Improving sterile male performance in support of programmes integrating the sterile insect technique against fruit flies

Authors


Rui Pereira (corresponding author), Insect Pest Control Section, Joint FAO/IAEA Division of Nuclear, Techniques in Food and Agriculture, Wagramerstrasse 5, PO Box 100, A-1400 Vienna, Austria. E-mail: R.Cardoso-Pereira@iaea.org

Abstract

Abstract The sterile insect technique (SIT) is being applied against fruit fly pests in many areas of the world. Currently, factories have a capacity to produce several billion sterile insects per week, to irradiate them and to make them available for shipment to their destinations. There the emerging flies are fed and maintained for various periods of time in fly emergence and release facilities, and then collected for release. While much research effort has been invested in improving mass rearing and quality control procedures at the fly factory level, the post-factory handling of sterile flies has received much less attention. A 6-year FAO/IAEA Coordinated Research Project (CRP) was initiated with the participation of 31 research institutes from 17 countries to assess and validate ways to improve sterile male performance through better management during the critical period that starts with the arrival of pupae at the fly emergence and release facility and ends with the release of the sterile flies in the field. This review summarizes the research that was conducted under the CRP focusing on fruit fly species from the genera Anastrepha, Bactrocera and Ceratitis against which the SIT is being applied. To increase the effectiveness of SIT programmes, exposure of sterile males to nutritional, hormonal and semiochemical supplements was assessed and improved handling and release methods tested. Incorporation of protein and juvenile hormone into pre-release diets significantly accelerated sterile male maturation and improved sexual performance among several species, while semiochemical treatments using ginger root oil or citrus oils in Ceratitis capitata and methyl eugenol in Bactrocera dorsalis complex species significantly increased sterile male mating competitiveness. Improved fly emergence, holding and release procedures were also assessed, together with the compilation of all this knowledge into a manual. Several of these findings have been transferred to and are being applied in operational programmes.

Introduction

Application of the sterile insect technique (SIT) as part of an area-wide integrated pest management (AW-IPM) approach against fruit fly (Diptera: Tephritidae) pests is gaining momentum, with active programmes targeting fruit fly pest species of economic importance in North, Central and South America, Europe, the Middle East, Asia, Africa and Australia (Enkerlin 2005). There are now fruit fly factories with the capacity to mass rear several billion sterile male insects per week. After irradiation, pupae are shipped to their destination, where they are processed in fly emergence and release facilities (FAO/IAEA 2007). Several days after emergence, the sterile flies are released in the field, where sterile males are expected to mate with and transfer sperm to wild females. Thus, an industrial process, consisting of numerous complex steps, is required to ultimately achieve a biological goal.

Although a number of quality control and quality assurance protocols have been developed to ensure that the released flies fulfil a series of minimum quality standards (FAO/IAEA/USDA 2003), the exigencies of the industrial process often affect the biological qualities of the final product. Additionally, suppression activities to reduce wild population levels are needed, as well as adequate overflooding ratios of sterile to wild males for the technique to be effective. Indeed, there is a general consensus that the SIT is an evolving science, and much can still be done to improve the efficacy of the technique.

Mass rearing of fruit flies up to the pupal stage, including sterilization, takes place in specially designed insect factories where insects are adapted to indoor conditions. As late-stage pupae, the mass-reared insects are irradiated and shipped to emergence and release facilities, where they are placed in containers for adult emergence, feeding and handling before they are released into target field areas. During this critical period at these facilities, there is potential to manipulate sterile males in a manner that will significantly improve their mating success in the field. This includes optimizing the provision of nutritional (Yuval et al. 2002), hormonal (Teal et al. 2000) and semiochemical supplements (Shelly 2001), as well as the holding conditions/release methods (Salvato et al. 2004; FAO/IAEA 2007; Tween and Rendón 2007).

To investigate these possibilities and to transfer the findings to ongoing programmes, the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture conducted a Coordinated Research Project (CRP) on ‘Improving Sterile Male Performance in Fruit Fly Sterile Insect Technique Programmes’ from 2004 to 2009. The research, involving 31 research institutes from 17 countries, focused on the fruit fly pest species targeted by SIT programmes (from the genera Anastrepha, Bactrocera and Ceratitis). The overall objective of this CRP was to increase the effectiveness of the SIT by improving the performance of mass-reared sterile males through the use of nutritional, hormonal and semiochemicals supplements and the improvement in handling and release methods.

