Proceedings of 6th International Fruit Symposium 6–10 May 2002, Stellenbosch, South pp. 367–381

Comparison of Mediterranean fruit fly ( capitata) () bisexual and genetic sexing strains: development, evaluation and economics

C. Caceres1, J.P. Cayol2, W. Enkerlin2*, G. Franz1, J. Hendrichs2 & A.S. Robinson1 1Entomology Unit, Agriculture and Biotechnology Laboratory, Agency’s Laboratories, A-2444 Seibersdorf, Austria 2Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, IAEA, Wagramerstrasse 5, A-1400, Vienna, Austria

In Medfly, , sterile technique (SIT) programmes, the use of genetic sexing strains (GSS) is now routine. The use of these strains in mass-rearing facilities enables them to produce only males for irradiation and release.The advantages of using these strains are described together with genetic approaches used for their construction. The early use of the strains in mass- rearingfacilitieshighlightedimportantlimitationsthatwerenotapparentduringtheirconstruction and small-scale evaluation. Using a combination of new genetic and rearing approaches the problems have essentially been solved. The potential use of one GSS in many different geographic locations raised concerns about mating compatibility. A very detailed field-cage study using populations from very diverse areas showed that these strains had no unexpected impact on mating compatibility. The economics of the use of GSS in SIT programmes is described in detail, covering both their mass production and use in the field. The analysis reveals a much improved cost/benefit ratio when GSS are used. Future improvements that will further enhance the use of GSS are also discussed.

BACKGROUND Laboratories at Seibersdorf in Austria, and the The (SIT) is an estab- funding of three Coordinated Research Projects lished technology for the suppression and/or (CRPs) (IAEA 1990, 1997; Genetica 2002). With eradication of selected key insect pests of man, his the completion of the final CRP, almost all of livestock and crops (Tan 2000). The technology the Medfly rearing facilities worldwide are using involves the mass production and release, on an these genetic sexing strains (GSS) for their SIT area-wide basis,of large numbers of sexually sterile programmes (Robinson et al. 1999; Table 1). into a field population. The released males Prior to the start of the first CRP very little was mate with the females in the field resulting in no known about the genetics of Medfly, and the task production of offspring. Following repeated of this CRP was to develop many of the essential releases of the sterilized insects, the field popula- genetic tools, e.g. mutations, polytene chromo- tion is suppressed and in certain circumstances some analysis and male-linked translocations, eradication can be achieved (Hendrichs 2000). which are essential for the construction of a GSS. To be effective,SIT does not require the release of Table 1. Medfly mass-rearing facilities using GSS with sterile females as they do not contribute to the the potential capacity in millions of males/week. transfer of sterility to the wild population. Thus, mass production of sterile females is unnecessary Country Strain Capacity and the technique requires that only sterile male insects are released (Knipling 1955; Robinson et al. Argentina SEIB 6 (VIENNA 7/Tol) 80 1999; McInnis et al. 1994; Franz & McInnis 1995; VIENNA 7/Mix 7 Hendrichs et al. 1995). In some cases the release SEIB 6 (VIENNA 7) 40 of females can have a negative impact. Greece (Crete) SEIB 7/Mix 3 As a result of this situation, the Joint FAO/IAEA VIENNA 7/Tol 1600 Division of Nuclear Techniques in Food and (CDFA) VIENNA 7/Tol 100 Agriculture initiated activities in 1981 to develop Peru VIENNA 7/Mix 100 special strains for the Medfly, Ceratitis capitata, Portugal (Madeira) VIENNA 7/Mix 50 that could be used to produce only males for area- Seibersdorf VIENNA 7-D53/Mix 25 wide SIT programmes. These activities included South Africa VIENNA 7-D53/Mix 7 Hawaii VIENNA 7/Tol 200 research and development at the FAO/IAEA (USDA/APHIS) *To whom correspondence should be addressed. Total 2212 E-mail: [email protected] 368 Proceedings of the 6th International Fruit Fly Symposium

