Augmentative Biological Control Using Parasitoids for Fruit Fly Management in Brazil

The history of classical biological control of fruit flies in Brazil includes two reported attempts in the past 70 years. The first occurred in 1937 when an African species of parasitoid larvae (Tetrastichus giffardianus) was introduced to control the Mediterranean fruit fly, Ceratitis capitata and other tephritids. The second occurred in September 1994 when the exotic parasitoid Diachasmimorpha longicaudata, originally from Gainesville, Florida, was introduced by a Brazilian agricultural corporation (EMBRAPA) to evaluate the parasitoid’s potential for the biological control of Anastrepha spp. and Ceratitis capitata. Although there are numerous native Brazilian fruit fly parasitoids, mass rearing of these native species is difficult. Thus, D. longicaudata was chosen due to its specificity for the family Tephritidae and its ease of laboratory rearing. In this paper we review the literature on Brazilian fruit fly biological control and suggest that those tactics can be used on a large scale, together creating a biological barrier to the introduction of new fruit fly populations, reducing the source of outbreaks and the risk of species spread, while decreasing the use of insecticides on fruit destined for domestic and foreign markets.

many situations can be found in orchards, together parasitizing up to 40% of flies [9]. Of these species, D. areolatus is the most abundant and frequent in Brazil [9].
The aim of this review is to discuss the history and current state of augmentative biological control of fruit flies in Brazil.

Native Parasitoids
Brazil is home to many species of native parasitoids (Table 1), including the efficient parasitoid D. areolatus which is widely distributed across the country. However, to date, the mass rearing of this and other native parasitoids has not been successful. Doryctobracon areolatus is only attracted to fruit fly larvae when they are associated with the fruit, thus increasing production costs and preventing their use in biological control in Brazil [14].

Tetrastichus giffardianus
Two classical biological control programs for the control of fruit flies have been reported in Brazil, and which preceded any form of augmentative release. The first occurred in 1937 when the African larval parasitoid Tetrastichus giffardianus Silvestri (Hymenoptera: Eulophidae) was introduced to control Ceratitis capitata Wiedemann and other tephritids. The second occurred in 1994 when the troduced the exotic parasitoid Diachasmimorpha longicaudata (Ashmead) to flies of the genus Anastrepha and C. capitata [11,61 63].
The use of T. giffardianus for the augmentative biological control of fruit flies has advantages and disadvantages. Advantages include the gregarious nature of this species, the high proportion of females among the offspring, and females which penetrate the fruit to lay eggs in the larvae, thus directly attacking the larvae that often escape from Opiinae parasitism, of which the larger fruit of the latter may be an issue, due to difficulties of the parasitoids ovipositor reaching deeply entrenched larvae [64]. Additionally, during the mass-rearing process, the time lag between the emergence of the flies and that of the parasitoids eliminates the problem of releasing these flies into the environment [63]. Disadvantages that may possibly limit the applicability of this parasitoid include its relatively low rate of parasitism and short life span when released. However, the low rate of parasitism would be offset by the high gross reproductive rate because more than one parasitoid is produced by the host. This parasitoid was introduced in the state of São Paulo, and 60 years later, it is no longer found in the southeast region. However, it persists in the states of Bahia, Rio Grande do Norte and Ceará, likely due to the warmer climate [63].