Results

Nutritional supplements

Both male and female tephritid fruit flies are anautogenous, emerging as adults with undeveloped gonads. Both sexes rely on foraging during adult life to ingest carbohydrates to fuel metabolic activities, as well as nitrogenous compounds for gonadal and accessory gland development and pheromone production (Epsky and Heath 1993; Drew and Yuval 2000). Thus, the survival and reproductive success of males in most species of economic importance is linked to access these nutrients. Nevertheless, a majority of fly emergence and release facilities do not include nitrogenous compounds in the pre-release diet of sterile males. Sterile males are usually offered a pre-release diet of sugar or highly concentrated sucrose, presented in agar blocks, which is also a source of water (FAO/IAEA 2007; USDA/APHIS 2009).

Studies on species from three tephritid genera indicate that providing yeast hydrolysate to males in the days following emergence can enhance male reproductive success, although the optimal dosage and form of presentation in an operational context still need to be established (Papadopoulos et al. 1998; Kaspi and Yuval 2000; Aluja et al. 2001; Pérez-Staples et al. 2007). The implementation of such a strategy depends on the specific understanding of the nutritional needs of males, trade-offs between sexual performance and survival, and operational costs and benefits.

The studies carried out in conjunction with the CRP reached different levels of understanding in the various tephritid species targeted by the SIT regarding the nutritional requirements of males and their effect on sexual success. The findings for all three genera (Anastrepha, Bactrocera and Ceratitis) provide evidence that wild males feed on sources of protein in nature. However, while providing yeast hydrolysates or other sources of protein together with sugar to wild males consistently improved the mating success in Anastrepha and Bactrocera, in Ceratitis, there is variance in results reflecting other factors affecting the outcome. The addition of protein to the pre-release diet of mass-reared sterile males increased their reproductive success and reduced wild female remating. The effect of protein-rich diets on male survival and longevity was complex, reducing in some cases male survival and dispersal. This adverse effect was reduced when the protein ratio in the diet was lowered. Moreover, there was evidence that the integrity of microorganisms of the male gut contributes to fly health and sexual performance.

The results on nutritional supplement assessments are described thematically in more detail, together with the relevant references, in table 1.

