Introduction. Development and improvement of rearing techniques for fruit flies (Diptera: Tephritidae) of economic importance

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1 International Journal of Tropical Insect Science Vol. 34, No. S1, pp. S1 S12, 2014 q icipe 2014 doi: /s Introduction Development and improvement of rearing techniques for fruit flies (Diptera: Tephritidae) of economic importance Carlos Cáceres, Jorge Hendrichs and Marc J.B. Vreysen* Insect Pest Control Sub-programme, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, International Atomic Energy Agency, Vienna, Austria (Accepted 18 March 2013; First published online 19 August 2014) Abstract. The papers presented in this special issue are focused on developing and validating procedures for artificial rearing of selected fruit fly species of economic importance for use in area-wide integrated pest programmes with a sterile insect technique (SIT) component. They are the result of a 5-year coordinated research project (CRP) on Development and Improvement of Rearing Techniques for Anastrepha and Bactrocera Fruit Flies that was coordinated by the Joint FAO/IAEA Programme of Nuclear Techniques in Food and Agriculture. Twenty-two CRP participants from 18 countries worked on both basic and advanced rearing procedures of different fruit fly species to overcome technical bottlenecks and to develop appropriate and relevant procedures for use in mass-rearing facilities. A variety of studies were undertaken for three groups of tephritid species: (1) those for which mass-rearing for SIT application was available, but further refinement would be useful; (2) those for which mass-rearing for SIT application was under development in the laboratory, but that had not reached mass-rearing status; and (3) those for which research to develop colonization and rearing methods was needed. Many of the protocols developed or improved during the CRP have been transferred to the different fruit fly mass-rearing facilities worldwide. Key words: Tephritidae, mass-rearing, diets, irradiation, quality control, colony management, fruit flies Introduction Fruit flies (Diptera: Tephritidae) are of major economic importance in nearly all tropical, subtropical and temperate countries worldwide. They are among the worst pests in agriculture, causing enormous devastation to both production and international trade of fresh horticultural commodities (Klassen et al., 1994; Hendrichs, 1996). They are * m.vreysen@iaea.org also among the causes of poverty and malnutrition in large areas of tropical developing countries, where climatic conditions are favourable for labourintensive fruit- and vegetable-based agroindustries (Waterhouse, 1993a,b; Allwood and Leblanc, 1996; Allwood, 2000). As insecticide-based control of fruit flies is not sustainable due to environmental, health and resistance problems, their study and management have received considerable national and international attention over the last four decades. International organizations of the UN family such

2 S2 as the Food and Agriculture Organization (FAO) and the International Atomic Energy Agency (IAEA) as well as others have been actively involved in developing more environmentally friendly control tactics, including the sterile insect technique (SIT), and in supporting Member States in their application (Dyck et al., 2005). Significant resources have been allocated to develop and validate the SIT against fruit flies of economic importance, e.g. the Mediterranean fruit fly Ceratitis capitata Wiedemann, the Mexican fruit fly Anastrepha ludens (Loew), the West Indian fruit fly A. obliqua (Macquart), the Caribbean fruit fly A. suspensa (Loew), the melon fly Bactrocera cucurbitae (Coquillett), the Queensland fruit fly B. tryoni (Froggatt) and some species of the Bactrocera dorsalis complex. As a result, in several countries, the SIT has been successfully integrated with other control tactics to suppress, eradicate, contain or prevent the establishment of these pests using area-wide integrated pest management (AW-IPM) approaches (Enkerlin, 2005; Hendrichs et al., 2005, 2007). To be effective, the SIT requires that large numbers of high-quality insects be released on a sustained and area-wide basis to suppress the reproductive capacity of a resident fertile population, leading to suppression or, in some situations, eradication (Dyck et al., 2005). The cost-effectiveness and applicability of the SIT against these fruit fly pests have been increasing due to the development of new, improved