Diachasmimorpha longicaudata
Four years after D. longicaudata et al. [65] conducted field surveys to evaluate the parasitoid s establishment and association with several species of fruiting trees including: the common guava, Psidium guajava L. (Mirtaceae); Surinam cherry, Eugenia uniflora L. (Mirtaceae); starfruit, Averrhoa carambola L. (Oxalidacea); and mango, Magnifica indica Linnaeus (Anacardiaceae). Subsequently, the exotic D. longicaudata was mass reared in the laboratory and released weekly on all of the aforementioned fruiting trees but at low levels of approximately 1,000 parasitoids. Surveys prior to these releases in a multi-species orchard revealed the presence of a number of native hymenopterans, including D. areolatus, Utetes anastrephae, Opius spp., Asobara anastrephae (Muesebeck) and Aganaspis pelleranoi. Surveys conducted after the release of D. longicaudata showed that these native species persisted and that although interspecific competition for oviposition sites existed, no loss of biodiversity was observed [66]. Diachasmimorpha longicaudata was detected 17 months post release from starfruit, guava and other environments, such as a hybrid species of umbu-caja, but was not recorded during the surveys conducted in 2004 and 2005 [66]. The ability of D. longicaudata to adapt to semi-arid (São Francisco water basin, State of Bahia, Brazil) and Amazonian (Oiapoque, State of Amapa, Brazil) ecosystems to parasitize hosts and complete its life cycle in a variety of different environments. Although the Amazon in particular presents complex ecosystems and interspecific competition with native species, this parasitoid was able to readily adapt [67]. Approximately 34,000 pairs of D. longicaudata were released on fifteen occasions over seven weeks in commercial guava orchards in the semi-arid northern region of Minas Gerais. Seven days post release, a survey recovered a total of 37 specimens of D. longicaudata, indicating that the species completed its life cycle in the local conditions and has the capacity to establish in the region [64,68,69].
In Botucatu, State of São Paulo, D. longicaudata were released in areas of riparian forest to suppress the density of Anastrepha fraterculus impacting nearby citrus orchards [70]. The release was timed to occur between insecticide applications, to minimize any effects of spray drift on released parasitoids. A total of 14 releases occurred between April and October, and a total of 560,000 parasitoids were released, which resulted in a 30% reduction in A. fraterculus infestations and a consequent reduction in the application of insecticides [70].

Mass Rearing and Release Requirements for Parasitoids in Brazil
Although Brazil has a diverse number of native fruit fly parasitoid species, difficulties have been found in the mass rearing of these species. Such difficulties led to the introduction of D. longicaudata. This parasitoid has been used because of its host specificity for the family Tephritidae and its ease of laboratory rearing. The parasitoid D. longicaudata originates from Indo-Australia where the natural hosts are flies of the genus Bactrocera [71].
When searching for a host, D. longicaudata is attracted to volatile substances released by the fruit and the vibrations produced by the fly larvae during feeding, mainly in fallen fruits rotting on the ground [72,73]. The average life cycle for D. longicaudata reared in the laboratory at a temperature of 22 26 °C, and 14-hour photoperiod on C. capitata larvae ranges from 14 to 16 days [73]. The pre-oviposition period is two days, and the oviposition period is 28 30 days. In mass-rearing facilities in the State of São Paulo, an average parasitism rate of 46.1% is achieved, with a ratio of three females to one male [73].
In Brazil, in studies with rearing cages placed under natural (outdoor) conditions, D. longicaudata was more efficient compared to other parasitoids and, in some cases, reached parasitism levels of 91.42%. This efficiency is true when reared on both C. capitata and Anastrepha sp. [14,67,74]. In the field, a significant reduction (40.0% 95.0%) was observed in trapped A. suspensa flies when the parasitoid was released at inundation [68,75 79].
Although D. longicaudata attacks both C. capitata, and A. fraterculus, when these species are present together in the same environment, the parasitoid demonstrates a preference for A. fraterculus [68].
In Brazil, Diachasmimorpha longicaudata is mass-reared in cages containing water, food (sugar and soy protein) and a means for oviposition (papaya fruit and artificial plastic fruit, although the flies will also oviposit through the cage screen) (Figure 1) [61]. The eggs are collected with a paintbrush, and the larvae are subsequently reared on an artificial diet. When the larvae reach third instar, they are exposed to adult parasitoids in a nursery container, simulating an infested fruit to facilitate access to the larvae. Adult parasitoids are fed an artificial diet (120 mL warm water, 0.8 g agar, 0.05 g ascorbic acid, 0.005 g Nipagin and 120 mL honey) and water. After 24 hours, the larvae are placed in containers containing vermiculite for pupation and adult emergence [61]. The parasitoid offspring are removed from the emergent cages with the aid of a vacuum adapted to provide weak suction [61]. Rearing of D. areolatus in Brazil is done in cages of varying sizes and always uses A. fraterculus as the host. The diet for this parasitoid is composed of honey, distilled water, agar, ascorbic acid, and sodium benzoate or honey and water. Mass production of this parasitoid has been unsuccessful because it is a species with a high risk of death due to behavioral attributes and genetic degeneration in the colonization process [14,21,80]. Additionally, the parasitoid T. giffardianus is a potential candidate for biological control in Brazil, but there is a lack of information about diet, mass-rearing methods and economics. On the other hand, D. longicaudata is easily reared and is the species most often utilized in Brazil. This species is primarily reared in larval C. capitata because they possess sterile lines, and the adults also utilize a diet based on honey, distilled water, agar, ascorbic acid and sodium benzoate (Table 2) [14,69,81,82]. In Brazil, there are few publications on biological parameters of parasitoids of fruit flies reared in the laboratory, which makes it difficult to determine the best technique. However, it is known that the mass-rearing technique used by the Center for Nuclear Energy in Agriculture, University of São Paulo (CENA/USP) produced more than 150 million specimens of D. longicaudata in Brazil from January 1995 to October 2001 [81].