Table 1. Effects of incorporation of nutritional supplements to pre-release diet on male performance of tephritids of the genera Anastrepha, Bactrocera and Ceratitis
Areas addressedMain findings
Natural foodMales fed on orange [Citrus sinensis (L.) Osbeck] or mango (Mangifera indica L.) exhibited improved sexual performance in Anastrepha ludens and Anastrepha obliqua (Liedo et al. 2013; Utgés et al. 2013)
Male pheromone production is quantitatively affected by natural food in A. ludens and A. obliqua (Liedo et al. 2013)
Wild Bactrocera tryoni males fed on natural sources of food matured much slower than males with access to yeast hydrolysate; effects of diet were far less pronounced in mass-reared males (Weldon and Taylor 2011)
Exposure to oranges and grapefruits (Citrus paradisi Macfad) improved mating by male Ceratitis capitata (Shelly et al. 2004a; Shelly 2009)
Dietary supplementsAddition of yeast hydrolysate (protein) to the pre-release diet significantly improved male sexual performance in Anastrepha fraterculus, A. ludens, A. obliqua, Anastrepha suspensa, Bactrocera correcta, Bactrocera cucurbitae, Bactrocera dorsalis, Bactrocera philippinensis, B. tryoni, B. zonata, C. capitata and Ceratitis rosa (Taylor and Yuval 1999; Shelly et al. 2005; Pérez-Staples et al. 2007, 2009; Yuval et al. 2007; Faria et al. 2008; Weldon et al. 2008; Pereira et al. 2009, 2010a,b, 2013; Haq et al. 2010a,b, 2013; Haq and Hendrichs 2013; Quilici et al. 2013; Liedo et al. 2013)
Addition of protein to the pre-release diet significantly improved male weight and body protein content for A. suspensa and B. cucurbitae (Pereira et al. 2011; Haq et al. 2010c)
Ratio of protein to carbohydratesThe ratio of protein to carbohydrates in dry diets provisioned early in adult life contributed to male sexual performance and affected survival in a dose-dependent manner in A. fraterculus, A. ludens, A. obliqua and B. tryoni (Prabhu et al. 2008; Gómez et al. 2013; Liedo et al. 2013; Segura et al. 2013; Utgés et al. 2013).
As little as 4-10% of protein content in the pre-release diet was sufficient to significantly enhance male sexual performance in A. fraterculus, A. ludens, A. obliqua and B. tryoni (Pérez-Staples et al. 2008; Gómez et al. 2013; Liedo et al. 2013; Pereira et al. 2013; Segura et al. 2013)
Optimal formulation/delivery systemA formulation based on soy whey protein and incorporating methoprene has been developed and tested for A. suspensa (Teal et al. 2013)
Concurrently, a commercial product (‘Mubarqui’) containing protein has been introduced in a dry formulation into release programmes for A. ludens and A. obliqua (Gómez and Teal 2010; Gómez et al. 2013)
Interaction between diet, dispersal and survivalProtein-rich diets were related to a lower resistance to starvation in A. fraterculus, A. ludens, A. obliqua and C. capitata (Levy et al. 2005; Carey et al. 2008; Segura et al. 2013; Utgés et al. 2013)
Protein-rich diets were related to lower recapture rates in protein-baited traps and to reduced mobility and longevity in sterile A. fraterculus, A. ludens, A. obliqua (but not in C. capitata) males (Maor et al. 2004; San Andrés et al. 2009; Segura et al. 2013; Utgés et al. 2013)
Protein-rich post-teneral diets did not adversely affect survival and movement in the field of sterile B. tryoni and C. capitata males (Yuval et al. 2007; Gavriel et al. 2010; Taylor et al. 2013)
In the laboratory, protein-fed males lived significantly longer than sugar-fed males of B. cucurbitae and B. tryoni (Haq et al. 2010b; Taylor et al. 2013). The same was found in A. ludens and A. obliqua when the sugar/yeast ratio was 9:1 or 24:1, but not with the 3:1 ratio (Liedo et al. 2013)
Inhibition of rematingFeeding on a nitrogen-rich food significantly improved the ability of sterile males to inhibit female remating in A. fraterculus, B. tryoni and C. capitata (Harmer et al. 2006; Radhakrishnan and Taylor 2007; Gavriel et al. 2009; Segura et al. 2013)
IrradiationIrradiation did not affect the activity of proteases and peptidases in the gut of male C. capitata (San Andrés et al. 2007)
Irradiation significantly diminishes the ability of the flies to tolerate protein deprivation in B. tryoni (Taylor et al. 2013)
Contribution of microorganismsDiversity of the microflora of laboratory reared and irradiated males is low compared with wild C. capitata males (Ben-Ami et al. 2010; Coronado-Gonzalez et al. 2008)
The contribution of microorganisms to fitness of non-sterile Bactrocera oleae and C. capitata was established (Behar et al. 2008a; Ben-Yosef et al. 2008a,b, 2010; Yuval et al. 2013)
Manipulating the microflora of sterile males suggests that probiotic supplements could improve male sexual performance in C. capitata (Niyazi et al. 2004; Behar et al. 2005, 2008a,b,c; Ben-Ami et al. 2010)

Hormonal supplements

Age is a significant factor affecting sexual signalling and reproduction in numerous tephritid species (Liedo et al. 2002). Being anautogenous, tephritid fruit fly pests require a considerable period (over a week in Ceratitis spp.; 2–3 weeks in Anastrepha spp. and Bactrocera spp.) under adequate nutritional conditions, to reach sexual maturation. Even though mass rearing inadvertently selects for accelerated sexual maturation, the discrepancy between standard holding periods at fly emergence and release facilities and time to reach sexual maturation poses a significant problem for SIT programmes. After release and before reaching full sexual maturation, sterile males suffer significant losses because of predation and other causes, resulting in much fewer males surviving to maturity and copulation (Hendrichs and Hendrichs 1998). Holding sterile males for more days increases the management costs, and additional investment is required to expand the infrastructure at fly emergence facilities (USDA/APHIS 2009). In addition, in those programmes where sterile males are released together with sterile females, longer holding time is not advisable in view that males will start mating before release, thereby wasting their limited sperm on sterile females.

Clearly, development of cost-effective methods to accelerate sexual maturity in released flies would have a significant positive impact on the efficacy of the SIT. Research on a number of tropical Anastrepha species showed that juvenile hormone is a critical hormone regulating sexual maturity and sexual signalling in these species and that application of juvenile hormone or the mimics, methoprene or fenoxycarb, can accelerate reproductive development and sexual signalling in sterile males (Teal et al. 2000). This finding, coupled with the relatively low cost associated with the purchase of methoprene, indicated that exploring ways under the CRP to incorporate hormone supplements into adult emergence protocols could have the potential to develop a cost-effective way to accelerate male maturation, thereby improving the efficacy of operational SIT programmes.