At the conclusion of the second CRP, the first sterile females) and thus released at a more transfer of a GSS to an operational SIT programme mature age, thereby reducing losses before in Guatemala took place. males reach full sexual capacity; Studies carried out during the second CRP e. simplified and more precise monitoring demonstrated the efficacy of an all-male release activities when using female attractants, in the field (McInnis et al. 1994) and included infor- as the recapture of sterile males is largely mation on a temperature-sensitive mutation, tsl, reduced, thus also significantly reducing the in Medfly (Franz et al. 1996). The use of this risk of mis-identification (plus the value of mutation has been central to the practical applica- not removing many valuable sterile males); tion of GSS in Medfly. Some of the most serious f. no damage in certain types of fruit due to problems to be confronted in the use of GSS in absence of oviposition stings by sterile females, operational programmes concerned the stability and reduced transfer of pathogenic fungi and of the strains under mass-rearing conditions. bacteria to such fruit; These have now been essentially solved by a g. increased applicability of SIT for Medfly combination of new rearing methods and the suppression in fruit growing regions, because use of more appropriate translocations. the reduced cost and absence of oviposition Initially it was assumed that the sex determina- damage to the fruit enables the routine use tion mechanism of Medfly would be very similar to of sterile males as a biological ‘insecticide’ to that of melanogaster. However, it is replace chemical bait-sprays during fruiting now known that in most tephritid fruit there is seasons; probably a single gene that initiates male sex h. increased bio-safety,as an accidental release of determination (Zapater & Robinson 1986; Will- non-irradiated flies would only include males, hoeft & Franz 1996) in contrast to D. melanogaster, and escaping females from mass-rearing where a chromosomal balance system operates. would have reduced fitness. This is particu- The use of polytene chromosome analysis larly important for mass-rearing facilities (Kerremans et al. 1990, 1992; Zacharopoulou located in fruit fly-free areas or areas where et al. 1991) has been essential for the develop- Medfly eradication is the objective. ment of stable GSS, by enabling both mutations In conclusion, the most important benefit is the and translocation breakpoints to be accurately increased efficiency of SIT, as it has been shown mapped (Franz & Kerremans 1993; Franz et al. in many Medfly studies that male-only releases 1994; Kerremans & Franz 1994, 1995). introduce 3–4 times more sterility into the target population than do bi-sexual releases (McInnis DEVELOPMENT,IMPROVEMENTS AND et al. 1986; Robinson et al. 1986; McInnis et al. REARING OF MEDFLY GENETIC SEXING 1994; Rendon et al. 2000). STRAINS FOR USE IN SIT Choice of sexing system Benefits of genetic sexing strains (GSS) The practicality and economics of GSS use The benefits of using GSS for Medfly SIT have depends on the choice of the appropriate been articulated many times and were summa- selectable marker in the sexing system. Initially rized by Hendrichs et al. (1995) as follows: the white pupae (wp) mutation (Rössler 1979) was a. economic savings in rearing, irradiation, used in combination with seed sorters to separate packaging, transport and release; white (female) and brown (male) pupae. However, b. increased male quality as male pupae can be it became clear that this type of selectable marker irradiated 24 h before emergence, instead of has two significant disadvantages. First of all, the 48 h when also irradiating females; sex separation can be achieved only after the costly c. several-fold increase in field effectiveness as mass-rearing step, i.e. expensive diet has to be the sterile sperm is not wasted in matings with wasted on rearing females and, in addition, the sterile females, and sterile males compete females had to be killed and disposed of in a safe better for wild females. Some studies also way. Second, the use of seed sorters is not easy. showed that sterile males disperse more in Such machines are very expensive, especially the absence of sterile females; considering the separation capacity needed in d. in the absence of sterile females in the fly most mass-rearing facilities; they are relatively emergence containers, sterile males can be complicated and are not very accurate (c. 5% held longer (without mating taking place with contamination with females in the ‘male-only’ Caceres et al.: Medfly bisexual and genetic sexing strains: development, evaluation and economics 369 product and a significant loss of male pupae). translocation breakpoint on that chromosome Furthermore, the sorting process can damage made it possible to significantly reduce the the pupae, resulting in flies with reduced flight impact of a particular genetic destabilizing ability. event. Acknowledging these severe disadvantages led There is now a clear understanding of the ge- to the development of an alternative sexing netic factors that are involved in the stability of system. This is based on a temperature-sensitive genetic sexing strains in Medfly, and using this lethal (tsl) mutation that allows females to be killed information their stability has been improved to a as early as the egg stage by applying elevated level enabling them to be mass-reared in a predict- temperatures. This is, in mass-rearing, the earliest able and reliable way (Caceres et al. 2000). and most practical stage (as large numbers of eggs Inversions are now being introduced into GSS are maintained for two days in plastic bottles) and with the aim of further increasing stability and the killing of the females requires only very simple facilitating the introduction of different genetic and cheap equipment (a water bath). By now, backgrounds, if specifically required. In addition, extensive experience with the tsl-based GSS is a phenotypic marker is being evaluated which available and an accuracy of 99.5% or better can be may remove uncertainties regarding identification achieved routinely even in very large mass-rearing of released flies in the field. However, when tens operations (Fisher 1998, 2000). or hundreds of millions of insects are being reared weekly, random biological events can still occur The problem of stability and cause problems that can only be solved by The first mass-rearing of Medfly genetic sexing introduction of large changes in the rearing strains revealed that stability was going to be a methods. major problem that would need to be solved before these strains could be transferred to opera- Improved rearing strategies tional SIT programmes (Robinson & van Heemert Instability occurs during mass-rearing of GSS 1982; Robinson 1984; Hooper et al. 1986). Solving following rare genetic recombination events. the problem involved two strategies, 1) inducing Occasionally, some of the different phenotypes, new strains with inherently improved genetic produced as a result of these events, accumulate characteristics (Franz et al.1994) and 2) developing because of a higher fitness in the colony, as new rearing procedures (Fisher 1998, 2000; Fisher traditional mass-rearing strategy returns these & Caceres 2000). By combining these strategies individuals back into the colony. In order to pre- the problem of stability has essentially been vent this accumulation, the Filter Rearing System brought under control. (FRS) was developed (Fisher & Caceres 2000). During the first large-scale mass-rearing of In this system, (Fig. 1), a small colony of the GSS is these strains, novel biological events occurred kept under relaxed rearing conditions and is which also impacted on stability. These unex- checked at every generation for the presence of pected genetic problems had also to be solved. unexpected flies which, if found, are discarded. In addition to controlling stability there has been a Eggs from this colony are used to produce a continuous improvement in the quality control large colony following a small number of amplifi- profile of GSS. cation steps. Eggs from this large colony are then used to produce males for release. No The solutions insects that have been through the mass-rearing procedure are returned to the small initial colony. Improved strains Production is essentially a one-way street. The Strain improvement has been achieved by: FRS has been successful in a number of large-scale • very accurate cytological mapping of the markers facilities that mass-rear tsl-GSS and has demon- used in the strains i.e. white pupae and tsl, strated its effectiveness in maintaining strain making it possible to select translocations with stability for many generations. breakpoints very close to the markers. This had The use of the FRS has another important the immediate effect of improving stability function in relation to the overall quality of the significantly, reducing recombination by c. 65%; mass-reared insects, both in GSS but also in • very accurate cytological mapping of the position bi-sexual strains (BSS). It is possible to design an of the male determining gene on the Y chromo- FRS in order to try to maintain conditions that are some and by analysing the position of the closer to the natural environment of the flies. 370 Proceedings of the 6th International Fruit Fly Symposium