and refined methodologies to: (a) mass-produce insects on an industrial scale (Parker, 2005), (b) standardize sterile fruit fly quality control (Hendrichs et al., 2002; Calkins and Parker, 2005; Cáceres et al., 2007), (c) release only sterile males as a result of genetic sexing of strains (Rendón et al., 2000a,b; Franz, 2005), (d) pack, ship, emerge and release sterile males after factory production (Enkerlin, 2007), and (e) apply to sterile males hormonal, semiochemical and nutritional performance-enhancing treatments before their release (Pereira et al., 2013). This has directly benefited ongoing fruit fly suppression and eradication programmes that integrate the SIT, which in turn has reduced insecticide use, improved fruit quality and facilitated international trade, stimulating investment and creating much employment. As the release of sterile insects is totally friendly to the environment, it has also added to the quality of life of rural communities and improved the agricultural environment. In addition, regions under high risk of introduction of exotic fruit fly pests have been using sterile male releases as a preventative measure to maintain their fruit fly-free status (Dowell et al., 2000; Hendrichs et al., 2005) as an alternative to a more reactive approach to deal with costly outbreaks ( C. Cáceres et al. Rationale for coordinated research project (CRP) The experience gained and the success achieved implementing AW-IPM fruit fly programmes that apply the SIT have demonstrated that it is feasible to colonize and mass-produce sterile insects and manage their quality to address local, regional or international pest fruit fly concerns (Enkerlin, 2005). The transboundary transport and distribution of sterile insects for these fruit fly pests are a reality today and have been made possible due to the above-mentioned improvements. However, there are many other fruit fly pests of economic importance against which the SIT would also be a valuable control tactic as part of an AW-IPM approach. Many of these pest insects are increasing their geographical and/or host crop range (e.g. Bactrocera carambolae Drew and Hancock, B. cucurbitae Coquillett, B. invadens Drew, Tsuruta & White, B. oleae Rossi and B. zonata Saunders). The lack of suitable technologies to mass-produce insects of these and other species to satisfy the demand for such AW-IPM programmes is a major bottleneck. An FAO/IAEA Consultants Meeting on Development and Improvement of Rearing Techniques for Anastrepha and Bactrocera Fruit Flies was held in Vienna, Austria, from 23 to 27 October 2002, and a list of major fruit fly pest species was developed in relation to the status of mass-rearing technologies (Table 1). Based on the degree of development and level of knowledge on the mass-rearing of these different fruit fly species, the list was divided into three categories: (i) species for which mass-rearing for SIT application was available, but refinement would be useful for some of these; (ii) species for which mass-rearing for SIT application was under development; this category included species that had been reared in some laboratories but not been brought to mass-rearing status; and (iii) species for which research to develop colonization and rearing methods was needed; this included species of regional or global economic importance where little or no technology for rearing on artificial diets was available. These species would require initial R&D to develop the basic rearing techniques necessary for eventual implementation of the SIT. Certain species of the genus Rhagoletis were also identified as potential candidates for the development of rearing methods to apply the SIT. However, the presence of a pupal diapause in these species and the lack of understanding of their physiology to manage them for continuous rearing present a unique challenge, and it was recommended that a future CRP should address specifically diapause and dormancy management in Rhagoletis and other pest species. Based on the recommendations of this Consultants Meeting, an FAO/IAEA CRP, implemented