Significant progress was made during the CRP in developing and evaluating hormonal therapies using methoprene to accelerate reproductive development and in some cases, further improving male sexual performance. It was found that in many, but not all species, sterile males become sexually mature significantly earlier when hormone therapy is included in pre-release holding protocols, but this optimum effect was only achieved when hormone treatment was coupled with a protein-enriched pre-release diet. Thus, irradiated males become sexually mature earlier and can be released earlier. This advantage is particularly important for SIT application against those species of Anastrepha spp. and Bactrocera spp., which have long pre-copulatory periods. The improvement in male sexual performance by the application of hormonal supplements was extensively studied in Anastrepha spp., Bactrocera cucurbitae and Ceratitis capitata, although in this last species, no benefits were found. In addition, considerable progress was made in developing delivery systems to treat large numbers of flies with methoprene in operational programmes.

Results on hormonal supplement assessments are described thematically in more detail, together with the relevant references, in table 2.

Table 2. Effects of pre-release application of hormonal supplements on male performance of tephritid fruit flies of the genera Anastrepha, Bactrocera and Ceratitis
Areas addressedMain findings
Sexual maturationAge at which laboratory reared and wild males of Anastrepha fraterculus, Anastrepha ludens, Anastrepha obliqua, Anastrepha serpentina, Anastrepha striata, Anastrepha suspensa, Bactrocera correcta, Bactrocera cucurbitae, B. dorsalis, Bactrocera philippinensis, Bactrocera tryoni, Ceratitis capitata and Ceratitis rosa become sexually mature was determined (Teal and Gómez-Simuta 2002; Faria et al. 2008; Pereira et al. 2009; Segura et al. 2009; Gómez et al. 2013; Obra and Resilva 2013; Orankanok et al. 2013; Quilici et al. 2013; Sookar et al. 2013; Taylor et al. 2013)
Improvement in reproductive maturation and sexual performancePositive effect of hormonal treatment on accelerating reproductive development and improving sexual performance found in A. fraterculus, A. ludens, A. obliqua, A. serpentine, A. suspensa, B. cucurbitae and B. tryoni (Pereira et al. 2009; Segura et al. 2009; Haq et al. 2010a, 2013; Gómez et al. 2013)
No effect on either B. dorsalis or C. capitata (Faria et al. 2008; Shelly et al. 2009)
Interaction of protein and hormone supplements on sexual performanceMales treated with hormone and fed a protein-rich diet effectively competed with mature wild males, but at significantly earlier ages for A. fraterculus, A. ludens and A. suspensa (Pereira et al. 2009, 2013; Segura et al. 2009, 2013; Gómez and Teal 2010; Gómez et al. 2013)
B. cucurbitae males treated with methoprene and access to a protein-rich diet had an accelerated maturation and improved mating performance (Haq et al. 2010a)
Interaction of irradiation and hormone supplements on sexual maturityInteraction of irradiation and hormone supplements has no negative effects on male acceleration of reproductive maturity of A. fraterculus, A. ludens, A. obliqua, A. suspensa and B. tryoni (Teal et al. 2007; Collins et al. 2008; Segura et al. 2009)
Optimal dose to accelerate maturationOptimal dose determined using topical application for A. fraterculus, A. ludens, A. obliqua, A. suspensa and B. cucurbitae (Teal et al. 2007; Pereira et al. 2009; Segura et al. 2009; Haq et al. 2010a)
Response in males and females to hormone supplementsA differential response in males and females to methoprene exposure was found in A. fraterculus that can act as a physiological sexing system minimizing matings between sterile males and sterile females (Segura et al. 2013)
No differential response to methoprene exposure found in A. ludens males and females (Pereira et al. 2013)
Development of delivery systems other than topical applicationAgar-based diet containing 5–10% protein along with 0.05% methoprene tested with A. ludens and A. suspensa produced large amounts of waste and is not cost effective (Teal et al. 2013; Pereira et al. 2013)
Pupal dipping in an acetone bath containing 0.05% methoprene tested with A. fraterculus, A. ludens and A. suspensa does not impact emergence or survival and accelerates reproductive development, although causes humidity problems when handling large volumes of pupae (Pereira et al. 2013)
Pupal dipping found impractical because of health risks, safety and disposal issues associated with the use of acetone (Pereira et al. 2013; Segura et al. 2013)
A dry sugar–protein pre-release diet containing 0.0015% methoprene for A. ludens and A. obliqua shown to be practical and effective in accelerating development when applied in operations of fly emergence and release facilities (Gómez et al. 2013)
Efficient large-scale methods for incorporation of JH analoguesThe dry sugar–protein–methoprene diet method assessed in a 3500-ha pilot area for A. ludens and A. obliqua in Mexico (Gómez et al. 2013)
The hormone and protein delivery system has been incorporated in the sterile insect technique ‘Moscafrut’ action programme to control A. ludens and A. obliqua fruit flies in Mexico (Gómez et al. 2013)