Fig. 1. Schematic representation for the operation of a filter rearing system (FRS) to maintain stability in a GSS.

Such a relaxed rearing environment for the small this sterility manifested itself at a relatively late initial colony may reduce strain deterioration stage of development, such that a significant pro- (e.g. less inbreeding as a result of a strong inadver- portion of the pupae produced were genetically tent selection of laboratory adapted traits that unbalanced, resulting in low adult emergence are not well fitted for field conditions), thereby and in reduced adult quality for this type of extending the viable life of a colonized strain genetically unbalanced pupae. This led to overall under mass-rearing conditions. reduced quality control values for emergence, flight ability and mating performance. Selecting Improved quality control profiles specific translocations for GSS has solved this The quality control profile of GSS can be nega- problem. The current GSS, VIENNA 7 and VIENNA tively influenced by the survival of genetically 8, were not only chosen because of their in- unbalanced individuals to different stages in the creased stability but also because their genetic developmental cycle. This problem is linked to structure is such that no, or only very few, geneti- the genetic structure of the GSS and is associ- cally unbalanced individuals survive beyond ated with the 50% sterility that has been character- egg/early larval stage. From that stage onwards istic of such strains. In earlier versions of GSS only the desired flies are reared resulting in qual- Caceres et al.: Medfly bisexual and genetic sexing strains: development, evaluation and economics 371 ity control values similar to standard BSS. These of the sexual behaviour, competitiveness and strains show a clearly better performance in mass- mating compatibility of Medfly GSS is reviewed rearing than older versions of GSS. below. First, however, we will review the informa- tion available on the sexual and other behaviours A problem that needs to be addressed of Medflies in the wild or in the laboratory, against GSS based on temperature sensitivity and which to assess GSS behaviour. translocations require a colony at least three times as large as a BSS colony to produce the same Baseline information on Medfly sexual number of eggs (see below). This is due to the behaviour fact that 1) the strains are semi-sterile due to the There is a solid base of information on mating translocation,and half the eggs are killed as a result competitiveness and sexual behaviour of wild and of the temperature treatment, and 2) female laboratory BSS against which the performance of Medflies of GSS are homozygous for the tsl GSS can be measured. Concerns about the sexual mutation and show a reduced viability. Inducing behaviour and competitiveness of mass-reared a particular type of translocation that will not be and laboratory Medfly strains were raised in the associated with semi-sterility could solve the 1970s. As a first step, Holbrook & Fujimoto (1970) first problem. Making some changes to the way assessed the mating competitiveness of irradiated adult colonies are maintained may help solve and non-irradiated Medflies. Rössler (1975) and the second. This could involve changes in cage Wong & Nakahara (1978) measured the ability of design and improved climatic/environmental laboratory-reared Medfly males to inseminate controls. The size of the colony is currently the females, in comparison with wild flies. Fried major disadvantage to the use of GSS involving the (1971), Boller & Chambers (1977), Boller et al. tsl mutation. Even so, as described below in the (1981), Chambers et al. (1983), Boller & Calkins sections on evaluation of competitiveness and (1984) and Orozco et al. (1983) published a collec- economics, the benefits of using these GSS in tion of quality control tests for fruit flies and a operational Medfly SIT programmes significantly quality control manual for Medfly mass-rearing outweigh the disadvantages during rearing. facilities. Prokopy & Hendrichs (1979) provided a description of the behaviour of wild EVALUATION OF SEXUAL BEHAVIOUR, Medflies and Zapien et al. (1983) carried out the COMPETITIVENESS AND COMPATIBILITY first Medfly quality control test on a field-caged OF MEDFLY GENETIC SEXING STRAINS host tree to assess wild female mate choice for In terms of the sexual behaviour and mating competing wild and sterile males. This was fol- compatibility of GSS there are two issues that have lowed by studies in the open field to confirm the been repeatedly raised in relation to the potential observed Medfly behaviours under natural condi- use of these strains in operational SIT programmes. tions (Hendrichs & Hendrichs 1990; Hendrichs The first concerns the genetic background of GSS. et al. 1991; Whittier et al. 1992, 1994, Shelly & This often differs from that of the target Medfly Whittier 1996). These observations were used to population, raising the question of the mating compare the behaviour of mass-reared insects compatibility among the different Medfly popula- with wild insects (Chambers et al. 1983; Orozco tions in the world. The second is the fact that et al. 1983; Zapien et al. 1983; Calkins et al. 1994). GSS carry mutations in the form of selectable In the 1980–90s, the various effects of mass-rear- genes and translocations, the impact of which, on ing (Calkins & Ashley 1989; Calkins 1991; Calkins behaviour, is unknown. et al. 1996), nutritional status (Blay & Yuval 1997; In view of these two considerations and the Bravo & Zucoloto 1998), and irradiation (Wong increasing demand for Medfly GSS for SIT suppres- et al. 1982, 1983) on the sexual competitiveness sion programmes, these strains had to undergo and mating behaviour (Liimatainen et al. 1997), thorough and systematic testing for sexual including female mating-induced changes (Jang compatibility and mating competitiveness with et al. 1998), were studied. Comparative assess- the target wild population before they could be ments of the mating competitiveness of various cleared for use in SIT programmes.In fact, very few BSS were also done, most notably in countries insect mass-reared strains, if any, have been as using this type of strain for large-scale SIT program- intensely and thoroughly tested as Medfly GSS, mes, such as Mexico (Liedo et al. 1996). All of especially tsl strains. these studies concluded that flies from standard The most relevant work done on the assessment BSS were less competitive in mating than their 372 Proceedings of the 6th International Fruit Fly Symposium wild counterparts, that some sequences of court- countries representing five continents, five differ- ship behaviour were shortened (Briceño & Eber- ent GSS (four tsl-based and one wp-based) and six hard 1998), and that these changes appeared to laboratory or mass-reared BSS under field-cage be mainly due to the mass-rearing conditions, the and/or laboratory conditions. The assessment of irradiation process and the age – or number of each strain’s mating compatibility and competi- generations – of the strain under mass-rearing tiveness (Cayol 2000a), together with the detailed conditions (Eberhard 2000). That the age of the slow-motion video analysis of their courtship strain has severe implications was also demon- behaviour (Lux et al. 2002; Cayol et al. 2002) con- strated in an extreme case in Hawaii,where the use cluded that no major differences could be found of one particular BSS (HiLab) in mass-rearing for among the strains, regardless of their genetic over 40 years without refreshment resulted background. The only exception was Medfly in ‘behavioural incompatibility’ between wild females from Madeira Island (Portugal), which and mass-reared flies (McInnis et al. 1996a). This were only partially compatible with males from represents the only case of ‘incompatibility’ docu- VIENNA 7-97 (Pereira unpubl. data; Cayol unpubl. mented in Medfly and the problem was solved data). It was concluded that BSS and GSS, regard- with a change of strain, emphasizing the need to less of their origin, could be used against different renew BSS often as is being done routinely in most Medfly populations, as well as for outbreaks of SIT programmes. unknown origin as in the cases of and In 