3 Rearing techniques for tephritid fruit fly pests S3 Table 1. Present rearing status of selected fruit fly species of economic importance Anastrepha species Bactrocera species Ceratitis species Mass-rearing for SIT application available A. ludens B. cucurbitae C. capitata A. suspensa B. dorsalis complex 1 A. obliqua 1 B. tryoni Mass-rearing for SIT application under development A. fraterculus 1 B. invadens C. rosa 1 A. serpentina B. latifrons C. fasciventris B. oleae 1 B. zonata Colonization and R&D needed 2 A. striata 1 B. pyrifoliae C. cosyra 1 A. zenilde Pacific region Bactrocera (non-methyl eugenol-responding spp.) SIT, sterile insect technique. 1 Species for which development or further improvement considered most urgent due to their potential threat and/or current level of economic losses. 2 Includes also Dacus ciliatus. under the IAEA Research Contract Programme ( was coordinated between 2005 and 2009 with the participation of 22 scientists from 18 countries. This special issue of the International Journal of Tropical Insect Science summarizes the results of the 5-year CRP on Development of Mass Rearing for New World (Anastrepha) and Asian (Bactrocera) Fruit Fly Pests in Support of Sterile Insect Technique. Table 2 lists the various species that were studied under the CRP, their economic and regional importance, and the countries involved. Table 3 lists the specific objectives of the CRP and the related outcomes, as reflected in the papers published during the CRP and in this special issue. This introductory paper highlights the key findings of the CRP related to the development or improvement of fruit fly mass-rearing methods and to nutrition and diets, including the role of symbionts and the effects of irradiation doses and handling procedures. It also aims to identify some gaps and areas for future research. The papers included in this special issue do not cover rearing protocols for all fruit flies of economic importance that could be addressed, but give an idea of rearing systems that could be adapted to rearing other species for which there might be an interest in applying the SIT. Specific outcomes Group I: species for which mass-rearing for SIT application was available, but further refinement would be useful Some participants of the CRP addressed specific issues that resulted in improved mass-rearing protocols for selected fruit fly species already under mass-production for SIT purposes and thereby increasing the quality or cost-effectiveness of the released insects. Hernández et al. (2014a) explored the possibility of using a generic artificial larval diet for rearing closely related Anastrepha species (A. ludens, A. obliqua and A. serpentina (Wiedemann)) at the Moscafrut mass-rearing facility located in Chiapas, Mexico, that provides sterile flies for the National Fruit Fly Campaign in Mexico. The application of the SIT for the control of A. ludens is a routine activity in northern Mexico and southern Texas, USA. Simple oligidic larval diets based on local cheap ingredients have been used to rear more than 400 million A. ludens sterile pupae per week in Mexico, Guatemala and the USA (Reyes et al., 2000). More recently, the Moscafrut facility has also been producing 100 million A. obliqua to be released to suppress populations or protect the areas that have already been freed of this pest in northern Mexico (Artiaga-López et al., 2004). The findings presented by Hernández et al. (2014a) indicate that with minor adjustments the A. ludens larval diet is a formulation that can also be used to rear A. obliqua and A. serpentina larvae. The availability of such a generic larval diet that responds to the nutritional needs of these three species maintained in multi-species mass-rearing facilities will optimize the cost-efficiency of their production without reducing their quality. Orozco et al. (2014) revised current rearing protocols for A. obliqua that is being produced at the Moscafrut facility in Chiapas, Mexico. An important finding of this research was the introduction and validation of the concept of relaxed rearing conditions for the adult colony to reduce the

4 S4 C. Cáceres et al. Table 2. Tephritid fruit fly species of economic importance in different regions of the world for which the sterile insect technique is or could be a key component for control and that were targeted during the coordinated research project (CRP) for the development and improvement of mass-rearing or related techniques Species Economic importance þ Regions of economic importance Origin of CRP participants and collaborators Anastrepha fraterculus High Tropical and subtropical Americas Argentina and Brazil Anastrepha ludens High North and Central America Mexico and Costa Rica Anastrepha obliqua High Americas Mexico Anastrepha serpentina Low Tropical and subtropical Americas Mexico Anastrepha striata Medium Tropical and subtropical Americas Mexico Anastrepha zenildae Medium Tropical and subtropical Americas Brazil Bactrocera correcta