Semiochemical supplements

Males of a majority of Bactrocera spp. and Ceratitis spp. are strongly attracted to natural compounds known as semiochemicals (Cunningham 1989). Some species are known to sequester these chemicals for use in pheromone synthesis; for example, ingestion of methyl eugenol (ME) from natural sources by males of the Bactrocera dorsalis complex results in the storage of metabolites in the rectal gland and their subsequent release as part of the pheromone during fanning performed in courtship (Tan and Nishida 1996). Assessing whether providing sterile B. dorsalis males with a source of ME to feed on before release could increase their mating competitiveness, Shelly et al. (2005) showed that males exposed to ME before release were as competitive as wild males. ME feeding may also confer a survival advantage, as metabolites appear to act as a very potent allomone to deter vertebrate predators (Wee and Tan 2001).

In the case of C. capitata, exposure to ginger (Zingiber officinale Roscoe) root oil or citrus oils (by contact or vapour) enhances considerably the mating competitiveness of wild or mass-reared males (Katsoyannos et al. 1997; Papadopoulos et al. 2001; Shelly 2001).

There are no known effective mating enhancing semiochemicals for Anastrepha; however, the dramatic mating enhancing effects seen with Bactrocera species and Ceratitis suggested that similar compounds may exist in the Anastrepha ecological sphere and await discovery.

The research was carried out under the CRP built on the aforementioned studies to understand these phenomena, to extend them to other species, and to transfer and validate them under large-scale conditions of action SIT programmes. Results obtained during the CRP have confirmed the compounds that have the potential to improve sterile male performance in the field and have identified some additional ones. These chemicals are the following:

  •  Methyl eugenol (ME), and holy (Ocimum tenuiflorum L.) and sweet (Ocimum basilicum L.) basil oils improve the mating competitiveness of several Bactrocera species, including B. dorsalis, Bactrocera philippinensis, Bactrocera zonata and Bactrocera correcta males;
  •  Cuelure (CUE) has the potential to enhance the performance of B. cucurbitae and Bactrocera tryoni males;
  •  Raspberry ketone and/or zingerone have the potential to enhance the performance of B. cucurbitae and B. tryoni males;
  •  Ginger root oil (GRO), α-copaene, and orange and other citrus oils enhance male performance of C. capitata, Ceratitis rosa and potentially Anastrepha fraterculus;
  •  Citrus and guava fruit volatiles have the potential to enhance the mating performance of male Anastrepha ludens and A. fraterculus;
  •  Manuka oil (rich in α-copaene) from the manuka tree (Leptospermum scoparium Forst & Forst) of New Zealand improves C. capitata male mating (Shelly et al. 2008c).

Methodologies for exposing large numbers of C. capitata males through GRO aromatherapy in large containers have been developed and are now routinely applied in a very cost-effective manner in ongoing SIT programmes in Australia, Brazil, Guatemala, Israel, Mexico, Portugal, Spain and USA, resulting in a significant improvement in mating performance of the released sterile males.

The results of semiochemical supplement assessments are described thematically in more detail, together with the relevant references, in table 3.