1998, on the basis of all the above studies, an in the U.S.A. However, it was again con- international manual was developed to harmonize firmed that long-term mass-rearing and irradiation quality control procedures, including the field- procedures reduce the mating competitiveness cage mating test (FAO/IAEA/USDA 2003). In view and shorten the courtship duration of labora- of the female-choice mating system in Medfly, tory-reared flies (Briceño & Eberhard 1998; Cayol the field-cage mating test is probably the most 2000b; Eberhard 2000). important component of quality control assess- ment in this species. Carried out under semi-natu- Assessment of the behaviour of Medfly GSS ral conditions and in the presence of wild females, this field-cage mating test allows the detection of First generation GSS: white pupae (wp) strains deviations from the ‘standard’ value of mating Robinson et al. (1986) assessed the mating com- performance expected from any mass-reared petitiveness and sexual compatibility of a GSS strain. based on wp with wild flies from Procida Island, Italy, and Hooper et al. (1986) assessed the Worldwide assessment of mating ‘behaviour’ of this strain under mass-rearing con- compatibility among Medfly populations ditions. The effectiveness of this wp GSS was In the early days of Medfly SIT, strains were successfully assessed in a large open-field trial in colonized from the local wild population, in- Israel in 1989/90 (Nitzan et al. 1993), resulting in creased in numbers, sterilized, and released in the only 0.4% of fruits with live maggots in a 500 ha same general area. In the early 90s, the increased test area. A similar strain was also tested in transboundary shipment of sterile Medflies southern Tunisia (Cayol & Zaraï 1999) and resulted and the availability of the first Medfly GSS for in Medfly being successfully suppressed in some large-scale use in SIT programmes, resulted in oases. the same strain being used in different countries, An improved version of the original wp GSS,SEIB regions or even continents. As these strains are 6-96, was tested under field-cage conditions in based on genetic material that often differs in the Argentina (Cayol et al. 1999). This strain was used geographical origin from that of the target wild for several years in Mendoza and various provinces population, concern was raised regarding their of Patagonia, where it has been very successful in sexual compatibility and competitiveness at other controlling Medfly population (De Longo et al. locations. 2000). These studies, together with the compara- From 1994 to 1999, within the framework of tive assessment of the detailed courtship behav- an FAO/IAEA Coordinated Research Project, the iour of wild flies (Cayol 2000a; Lux et al. 2002), mating compatibility of Medflies from wild and concluded that there were no major differences laboratory- or mass-reared BSS and GSS from between the wild flies and the GSS flies. Conse- various origins was compared. This resulted in a quently, the use of wp GSS represented a major comparative assessment of wild flies from nine step forward, even though it still involved the Caceres et al.: Medfly bisexual and genetic sexing strains: development, evaluation and economics 373 production and mechanical separation of females was three to five times more effective at inducing at the pupal stage. egg sterility in the field than was the BSS. Kaspi & Yuval (2000) measured how adult Second generation GSS: temperature-sensitive protein-feeding increased the mating competitive- lethal (tsl) strains ness of tsl GSS males from Guatemala. McInnis The first tsl-GSS was made available in the early et al. (2002) compared the remating frequency of 1990s (Franz et al. 1994). This represented a major this tsl GSS with that of BSS under field-cage condi- breakthrough and an increase in cost-effective- tions in Guatemala. The authors did not find any ness of Medfly SIT since it allowed sex separation significant differences between BSS and tsl GSS. at the egg-stage. In view of the fact that this type Mass-reared males (either BSS or GSS) had a similar of strain carries two mutations, wp and tsl, in addi- reduction in mating compared to their wild coun- tion to the translocation, it was thoroughly tested terparts. with respect to sexual behaviour, competitive- After confirming mating compatibility, tsl GSS ness, and field effectiveness. The original VIENNA pupae are being shipped weekly from the El Pino 42, and its improved ‘successors’, have been facility in Guatemala to Israel and Jordan, and tested at all levels in the laboratory, in field cages have been proved successful in suppressing the and in the open field. Medfly population in the Arava/Araba Valley south Hendrichs et al. (1993) tested the dispersal and of the Dead Sea (Rössler et al. 2000). From the end survival of VIENNA 42 in citrus orchards in Greece of 1999 and following preliminary feasibility and and showed that these flies dispersed and survived mating compatibility studies (Barnes & Eyles 2000), as well as those from a BSS. The mating competi- pupae from the same tsl GSS have been shipped tiveness of this strain was then tested under field- from Guatemala to be released for routine Medfly cage conditions against wild flies from Greece suppression over the Hex River Valley of the (Hendrichs et al. 1996). Again, tsl males proved to Western Cape Province of South Africa. This has be as competitive as males of a BSS, although less resulted in the elimination of insecticide used for competitive than wild flies. The VIENNA 43/44 tsl Medfly suppression and a halving in the number was then tested in comparison with a wp GSS over of table grape boxes rejected for export due to nearly 300 ha in southern Tunisia for 11 months the presence of Medfly larvae (B.N. Barnes, unpubl. (Cayol et al. 1997; Cayol & Zaraï 1999). Both GSS data). were found to be equally competitive and their VIENNA 7-D53/Mix-2001, which was very recently use in Tunisia resulted in the successful suppres- made available by the FAO/IAEA Laboratories in sion of the resident Medfly population. Seibersdorf, represents one of the latest develop- The VIENNA 4/Tol-94 tsl was then developed ments of tsl Medfly strains. Compared to its based on a local genetic background from predecessor tsl strains, the genetic stability of Guatemala. The performance of this strain was VIENNA 7-D53/Mix-2001 has been improved by first monitored under mass-rearing conditions the addition of an inversion, and the strain is (Caceres et al. 2000) and then the behaviour in the based on genetic material of a mixed wild-type laboratory, in field cages in Guatemala (Rendon strain. VIENNA 7-D53/Mix-2001 has already been et al. 1996a), in greenhouses in Vienna (Cayol et al. tested successfully in field cages against wild flies 2002), and in the open field (Rendon et al. 1996a,b; in Israel (Gazit & Rössler 2001) where it has been McInnis et al. 1996b). The relative mating competi- as competitive as VIENNA 4/Tol-94, achieving tiveness of VIENNA 4/Tol-94, VIENNA 42 and two about one third of wild female mates when com- standard BSS (HiLab and Petapa) was assessed in peting with wild males. From November 2001, field-cage experiments in Hawaii and in Guatemala the weekly releases of eight million tsl GSS from (Lance et al. 2000). The authors reported that Guatemala is being supplemented with an addi- even though quantitative differences could be tional five million pupae of this new strain from found between wild and laboratory flies, no major the FAO/IAEA Laboratories in Austria. qualitative differences were found between BSS Because of their genetic composition and their and GSS. Most importantly, VIENNA 4/Tol-94 was wide use in large-scale SIT programmes in various also compared against the standard Petapa BSS, countries and continents, the Medfly GSS, and during three consecutive years in a large open especially the tsl strains, have been thoroughly field trial in Guatemala, involving routine opera- tested. To date, much more is known on behav- tional SIT activities over large areas (Rendon et al. iour, field competitiveness, genetics and morpho- 2000). This comparison showed that the tsl GSS metrics of GSS strains than what was known for 374 Proceedings of the 6th International Fruit Fly Symposium