Bio-Fabrication
Today, the biological control of fruit flies in programs across Brazil is largely based on the augmentative release of introduced parasitoids to achieve effective suppression of pest fly populations on an area-wide basis [62]. Consequently, for these programs to be successful, a large number of agents must be available. In Brazil, bio-factories mass produce and commercially provide a range of biological control agents. The bio-factory Moscamed Brazil was created in Juazeiro, State of Bahia, in 2005, with the main objective of producing sterile insects for the release and eradication of C. capitata. This factory has a production capacity of 100 million sterile males per week. In addition, the factory also produces 10 million D. longicaudata/week for the biological control of Anastrepha spp. and C. capitata [14]. The factory was made possible through investments by federal and state governments and international partnerships (International Atomic Energy Agency IAEA).

Conclusions
In Brazil, the most effective parasitoid for use in augmentative biological control programs for Anastrepha spp. and C. capitata is D. longicaudata. This species is preferred mainly due to its ease of rearing and high parasitism levels. Successful control programs with this parasitoid require repeated releases until the parasitoid becomes fully established in the area [62].
Worldwide, D. longicaudata is an important parasitoid of fruit flies for several reasons including its ease of rearing, intensive foraging and high activity of resource exploitation by females. However, several studies have shown low indices of parasitism and recovery of specimens of D. longicaudata released in Brazil. Evidence from these studies suggests that the removal of fruit from the field during sampling may have reduced the susceptibility period of larvae to the parasitoid, therefore generating an underestimation of the parasitism index. Moreover, D. longicaudata also demonstrates a low tolerance to cold, inferred from its decreased effectiveness during the winter in southern Brazilian states, despite continued effectiveness in warmer regions of the country [63].
Studies that seek alternatives to using exotic introduced parasitoids in Brazil are greatly lacking, with more technically and economically feasible efforts required for the development of research aimed at the mass rearing of native parasitoids, particularly D. areolatus. Another parasitoid that requires research attention for its potential against a range of flies in Brazil is Fopius arisanus (Sonan). This parasitoid develops in the egg and early larval instar of fruit flies, and emerges from the pupae [83] and has surpassed D. longicaudata in all competition studies [84 86].
The successful use of parasitoids for the suppression of fruit fly populations depends on studies that evaluate the behavior of these insects and the use of such methods in conjunction with other area-wide control techniques on an integrated pest management basis (IPM). In Brazil, biological control and the sterile insect technique (SIT) are expected to be used across large areas, suppressing fly populations, reducing the source of infestations and the risk of spread of pest species, reducing the use of insecticides in fruit destined for domestic and foreign markets, and ultimately creating a biological barrier to fruit fly incursions in major horticultural regions.
The augmentative biological control of fruit flies is now a reality in Brazil, along with other biological control techniques, including SIT, which, when used in combination, is believed capable of increasing the overall efficiency and effectiveness of control of pest fruit flies in Brazil [87].