Table 3. Effects of pre-release application of semiochemical supplements on male performance of tephritids of the genera Anastrepha, Bactrocera and Ceratitis
Areas addressedMain findings
Search for semiochemical compounds that affect Anastrepha spp. male sexual performanceEffect of orange and mango fruit on male mating competitiveness and pheromone composition of Anastrepha ludens and Anastrepha obliqua (Liedo et al. 2013)
Exposure to guava fruit volatiles resulted in a positive effect on male mating performance of Anastrepha fraterculus (Vera et al. 2013)
Exposure to ginger root oil (GRO) and lemon fruit volatiles enhanced male mating performance for A. fraterculus; however, some of the tests resulted in detrimental effects (Vera et al. 2013)
Assessment of various semiochemicals in terms of enhancing Bactrocera dorsalis complex spp. male performanceHoly and sweet basil were found to be natural sources of methyl eugenol (ME) (Obra and Resilva 2013)
Exposure to basil oil had a positive effect on sterile male mating performance of Bactrocera philippinensis (Obra and Resilva 2013)
Exposure to ME significantly improved sterile male mating performance of Bactrocera correcta and B. dorsalis (Shelly et al. 2005, 2008b, 2009, 2010a; Shelly and Edu 2008; Ji et al. 2013; Orankanok et al. 2013)
Exposure to commercial ME and interaction with post-teneral adult diet demonstrated positive effects of male mating performance for B. correcta and B. dorsalis. For both species, ME-treated sterile males are now being released in operational sterile insect technique (SIT) programmes in Thailand (Orankanok et al. 2013)
Exposure to ME did not affect dispersal or survival of sterile flies of B. correcta and B. dorsalis (Orankanok et al. 2013)
Assessment of various semiochemicals in terms of enhancing other Bactrocera spp. male performanceExposure to both cuelure (CUE) and raspberry ketone enhanced male mating performance in Bactrocera cucurbitae, however, only for 1 day post-feeding (Shelly et al. 2013)
Exposure to CUE and raspberry ketone increased mating competitiveness of Bactrocera tryoni; however, young males failed to respond to CUE, orange oil (OO) and GRO (Weldon et al. 2008)
Optimal age and dose of feeding for Bactrocera spp. male fliesThe optimal age and duration of ME exposure, and the diurnal pattern of ME feeding, were determined for B. philippinensis (Obra and Resilva 2013)
Optimal age of ME exposure, in combination with nutritional supplements, was determined for sterile B. correcta and B. dorsalis males (Orankanok et al. 2013)
Effect of semiochemical supplements on C. capitata sexual signalling (pheromone calling)Ginger root oil (GRO)-exposed males exhibit higher rates of sexual signalling compared with unexposed C. capitata males (Shelly et al. 2004b, 2007a, 2010b; Papadopoulos et al. 2006; Briceño et al. 2007; Shelly 2008; Juan-Blasco et al. 2013; Paranhos et al. 2013; Steiner et al. 2013)
GRO-exposed sterile males exhibit during sexual courtship the same wing beat duration as wild males, and this was shorter than for unexposed sterile males (Morelli et al. 2013)
Exposure to orange oils (OO) and its components increased sexual signalling of males, and this effect was more pronounced in protein-fed C. capitata flies (Katsoyannos et al. 2005; Papadopoulos et al. 2006; Kouloussis et al. 2013)
Identification of active compounds other than GRO and their effects on sexual performance in C. capitataExposure to fruits of five citrus species revealed that sweet oranges conferred the highest increase in male mating performance of C. capitata males. Exposures to commercial citrus oils were found to be similarly effective (Kouloussis et al. 2013)
Positive effect of OO components (limonene, b-myrcene, and linalool) was found in protein/sugar-fed males, but not in sugar-only-fed males (Kouloussis et al. 2013)
A mixture of limonene, b-myrcene, linalool, a-pinene and geraniol (1:1:1:1:1) was proven to be very effective for both wild and sterile C. capitata males (Kouloussis et al. 2013)
Attractiveness of GRO and OO to C. rosaThe attractiveness of GRO and OO investigated for C. rosa. Sugar-only-fed males exposed to GRO and OO increased their mating performance. However, only GRO increased mating performance of protein/sugar-fed males (Quilici et al. 2013)
Effect of semiochemical supplements on C. capitata female remating frequencyThe remating frequency of C. capitata females mated to sterile males treated with 0.1 ml/m3 GRO similar to those mated with wild males and lower than that for females mated with sterile unexposed males (Morelli et al. 2013)
Effect of semiochemical supplements on C. capitata dispersal and survivalField dispersal and survival rates of GRO-exposed sterile males were similar to unexposed C. capitata males (San Andrés et al. 2009; Juan-Blasco et al. 2013)
Determination of semiochemical exposure dose for C. capitataThe optimal doses of exposure determined for OO compounds showed positive effects increasing C. capitata male performance (Kouloussis et al. 2013)
The optimal dose of OO exposure was determined for both protein–sugar and sugar-only C. capitata adult diets. Positive effects of OO in protein–sugar diet were found, but no effects in the sugar-only diet (Kouloussis et al. 2013)
Optimal delivery system for large-scale semiochemical application in Ceratitis spp. SIT programmesThe cost-effective GRO exposure to sterile males established and now in operational used in a number of C. capitata SIT action programmes (Shelly et al. 2007b,c, 2008a; Paranhos et al. 2008, 2013; Juan-Blasco et al. 2013; Silva et al. 2013; Steiner et al. 2013)