High South and South East Asia Thailand Bactrocera cucurbitae High South and South East Asia, Sub-Saharan Africa and Pacific Ocean Bangladesh, Mauritius and Sri Lanka Bactrocera invadens High South Asia and Sub-Saharan Africa Kenya Bactrocera kirki Low Pacific Ocean Samoa Bactrocera oleae High Mediterranean, USA, Mexico Greece, Italy and USA and South Africa Bactrocera philippinensis þ High South East Asia Philippines Bactrocera pyrifoliae High South Asia Vietnam Bactrocera tryoni High Australia and Pacific Ocean Australia Bactrocera xanthodes Low Pacific Ocean Samoa Bactrocera zonata High South Asia, Middle East, North Africa Mauritius and Pakistan and Indian Ocean Ceratitis fasciventris Medium Sub-Saharan Africa Kenya Dacus ciliatus High Sub-Saharan Africa, Middle East and Indian Ocean Israel þ B. philippinensis is now considered a morphotype of B. dorsalis. inadvertent selection for colony adaptation and therefore loss of competitiveness (Liedo et al., 2007). Trials using not only different adult fly densities in colony cages but also different larval densities in the diet revealed that a reduction from 100,000 to 60,000 adults per cage and a reduction from six to three larvae/g of diet for colony maintenance resulted after some generations in increased egg-to-pupa conversion and an increase in pupal weight. Relaxed rearing conditions not only can increase rearing efficiency and the quality of immature stages and adults, but can also improve male sexual performance as already demonstrated in C. capitata (Liedo et al., 2007). This will result in increased efficiency of operational programmes with an SIT component against Anastrepha species. Rull et al. (2014) evaluated the effect of different irradiation doses on the overall competitiveness of A. ludens. The main finding of this study was that in conventional field cage experiments, A. ludens males irradiated at a lower dose tended to induce more sterility in wild females than those irradiated at a higher dose when competing with wild males. When in competition with wild males for wild females, mass-reared flies (male and female) irradiated at 40 Gy and wild non-irradiated flies mated randomly, while flies irradiated at the high standard dose of 80 Gy mated assortatively. This finding can help insect pest control managers to revise current irradiation protocols used in fruit fly programmes with an SIT component and to create awareness that for other species also optimal doses to induce sterility are not those that cause the highest sperm sterility (Robinson and Hendrichs, 2005; Rull and Barreda-Landa, 2007) and furthermore that the optimal dose might be different when selecting a suppression, eradication or preventive control strategy. Gilchrist and Meats (2014) addressed alternatives for improving insect quality that is reduced as a result of the colonization process. Insect colonization can induce homozygosis due to genetic drift that results in genetic diversity loss and poor colony insect competitiveness with their wild counterparts in the field (Shelly, 2001; Zygouridis et al., 2013). Gilchrist and Meats (2014) demonstrated that crossing two inbred lines of B. tryoni already adapted for several generations to massrearing conditions induced heterozygosis and increased offspring quality. In comparison, strains only refreshed with wild material collected directly from the field had diminished values for some of the traits assessed. Mating competitiveness with wild insects was not compared and should be a

5 Table 3. Specific objectives and outputs of the coordinated research project (CRP) Objectives of the CRP To develop/improve mass-rearing diets used in the rearing of selected Anastrepha and Bactrocera fruit fly pests To assess the impact of biotic and abiotic factors on the production and quality of mass-reared fruit flies To increase the efficiency and cost-effectiveness of mass-rearing of fruit flies for SIT application To develop improved handling, process control, sterilization and automation of mass-reared fruit flies SIT, sterile insect technique. Specific outputs of the CRP Artificial larval and adult diets formulated and developed, modified, tested and validated for several species of fruit fly pests Advances made in the development and implementation of the liquid diet technology to suit locally available ingredients and materials Proper ratio of protein to sugar for the adult diets and quantity of adult diet consumption determined for some species Probiotic bacterial formulations evaluated for some fruit fly