BSS used in large SIT programmes for many years. smallest proportion of the overall production, the It can be concluded that there is strong evidence difference in cost between the two systems is no that different Medfly populations in the world have more than 15%. not evolved mating incompatibilities, with the possible exception of the population on Madeira Adult colony Island. Furthermore, there is no evidence that the Compared to the rearing of BSS, different proce- GSS available today behave any differently than dures are required for tsl-based GSS due to the BSS, and both types of strains are affected equally genetic composition of such strains (Caceres et al. by mass-rearing, irradiation and excessive long- 2000). For a tsl-based GSS a large adult colony is term rearing. required because 50% of all eggs produced are rendered unviable by the partial genetic sterility ECONOMIC COMPARISON OF THE USE OF of GSS males. Of the remaining eggs, 50% repre- MEDFLY BISEXUAL AND GENETIC SEXING sent the temperature-sensitive females and these STRAINS are killed by the temperature treatment. As a con- Medfly GSS, in particular the tsl-based GSS, have sequence,only 25% of all eggs produced will result been increasingly used in large operational SIT in males for release. An additional reduction in projects since the 1990s. Thus, sufficient informa- the overall egg production per cage is caused by tion on routine costs is available to make some the sensitivity of the tsl females to stresses such general comparisons of the economics of their use as high densities in the cages. These factors compared with that of standard BSS. together result in a three-fold increase in the Intuitively, the production of male-only tsl- space required for the adult colony rooms, a six- based GSS would appear to be more economical fold increase in number of cages and three-fold compared to the cost of producing males and increase in labour (Table 2). An increase in space females in the case of BSS. The fact, however, is brings an increase in the basic utilities required that producing only males of tsl-based GSS is such as water and power, as well as a cost in- currently slightly more expensive because overall, crease in maintenance and depreciation of the the operational and capital costs of male-only additional equipment and infrastructure. tsl-GSS rearing facilities are greater than that of the normal BSS rearing facilities. Nevertheless, Larval and pupal rearing when the costs of all programme operations are For the maintenance of a tsl-based GSS colony, included in the estimates,it becomes clear that the larval trays are seeded at half the egg density utilization of tsl-based GSS is much more cost-ef- used for a BSS. This has to be done to protect the fective than using BSS. temperature-sensitive females against the high temperatures resulting from larval overcrowding. Rearing process In addition, the trays have to be kept for four For comparisons of the two production systems additional days due to the lower temperatures (i.e.tsl-based GSS and BSS) the production process and the resultant slower female development has to be separated into two major parts: 1) adult rate. This results in a four-fold increase in diet use colony and 2) larval and pupal rearing. While for and a five-fold increase in space required for lar- the tsl-GSS the proportion of costs between the val production for colony maintenance, compared colony and larval rearing and pupal handling is to the larval production for a BSS colony. The approximately 1:4 (20% colony; 80% larval and increased amount of diet required to maintain the pupal rearing), for the BSS the proportion is 1:9 breeding colony for the tsl-based GSS system is (10% colony and 90% larval and pupal rearing). compensated by the 20% less diet required for When comparing the two rearing systems, the rearing the male larvae when compared to the major cost differences in rearing procedures are amount required for rearing both males and observed in the adult colony (egg production). females for BSS field releases (Table 2). Comparing Although for the larval and pupal rearing there are the overall volume of diet required to rear either a also differences in some of the basic requirements, tsl-based GSS or a BSS, both systems are more or such as the amount of diet and space, the differ- less equal (Table 2). Although the savings in diet ences in the two systems compensate each for adult release in the case of a GSS are small in other as will be explained in the following sections. absolute numbers (20%), they affect the major Since maintenance of the adult colony, compared part of diet use, i.e. for larval rearing for release. to the larval and pupal rearing, represents the Here, 2.6-fold more diet is used than in the larval Caceres et al.: Medfly bisexual and genetic sexing strains: development, evaluation and economics 375