Abiotic environment and release methods

The environmental conditions in which sterile flies are held at fly emergence and release facilities are potentially critical (FAO/IAEA 2007; USDA/APHIS 2009). Parameters such as light intensity, the light/dark cycle (if any), temperature, humidity and barometric pressure may all play important roles in conditioning the flies before their release. Much remains unknown about the effects of manipulating these environmental conditions, either separately or in combination, on subsequent male field performance.

The type of holding container will depend on the release method, as they can be distributed by ground or by air. Fly density and the surface area available per fly to rest in the holding containers are important factors to avoid overcrowding and stressing the flies. Containers for ground release are typically small bags or boxes. An advantage of ground releases is that they require no further handling, except perhaps to add additional food or water, because flies are released from the same containers into which the pupae are packaged when they are received at the fly emergence and release facilities.

The aerial release of sterile flies, a necessity in larger scale programmes, is usually more complicated, except when using small bags or boxes, which can be loaded directly into planes and torn open at the time of release. Normally, the pupae arriving at fly emergence and release facilities will be held in temporary containers, such as plastic adult rearing container (PARC) boxes or fly emergence towers (racks with trays to collect emerging fruit flies), until just prior to aerial release (FAO/IAEA 2007). Flies must be extracted from these containers by temporarily anaesthetizing them, usually with cold, and then transferring them to aerial drop machines, which are loaded in the aircrafts (FAO/IAEA 2007). The effect of cold anaesthetizing on sterile male performance, including pheromone quantity or quality, needs to be investigated.

One of the goals of the CRP was to integrate improved pre-release environmental conditions, holding containers, fly collection and release methods, with the nutritional, hormonal and semiochemical manipulations discussed earlier, tailoring them to the biology of each fruit fly species.

The results of the assessment of environmental holding conditions and release methods are described thematically in more detail, together with the relevant references, in table 4.

Table 4. Effects of abiotic environment/release methods at fly emergence and release facilities on male performance of tephritids of the genera Anastrepha, Bactrocera and Ceratitis
Areas addressedMain findings
Compilation of procedures to ship, emerge, feed, hold, collect and releaseCurrent procedures in different parts of the world to ship, emerge, feed, hold, chill, collect and release sterile flies were compiled and published (FAO/IAEA, 2007)
Effects of varying temperature, relative humidity and ventilation during fly holdingEffects of holding conditions and ventilation in ground release containers on Ceratitis capitata sterile male performance, indicating the importance of proper air exchange at higher densities (B. Barnes, personal communication)
Evaluation of different fly emergence systemsEvaluation of different fly emergence systems, including densities, volumes and ventilation on the male performance, was carried out for Anastrepha ludens and C. capitata (Shelly et al. 2006; Gómez et al. 2013; Silva et al. 2013)
Parameters of sterile fly quality, space, labour and economics assessed for eight fly emergence and release facilities were compared in Guatemala, Mexico and USA (USDA/APHIS 2009)
In conjunction with the extensive evaluations under the CRP, the California C. capitata programme moved its emergence, feeding and holding operations from boxes to towers (USDA/APHIS 2009)
Interaction of different holding conditions and hormonal, nutritional and/or semiochemical supplementsAssessment of the interaction of different holding conditions and hormonal, nutritional and/or semiochemical supplements on sterile fly performance resulted in the routine application of GRO under large-scale holding conditions in operational C. capitata sterile insect technique programmes (FAO/IAEA 2007; USDA/APHIS 2009)
Holding conditions in relation to fly age and sexual maturation indicate that in C. capitata sexing strains, the release of older males is preferable resulting in more flies reaching mating age in the field (McInnis et al. 2013)
Species with longer pre-copulatory period would benefit more from longer pre-release holding periods (Gómez et al. 2013; Pereira et al. 2013)
Design for a fly holding system that optimizes methyl eugenol feeding and fly holding in space and time was completed for Bactrocera dorsalis. Preliminary tests on interactions of males with the semiochemical feeding structure were tested in a small-scale prototype (Tan and Tan 2013b)
Comparison of males from standard conditions with males pre-conditioned to specific field conditionsPre-conditioning of sterile males to specific field conditions was assessed in Bactrocera tryoni and C. capitata for temperature (Steiner et al. 2013)
Effects of cold knockdownEffect of cold knockdown varies with C. capitata density. At density typical of emergence tower, cold lessens flight ability and mating competitiveness; though, these effects may be temporary (Shelly et al. 2013)
Effect of cold knockdown was found to affect pheromone production and the mating performance during the first 3 days after chilling for A. ludens and Anastrepha obliqua; however, flies recovered thereafter (Liedo personal communication)
Effects of different sterile fly release systemsThe effects on fly performance of different sterile fly release systems, and the interaction with hormonal, nutritional and/or semiochemical supplements was assessed for some aerial and ground release systems for C. capitata in terms of dispersal and recapture (Paranhos et al. 2013; Silva et al. 2013)
Hormonally and nutritionally treated A. ludens and A. obliqua released by air were evaluated in terms of quality and field survival (Gómez et al. 2013)
Ground release machine was developed and evaluated (Bjeliš et al. 2013)
Comparison of different alternative aircrafts was carried out in terms of their suitability and economics for sterile fly aerial release (Tan and Tan 2013a)