pests Progress made in identifying symbionts and associated bacterial communities and the environmental factors affecting the composition of the communities and their importance in rearing and adult release Strategies to avoid or minimize the use of antibiotics and preservatives assessed and recommendations made Suitable temperature, relative humidity and other environmental physical conditions determined for the establishment of new laboratory cultures of fruit fly pests Physical and chemical systems to stimulate egg-laying identified and assessed Recommendations to improve egging devices, cage design and pupal substrates made Recommendations to avoid inbreeding and its negative effect on male mating performance made Optimization and simplification of diet ingredients using cheaper, locally available ingredients achieved and implemented for different species of fruit fly pests Sterilization doses for several species revised and new recommendation to lower irradiation doses made Introduction and validation of relaxed rearing system for adult colony management Reduction of space and labour requirements resulted from the adoption of simplified rearing systems Papers published during the CRP and this special issue Braga (2014), Chang (2009a,b), Ekesi et al. (2007) Chang (2009a), Chang et al. (2004, 2006, 2008, 2011), Chang and Vargas (2007), Chang and McInnis (2008), Ekesi et al. (2014), Hernández et al. (2014a,b), Khanh et al. (2014), Resilva et al. (2014), Sookar et al. (2014), Vera et al. (2014) Nemny-Lavy and Nestel (2014), Zur et al. (2009) Meats et al. (2009) Estes et al. (2014), Rempoulakis et al. (2014), Sacchetti et al. (2008) Rempoulakis et al. (2014) Hernández et al. (2014a,b), Orozco et al. (2014) Sookar et al. (2014) Braga (2014), Resilva and Obra (2014) Gilchrist and Meats (2014), Rull et al. (2007, 2014) Chang (2009a,b), Chang et al. (2004, 2006, 2007, 2008, 2011), Chang and Vargas (2007), Chang and McInnis (2008), Chang et al. (2011), Ekesi et al. (2007, 2014), Hernández et al. (2014a,b), Khanh et al. (2014), Nemny-Lavy and Nestel (2014), Resilva et al. (2014), Sookar et al. (2014), Vera et al. (2014) Rull et al. 2007, 2014), Rull and Barreda-Landa (2007), Resilva and Pereira (2014) Orozco et al. (2014) Chang et al. (2004, 2006, 2008) Rearing techniques for tephritid fruit fly pests S5

6 S6 subject of future studies. This alternative, although requiring that two inbred strains be reared in parallel in a mass-rearing facility, can be particularly useful for those fruit fly species for which genetic sexing strains have already been developed in view that the availability of sex separation mechanisms can facilitate the directed mating of males of strain A and females of strain B and vice versa to produce F 1 males for sterilization and release as previously suggested by Robinson and Hendrichs (2005). Strategic options of colony management such as strain replacement, strain refreshment, hybrid vigour and relaxed mother colony holding still require more investigation, but are all potential alternatives to improve insect quality to counter deterioration of their biological attributes induced by mass-rearing for many generations (Robinson and Hendrichs, 2005; Liedo et al., 2007; Rull and Barreda-Landa, 2007; Rull et al., 2007). Resilva and Obra (2014) reported on improved rearing protocols for B. philippinensis Drew and Hancock, a member of the B. dorsalis complex, that consisted of new adult oviposition cages that had perforated egging tubes and the replacement of sweet potato as a bulking agent in the larval diet with sugarcane bagasse. In a separate paper, Resilva et al. (2014) assessed the potential of rearing B. philippinensis on a liquid larval, and although initial results look promising, more work is needed, especially to increase larval yield. Resilva and Pereira (2014) used the degree-day model of Ruhm and Calkins (1981) to derive the correct physiological age for irradiation of pupae held at different temperatures based on the correlation with pupal eye colour for bisexual and genetic sexing strains of C. capitata (Resilva et al., 2007). Using this correlation, they also developed models timing irradiation for 11 other tephritid species: Anastrepha fraterculus (Wiedemann), A. ludens, A. obliqua, A. serpentina, B. cucurbitae, B. dorsalis (Hendel), B. invadens, B. oleae, B. philippinensis, B. tryoni and B. zonata. Synchronization and timely exposure of mature pupae to the irradiation treatment at an optimal age are crucial to obtain a desirable level of sterility