Table 2. Comparison of rearing requirements between a tsl-GSS and a BSS to produce 100 million flying Medfly males/week.

Requirements Ratio Number/quantity BSS:GSS BSS GSS

Adult colony Colony size (million of adults) 1.0:1.8 12 22 Colony replacement (millions of pupae) 1.0:3.7 16 60 No. cages for male only production 1.0:3.3 50 165 Egging room (m2); male only 1.0:3.3 133 437 No. cages for colony maintenance 1:6 4 24 Egging room (m2); colony 1:7 11 78 Adult diet (sugar) (kg/day) 1.0:2.7 9 24 Adult diet (yeast hydrolysate) (kg/day) 1.0:2.7 3 8 Workers (colony + filter) 1:3 3 9 Larval rearing/pupal handling Workers 1:1 10 10 Larval diet/day (kg) for colony 1.0:4.2 300 1265 Larval diet/day (kg) for release 1.0:0.8 4100 3358 Subtotal 1.0:1.1 4400 4623 Rearing area for colony (m2) 1.0:5.2 60 310 Rearing area for release (m2) 1.0:0.6 606 390 Subtotal 1.0:1.1 666 700 Rearing area (total) 1.0:1.5 810 1215 Total pupal production/week (millions) 1.0:0.7 300 210 Millions of flying males/week 1:1 100 100 rearing for colony maintenance. The rearing of a In summary, for the larval and pupal rearing tsl-based GSS requires more diet for colony rearing process for release, which accounts for the larg- but this represents the smaller proportion of the est proportion of the production costs, there is a overall diet use. These relationships virtually lead significant difference in costs between the two to equal diet consumption for both systems. rearing systems. At the end, the tsl-based GSS In terms of space requirements a similar relation- system is currently slightly more expensive with ship holds true. The tsl-GSS system requires five respect to colony maintenance. Overall, the cur- times as much space for rearing the larvae and rent cost per million tsl-males is not more than pupae that will maintain the breeding colony. 10–15% higher than for the BSS system. However, However,in contrast,the amount of space required a new tsl-GSS has recently been developed and for rearing of the male larvae for adult release is tested at the Seibersdorf Laboratory, in which nearly twice as large for the BSS. This is due to a mortality of genetically unbalanced individuals more synchronized development of male-only occurs much earlier during development. As a larvae that allows retrieval of all the larvae in result the amount of larval diet required for this one-and-a-half days compared to the BSS rearing strain for male-only larval rearing is not 0.2 but at system that requires three days. As in the case of least 0.4 times lower than the amount required the diet, if the space required for the two systems for rearing BSS. This and other recent improve- is summed, the comparison between GSS and ments in GSS rearing are discussed below. BSS comes close to a 1:1 ratio (Table 2). Given that the space for larval and pupal rearing is very Post-production process similar for both systems, and considering that There are a number of direct and indirect costs the type of activities required are in general also of using the BSS that are not incurred when using very similar, we can infer that the basic utilities the tsl-based GSS, and these are transformed (i.e. water, power, etc.) and maintenance costs are into benefits for the programme when a tsl-based practically the same. GSS is used.The major sources of direct cost reduc- 376 Proceedings of the 6th International Fruit Fly Symposium