Impact of the research conducted

The CRP confirmed through extensive laboratory and semi-field tests that pre-release diets nutritionally enriched with yeast hydrolysate will improve sterile male performance and therefore result in increased SIT efficiency for a majority of fruit fly species tested in the three genera, and thus can be adopted by programme managers. Also hormone therapy coupled with feeding protein to adults significantly accelerates male maturation and sexual performance among Anastrepha species and some Bactrocera species with no negative side effects (survival and competitiveness). This can represent significant cost savings associated with reduced holding periods for sterile males prior to release and/or fewer males dying in the field before reaching sexual maturity. The validation in a number of fruit flies has resulted in the incorporation of these major breakthroughs into action programmes against several of these pest species.

The development of cost-effective semiochemical treatments that improve sterile male sexual signalling and attractiveness to wild females and thus their overall mating performance has been highly desirable. Implementation of aromatherapy has significantly increased the effectiveness of ongoing SIT programmes against C. capitata. While the incorporation of semiochemical treatments for Bactrocera species appears feasible, it still remains to be implemented operationally. In combination with the simultaneous application of male annihilation to eliminate wild males, incorporation of ME pre-release treatment has considerable potential to drastically increasing sterile-to-wild male overflooding ratios (Robinson and Hendrichs 2005).

Improved fly emergence, holding and release procedures, and the compilation of all this knowledge into a manual, have benefited all operational fruit fly SIT programmes. Knowledge gained and practical procedures developed during this CRP are transferable, at least in part, to other insect pest species with control programmes that include an SIT component. Much remains unknown about the effects of manipulating the environmental holding conditions, either separately or in combination with nutritional, hormonal and semiochemical treatments, on subsequent male quality in the field. Release methods, while operationally convenient, are not always optimal in terms of sterile male performance. Therefore, the effects of the different processes and systems need to be further assessed, tailoring them to the biology of each fly species.

This article was published online on 28 November 2011. The references for Bjeliš et al. (2013), Gómez et al. (2013), Haq and Hendrichs (2013), Haq et al. (2013), Ji et al. (2013), Juan-Blasco et al. (2013), Kouloussis et al. (2013), Liedo et al. (2013), McInnis et al. (2013), Morelli et al. (2013), Obra and Resilva (2013), Orankanok et al. (2013), Paranhos et al. (2013), Pereira et al. (2013), Quilici et al. (2013), Segura et al. (2013), Shelly et al. (2013), Silva et al. (2013), Sookar et al. (2013), Steiner et al. (2013), Tan and Tan (2013a,b), Taylor et al. (2013), Teal et al. (2013), Utgés et al. (2013), Vera et al. (2013) and Yuval et al. (2013) have now been updated to show the correct citation details for J. Appl. Entomol. Vol. 137, Suppl. 1.

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