with minimal negative somatic effects on sterile male quality. Group II: species for which mass-rearing for SIT application was under development in the laboratory, but that had not reached mass-rearing status Braga (2014) and Vera et al. (2014) demonstrated that A. fraterculus larvae can be reared on a diet that has agar replaced (Salles, 1995; Jaldo et al., 2001) either with organic bulking agents such as sugarcane bagasse or with synthetic sponges. Both types of diets led to reasonable numbers of larvae C. Cáceres et al. that yielded adults in numbers and quality above the minimum requirements for this parameter as stipulated in the FAO/IAEA/USDA quality control manual for this species (FAO/IAEA/USDA, 2003). However, more research is required to assess what is logistically and economically feasible for the utilization of these diets in mass-rearing. For SIT effectiveness, it is essential that artificial rearing does not reduce male mating competitiveness. Gómez-Cendra et al. (2014) compared several morphometric traits to detect differences between a wild population and a laboratory-adapted strain of A. fraterculus that originated from the same wild population. Eight morphometric traits were analysed as indicators of body size, head shape and mobility. Findings indicated important morphometric differentiation between wild and laboratoryadapted strains of A. fraterculus that could be due to environmental and genetic factors or a consequence of genetic drift during colony establishment and maintenance. Short-distance interactions during courtship among individuals are more frequent in colony insects maintained at high colony densities, which might have favoured increased facial trait sizes as a result of sexual selection. Future studies are recommended to further assess whether these traits can be associated with mating success of sterile insects. With respect to species members of the genus Bactrocera, good progress was made with the rearing of B. invadens and B. zonata. Ekesi et al. (2014) reported successful rearing of B. invadens on a liquid larval diet with values for certain larval quality control and production parameters often exceeding those obtained for larvae reared on a carrot-based solid diet (Chang et al., 2004, 2006, 2008, 2011; Chang and Vargas, 2007; Ekesi et al., 2007; Chang and McInnis, 2008; Chang, 2009a,b). The use of a liquid larval diet could be a potential alternative to rearing B. invadens for possible future programmes with an SIT component. Sookar et al. (2014) and Shah et al. (2014) reported on the development of mass-rearing protocols for B. zonata that were slightly modified from those used for rearing B. dorsalis (Vargas and Chang, 1991). These included the use of perforated plastic egging devices and the use of water to stimulate egg-laying inside the oviposition receptacles instead of using commercially available papaya, mango, peach and guava juices. The use of solid larval diets that were based on sugarcane bagasse and ground maize as bulking agents resulted in the expected number of good-quality larvae. In addition, Sookar et al. (2014) reported that there is potential for using the liquid larval diet technology for B. zonata, but further improvements need to be made. The use of the SIT as a control tactic against the olive fruit fly has in the past been hampered due to

7 difficultiesinmass-rearinghigh-qualityinsectson an artificial diet (Estes et al., 2012). Rempoulakis et al. (2014) present attempts to overcome some of these problems. Their research confirmed that the practice of adding antibiotics to the diet is not required and even counterproductive in view of the presence of essential symbionts in the gut of olive fly larvae (Dimou et al., 2010). It was clearly demonstrated that the gut microflora of laboratory flies was different from that of wild flies and that the microflora of wild flies varied with the different varieties of olives used as host in the field. The use of a liquid larval diet could be a potential alternative to avoid the use of cellulose as a bulking agent in the larval diet. Estes et al. (2014) reported that the olive fruit fly symbiont Candidatus Erwinia dacicola was present in both wild and laboratory flies, but the symbiont was found in significantly fewer individuals in laboratory populations than in wild populations. The presence of C. E. dacicola did not influence the mating behaviour of the olive fruit fly in field cage studies. Future experiments were recommended to assess additional fitness parameters to better