Table 3. Comparison of cost ratios between the use of Overall economic advantage of using tsl-GSS a BSS and a tsl-GSS. in SIT operations Using as a hypothetical case the shipment from Cost Item BSS:GSS Guatemala and release of sterile males in Panama, and transforming the above ratios into actual costs Direct costs in US$/treated km2/week, the cost for the tsl-GSS Rearing 1.0:1.2 was estimated at US$40/km2/week, compared Sterilization 1.0:0.5 with US$ 146/km2/week for the BSS, a 3.5-fold Shipping 1.0:0.5 cost reduction (Table 4). This difference could be Holding and emergence 1.0:0.5 even greater if the other cost factors shown in Table 4 (the non-quantified indirect costs) were Release 1.0:0.5 added. Indirect costs A range of costs/km2/week, including those Male competitiveness 1.0:0.3 estimated for the two sexing systems, was entered Sterile:fertile fly identification 1.0:0.5 in a fruit fly economic model (FAO/IAEA and Centre False ID of released sterile flies 1.0:0.5 for Environmental Technology Imperial College of Outbreak following escape of 1:0 Science, Technology and Medicine 2001). The non-irradiated adults model estimates a number of economic indices for Quality loss due to female stings 1:0 the utilization of the tsl-GSS and BSS.The economic indices were estimated by computing the costs and benefits of a Medfly area-wide integrated tion are related to irradiation, packing, shipping, control programme, again using Medfly control in emerging, feeding, holding, collecting and release. Panama as an example, where the use of sterile This is due to the fact that for tsl-based GSS only insects would be a significant component. The half of the pupae and adult fly volume is handled costs and benefits were transformed into net throughout the process from irradiation and packing present value and were projected over a 15-year to release, compared to BSS where pupae of both time frame. Results indicate that when using the sexes have to be irradiated, transported, fed, tsl-GSS system instead of a BSS,the initial project in- handled and released. When transforming these vestment is paid one year earlier. The benefit:cost cost factors into ratios, the differences between ratio of the programme when using the tsl-GSS is the tsl-based GSS and BSS systems become obvi- 4.9:1 compared to 3.7:1 when the BSS is used ous (Table 3). (Fig. 2). Furthermore, the net revenues for the fruit In addition, there are several sources of indirect industry in the country would be greater by an costs which have to be taken into account (Table 3). amount of US$19 million over 15 years if a tsl-GSS These indirect costs in favour of tsl-based GSS are: was used, which is a significant difference for a 1) significantly improved competitiveness of sterile small fruit industry. This example demonstrates males in the absence of sterile females (Robinson the economic advantages of using a Medfly et al. 1986; McInnis et al. 1994; Rendon et al. 2000); tsl-GSS. These advantages would apply to any 2) cost savings in the identification of trapped flies situation where the improved Medfly strain is to discriminate between sterile and fertile individu- used.mm als; and 3) more sensitive survey systems based on In conclusion, governments and modern fruit the use of specific female lures result in signifi- industries are increasingly aware not only of the cantly reduced recapture of sterile males. In addi- trade and environmental benefits of using SIT but tion, there are other more intangible and indirect also the economic benefits. The development of benefits of using tsl-based GSS such as: 4) avoid- tsl-GSS has already helped reduce the costs of ing the false identification of unmarked released area-wide application of SIT against Medfly to the sterile females, representing savings when not re- point where SIT is now economically viable for use sponding to false outbreaks; 5) significantly in- as a ‘biological insecticide’ for routine Medfly creased safety in the event of an escape of suppression, rather than to be applied only for non-irradiated adults into -free areas, as a eradication or barrier purposes. There are already result of the non-viability of tsl females; and 6) viable suppression projects in progress in various avoidance of quality loss in some fruit commodi- locations, including South Africa (Barnes et al. ties due to the absence of fruit stinging by sterile 2001) and Israel (Nitzan et al. 1993; Rössler et al. females. 2000). The utilization of the SIT will continue to Caceres et al.: Medfly bisexual and genetic sexing strains: development, evaluation and economics 377

Table 4. Comparative cost per km2 per week in US$ for the BSS and the tsl-GSS using current approximate costs from Central American SIT operations.

Cost Item BSS GSS

Direct costs Sterile males1 25 27.5 Shipping 8 4 Holding and emergence 5.2 2.5 Release 8.5 4.3 Subtotal 46.5 38.3 Indirect costs Male competitiveness2 93 0 Female sterile:fertile identification 1.5 0 Subtotal 94.5 0.0 Non-quantified indirect costs False ID of released sterile flies * * Outbreak as a result of escape of non-irradiated adults * * Quality loss due to sterile female stings Unknown Unknown Total 146 40

1Assuming a release density of 1000 males per ha or 100 000 per square kilometre. 2Refers to induction of sterility in the field, which is increased three fold by the release of males only (calculated as two additional times direct costs). *Not significant on a km2 per week basis.

