 December 2011. The references for Kouloussis et al. (2013) and Paranhos et al. (2013) have now been updated to show the correct citation details for J. Appl. Entomol. Vol. 137, Suppl. 1 and the reference for Shelly (2012) has been updated to show the correct citation details for J. Appl. Entomol. Vol. 136.

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