understand the benefits of the presence of C. E. dacicola in relation to insect quality. Earlier research has indicated some mating time asynchrony between laboratory and wild olive flies (Economopoulos, 1972) and, therefore, the release of only sterile males could be the solution to this problem. However, until now, there has been no genetic sexing strain developed for the olive fruit fly. Transgenic approaches could be an alternative to generate such strains needed for improving the SIT for the olive fruit fly. Rempoulakis et al. (2014) assessed the quality parameters of the first green fluorescence protein transgenic strain developed for the olive fruit fly, but all standard quality control tests revealed inferior results for the transgenic strain when compared with a standard mass-reared strain. Ekesi et al. (2014) addressed the improvement of the larval diet for Ceratitis fasciventris (Bezzi), a fruit fly species that is reared in the laboratory on a solid diet that includes carrot powder as a bulking agent. The research focused on an assessment of the possibility of rearing C. fasciventris on a liquid larval diet. The results revealed that C. fasciventris larvae were able to develop on the liquid diet, but in contrast to those of C. capitata and other species of fruit flies (Chang et al., 2004, 2006, 2007, 2008, 2011; Chang and Vargas, 2007; Ekesi et al., 2007; Chang and McInnis, 2008; Chang, 2009a,b), the values of several parameters assessed indicated that the performance of C. fasciventris flies maintained on a liquid larval diet was significantly lower over five generations than that of flies maintained on a solid diet. This is evidence that success in adapting fruit flies to an artificial diet is dependent on the fly species and diet type (Souza Rearing techniques for tephritid fruit fly pests S7 et al., 1988). This should be taken into account when improvements for mass-rearing are intended or when new species are to be adapted to massrearing conditions. Group III: species for which research to develop colonization and rearing methods was needed To assess the rearing potential for Anastrepha striata (Schiner), Hernández et al. (2014b) determined demographic, rearing and quality parameters for this difficult-to-rear species. Acceptable values for rearing and quality control parameters of laboratoryadapted flies were obtained when compared with those reported for the F 1 generation of wild insects collected in the field from infested guavas. However, some rearing aspects such as larval survival and egg production still need to be improved to make massrearing of this species economically viable. Anastrepha zenildae Zucchi coexists sympatrically with A. fraterculus and is an endemic pest of fruits in tropical areas of northern Brazil. Braga (2014) reported good progress developing and establishing rearing protocols for this species that in principle are similar to the protocols developed for A. fraterculus (Braga, 2014; Vera et al., 2014). The best oviposition device was an agar panel over a glass bottle. Post-zygotic compatibility of several populations of A. fraterculus and A. zenildae was assessed, but no viable eggs were produced, which confirmed that the populations tested belonged to separate species. Nemny-Lavy and Nestel (2014) assessed the protein requirements of the adult Ethiopian fruit fly Dacus ciliatus (Loew). Yeast hydrolysate at a concentration of 16 50% relative to sugar resulted in the best reproduction rate in terms of pupa production. However, the development of a functional artificial larval diet for D. ciliatus remains the major constraint. Khanh et al. (2014) assessed the basic biology and artificial rearing techniques for Bactrocera pyrifoliae (Drew and Hancock), a pest of peaches and plums in higher-elevation areas of northern Vietnam and Thailand. This species is not attracted to traps baited with methyl eugenol or cuelure, but responds to protein baits in McPhail traps (Khanh et al., 2007; Khanh and Hien, 2008). As insecticide bait sprays are solely used to suppress this pest, the SIT could be an environmentally friendly alternative for its control. An important result of this research was the successful adaptation of this species to the rearing on a carrot and corn powder-based larval diet. Additional research will be necessary to develop full rearing and quality control protocols for this species based on experience and methods developed for other species of Bactrocera (Vargas and Mitchell, 1987; Vargas et al., 1990; Vargas and Chang, 1991).