The FRS can be used to introduce a more relaxed or natural rearing environments to re- duce strain deterioration. This would enable the viable life of a colonized strain to be extended under mass-rearing conditions, thus requiring strain renewals less often. 3) Development of protocols for long distance shipment of Medfly eggs Fig. 2. Economic returns for SIT when using a GSS Improved productivity as described above strain (tsl ) and a normal bisexual strain (BSS). may allow facilities to achieve increased econo- mies of scale and invest in increased egg pro- increase as the cost-effectiveness of existing SIT duction capacities in order to ship the excess technology continues to be optimized. eggs to facilities in other locations.These facili- ties could produce tsl males for sterilization ONGOING IMPROVEMENTS AND and release without the need to maintain filter, FUTURE PROSPECTS amplification and release stream colonies. A number of recent research findings indicate 4) Use of a morphological marker 2 that the 3.5-fold reduction of Medfly SIT cost/km / Sergeant (Sr2) is a dominant marker that week that is achieved when using GSS rather than produces an extra stripe on the abdomen of BSS (Table 4), can be even greater once the follow- the adult fly. It can be introduced into a GSS ing improvements are introduced: so that males will show the phenotype while 1) Improved productivity females will be wild type at this locus. Initial Increasing the egg seeding density on larval field tests have not revealed any reduction in diet due to early mortality of genetically unbal- the mating competitiveness of flies carrying anced individuals as explained above, and this marker, and it is now being introduced modifying the egging cages used for the re- into a GSS for further evaluation under mass- lease stream, can potentially increase egg rearing conditions. Releasing sterile males production 2.5–3 times. that carry this marker would facilitate the 2) Improved use of the Filter Rearing System sterile:fertile identification process and would 378 Proceedings of the 6th International Fruit Fly Symposium

Table 5. Comparison of rearing requirements between a tsl-GSS and BSS to produce 100 million flying Medfly males per week assuming that the improvements in the GSS rearing system have been incorporated to the production process.

Requirements Ratio Number/quantity BSS:GSS BSS GSS

Adult colony Colony size (million of adults) 1.0:1.5 12 18 Colony replacement (millions of pupae) 1.0:2.5 16 40 No. cages for male only production 1.0:2.8 50 139 Egging room (m2) 1.0:2.8 133 371 No. cages for colony maintenance 1.0:4.2 4 17 Egging room (m2) 1.0:5.1 11 56 Adult diet (sugar) (kg/day) 1.0:2.2 9 20 Adults diet (yeast hydrolysate) (kg/day) 1.0:1.7 3 5 Workers (colony + filter) 1:3 3 9 Larval rearing/pupal handling Workers 1:1 10 10 Larval diet/day (kg) for colony 1.0:2.6 300 785 Larval diet/day (kg) for release 1.0:0.5 4100 1890 Subtotal 1.0:0.6 4400 2675 Rearing area (larvae+pupae) for colony (m2) 1.0:3.8 60 226 Rearing area (larvae+pupae) for release (m2) 1.0:0.4 606 247 Subtotal 1.0:0.7 666 473 Rearing area (total) 1.0:1.1 810 901 Total pupal production/week (millions) 1.0:0.6 300 170 Millions of flying males per week 1:1 100 100

provide greater confidence in the identifi- rearing of this new GSS could be at least 10% cation, thus saving substantial amounts of more economical than a BSS. money in unnecessary control and regulatory In comparison with a BSS, use of the new GSS actions. Furthermore, such a marker would rearing system could reduce the costs of SIT per improve the quality of the sterile released square kilometer even further than the current insects since no dye would be necessary to estimate of 3.5- to 4.2-fold less expensive. This mark the sterile insects before release. further reduction in operational costs would 5) A new GSS make the economic returns of SIT application A new strain is being evaluated which has a even more favourable. greatly improved quality control profile proba- bly due to the properties of the translocation REFERENCES used in the strain. Transferring this new GSS to BARNES, B.N. & EYLES, D.K. 2000. Feasibility of eradicat- ing Ceratitis spp. fruit flies from South Africa by SIT. mass-rearing facilities and incorporating the 449–455. In: Tan, K.H. (Ed.) Area-wide Management of new operational procedures to large-scale Fruit Flies and Other Insect Pests. Universiti Sains mass-rearing will have immediate economic Malaysia Press, Penang, Malaysia. implications. The cost per million tsl-males will BARNES, B.N., EYLES, D.K. & SPIES, A. 2001. Fruit fly con- be at least equal to, but probably lower than trol with the sterile insect technique – How high will it fly? In: Olckers, T. & Brothers, D.J. (Eds) Proceedings BSS production due to the fact that the space of the 13th Entomological Congress. Entomological for larval and pupal rearing would be 30% Society of Southern Africa, Pretoria, South Africa. less than for BSS. In addition, the required BLAY, S. & YUVAL, B. 1997. Nutritional correlates of repro- volume of diet would be 40% less than for BSS ductive success of male Mediterranean fruit flies (Diptera: Tephritidae). Behaviour 54: 59–66. (Table 5). Under these conditions it can be BOLLER, E.F. & CALKINS, C. 1984. Measuring, monitoring inferred that, as larval rearing activities repre- and improving the quality of mass-reared Mediterra- sent around 80% of the total rearing cost, nean fruit flies, Ceratitis capitata Wied. 3. Improve- Caceres et al.: Medfly bisexual and genetic sexing strains: development, evaluation and economics 379

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