8 S8 Conclusions Developing and improving technologies for mass-rearing fruit flies are fundamental to ensure the success and cost-effectiveness of AW-IPM programmes that include an SITcomponent against these tephritid pests. This CRP motivated and supported the development and improvement of larval and adult diets, rearing and sterilization procedures that are required for the mass-production of selected Anastrepha, Bactrocera, Ceratitis and Dacus fruit fly pests. While a number of studies have resulted in significant improvements in the rearing of some species that can or have already been transferred to operational programmes, others will require more research before they can be implemented in fruit fly mass-rearing facilities (see conclusions on identified gaps below). In addition, this CRP has succeeded in bringing together an international group of researchers with similar focus and objectives, who through networking shared specialized technical know-how and researched mass-rearing protocols for SIT implementation against an expanded group of fruit fly species of major economic importance. The major outcomes of this research network can be summarized as follows (Table 3):. Artificial larval and adult diets were developed or improved for several species of fruit fly pests using locally available low-cost ingredients.. The use of a liquid larval diet as a novel technology was tested and adopted for some species using locally available ingredients and materials.. Progress was made in assessing the importance of various symbionts and associated bacteria and in identifying some factors that affect their composition and potential to improve the quality of the released insects.. For some species, new or improved rearing equipment was designed for use in mass-rearing facilities.. It was demonstrated that in certain species, the irradiation doses can be reduced to increase overall sterile insect quality while increasing induction of sterility in the field.. Strategies were identified to improve colony management and to reduce the effect of inbreeding and, hence, increase insects vigour and male mating performance. Although good progress was made during the CRP to develop or improve mass-rearing technologies for selected fruit fly pests, there are still various gaps that became apparent during the implementation of the CRP. These gaps need to be addressed in the future to enable stable insect production and improved overall sterile insect quality and SIT efficiency: C. Cáceres et al.. In some cases, it is still not possible to maintain a steady and reliable mass-production of insects for SIT application. In species such as A. fraterculus, A. striata, B. oleae and B. pyrifoliae, additional research is required to better understand the abiotic and biotic factors that are causing periodic low productivity that can even lead to the loss of colonies.. Past efforts to improve sterile fruit fly quality have focused on aspects of colonization, massrearing, quality control and pre-release handling. More evidence that associated micro-organisms could improve male sexual performance is being found (Ben-Yosef et al., 2008). Therefore, it is necessary to better understand the dynamics and the role of micro-organisms and symbionts to be able to exploit these to develop more efficient rearing systems and be able to release sterile males of higher biological quality.. Numerous data demonstrating that male sexual performance can decrease progressively with the number of generations under mass-rearing are available (Shelly, 2001), while increasing evidence indicates that relaxed rearing conditions for the adult colony can effectively counter the inadvertent selection for colony adaptation and therefore male competitiveness loss (Orozco et al., 2014). However, there is still a need to compile all available information to develop protocols for effective colony establishment, maintenance, replacement or refreshment that would sustain the sexual competitiveness of the sterile males.. Despite the good progress made with the development of efficient liquid diets, most fruit fly mass-rearing facilities still use bulking agents that are subproducts of the agroindustry, which are difficult to acquire with stable quality in terms of physical and chemical properties and micro-organism load. Additional research is still needed to replace these bulking agents with either liquid or gelid formulations that could allow standardization of the required properties of the larval diet.. Currently, yeast is being utilized as a source of essential nutrients and protein for all larval and adult diet formulations to rear fruit flies. However, yeast is the most expensive component and therefore it remains very desirable to reduce or fully replace it with micro-organisms or other alternatives to decrease the cost of diets and consequently to further diminish the cost of production of sterile fruit flies and SIT application. To support continued rapid progress in some of the identified R&D areas, two new 5-year FAO/

9 IAEA CRPs have been initiated, involving scientists from the world s main fruit fly research centres and major fruit fly SIT programmes. In 2010, a CRP was initiated on Resolution of Cryptic Species Complexes of Tephritid Pests to Overcome Constraints to SIT Application and International Trade, and in 2011, another CRP was started on Use of Symbiotic Bacteria to Reduce Mass-Rearing Costs and Increase Mating Success in Selected Fruit Pests in Support of SIT Application. Acknowledgements The authors thank all their colleagues who enthusiastically took part in this CRP, the group of consultants who participated in formulating the CRP (Andrew Jessup, Aristides Economopoulos, Eric Jang, Pedro Rendón and John Worley), and all their colleagues who contributed to reviewing the papers presented in this special issue. References Allwood A. 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