Pupal parasitoids associated with Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) in a semiarid environment in Brazil

Fruit flies (Diptera: Tephritidae) are important pests that affect the fruit-growing worldwide. In the northeastern Brazil, where a semiarid climate predominates and the production of tropical fruits for export is concentrated, some fruit flies, including Anastrepha spp. and Ceratitis capitata (Wiedemann), are considered pests due to economic damage and quarantine restrictions. In several parts of the world, fruit fly population regulation is carried out with the support of hymenopteran parasitoids. In Brazil, some information exists about larval parasitoids of fruit flies, but little is known about pupal parasitoids of these tephritids, especially in Brazilian semiarid environments. Therefore, the main objective of this study was to know the pupal parasitoids associated with C. capitata in a semiarid environment in Brazil. The parasitoid survey was carried out in a semiarid region in the states of Rio do Norte and Ceará. To obtain the parasitoids, pupae (sentinels) of C. capitata from the laboratory, Applied Entomology Laboratory of Universidade Federal Rural do Semi-Árido, were exposed to natural parasitism in the field. Six parasitoid (Hymenoptera) species were obtained: Pachycrepoideus vindemmiae (Rondani), Spalangia simplex Perkins, Spalangia gemina Boucek, Spalangia leiopleura Gibson, and Spalangia impunctata Howard (Pteromalidae); Trichopria anastrephae Lima (Diapriidae). These are the first records of pupal parasitoids associated with the fruit fly C. capitata in Brazil. A R T I C L E I N F O Article history: Received 03 October 2019 Accepted 23 March 2020 Available online 15 May 2020 Associate Editor: Diana Grisales Ochoa


Introduction
Fruit flies (Diptera: Tephritidae) are considered pests that affect the fruit-growing worldwide (White and Elson-Harris, 1992;Szyniszewska and Tatem, 2014). The fruit flies oviposit inside the fruits and their larvae feed on the pulp, making them unsuitable for fresh consumption and industrialization. After completing their development, the larvae leave the fruits and bury themselves in the soil to pupate, after a few days the adults emerge (White and Elson-Harris, 1992). In Brazil, Ceratitis capitata (Wiedemann) and several species from genus Anastrepha Schiner are important, due to economic damages and quarantine restrictions (Malavasi and Zucchi, 2000;Vilela and Zucchi, 2015). Some Anastrepha species are endemic to Brazil, whereas C. capitata was first detected in the country in 1901 (Zucchi, 2000).
In semiarid regions of Brazil, where the Caatinga biome predominates and tropical fruit production is concentrated, C. capitata was detected in the early 1990s and is currently considered one of the region's key fruit pests . The Caatinga biome is characterized by its vegetation with xerophyte and deciduous plants. The climate is predominantly hot semi-arid (BSh type), with sparse and irregular rainfall (average annual rainfall of 600 mm), high average temperatures (± 28ºC) and low air humidity (± 65%) (Sousa et al., 2019).
For the control of C. capitata populations, it is important to know its natural enemies that can be used in integrated management programs, and the parasitoids are among the main natural enemies of this fruit fly (Aluja et al., 2014). However, as most of the life cycle of C. capitata occurs protected inside the fruit (egg and larval stage) and buried in the soil (pupal stage), the control of this fruit fly and the survey of its parasitoids, especially pupal parasitoids, is difficult. The methodology currently used for the survey of fruit fly parasitoids, which is based on the collection of fruits, allows only the knowledge of the egg and larval parasitoids, but it is not possible to know the pupal parasitoids.
In Brazil, fruit fly parasitoid surveys have been practically restricted to larvae parasitoids, and these surveys have indicated that hymenopteran parasitoids (Braconidae and Figitidae) are the most common in several regions of the country and are associated mainly with species of Anastrepha (Garcia and Corseuil, 2004;Souza-Filho et al., 2009;Silva et al., 2010;Souza et al., 2012;Taira et al., 2013).
Little is known about pupal fruit fly parasitoids and this information is concentrated to Brazil's central and southern regions, where some hymenopteran parasitoids, including Pachycrepoideus vindemmiae (Rondani), Spalangia simplex Perkins, Spalangia endius Walker, and Spalangia gemina Bouček (Pteromalidae); Trichopria anastrephae Lima and Coptera haywardi Loiácono (Diapriidae), were found parasitizing pupae of Anastrepha (Marchiori et al., 2000;Aguiar-Menezes et al., 2003;Uchôa-Fernandes et al., 2003;Garcia and Corseuil, 2004;Cruz et al., 2011). There are no records of pupal parasitoids associated with C. capitata (Paranhos et al., 2019). In semiarid regions of Brazil, the obtained information is related only to fruit fly larvae parasitoids (Araujo and Zucchi, 2002;Alvarenga et al., 2009;Araujo et al., 2015). However, knowing pupal parasitoids is important because these can be used to regulate the population of C. capitata. Therefore, this study's main objective was to know the pupal parasitoids associated with C. capitata, in a semiarid environment in Brazil.

Material and methods
To collect the parasitoids, live C. capitata (strain Vienna 8) pupae were exposed to parasitism throughout the studied zone using the sentinel pupae technique (Petersen and Watson, 1992). Ceratitis capitata (strain Vienna 8) pupae were obtained from the Applied Entomology Laboratory of Universidade Federal Rural do Semi-Árido (UFERSA) (Mossoró, Rio Grande do Norte, Brazil), as unirradiated 48 hours-old pupae.
For each studied location, each 15 days one parasitism units was exposed in the field for 48 hours, during the period from May to September 2016. Then, a total of 90 parasitism units were used, with one parasitism unit per location and sampling date. To avoid predation by soil insects, the parasitism units were placed inside plastic trays (51 x 30 x 9.5 cm [l x w x h]) containing a solution of water and detergent (10%).
After field exposure, the parasitism units were retrieved to the laboratory, and the pupae were placed inside Petri dishes (10 cm in diameter x 1.5 cm in height) under a thin layer (4 cm) of moist vermiculite. The Petri dishes were then closed with voile fabric and kept in a climate-controlled room at a temperature of 25 ± 2ºC, relative humidity of 70 ± 10% and a photoperiod of 12:12 h (L:D), where they remained until the emergence of adults (flies or parasitoids). Parasitoids that emerged were counted and fixed in 70% alcohol for later specific identification.
The parasitoids were identified based on Bouček and Heydon (1997), Gibson (2009) and Rueda and Axtell (1985) taxonomical keys. Voucher specimens were deposited in the parasitoid hymenoptera collection at the Biological Institute, Campinas, São Paulo, Brazil.
The considerable diversity of Pteromalidae (Chalcidoidea) collected is possibly related to the fact that the main genera of pupal parasitoids of fruit flies in the world belong to this family (Noyes, 2014). Although most parasitoid species collected in this study had already been registered in Brazil, this is the first report of S. impunctata in the country. Among the pupal fruit fly parasitoids reported in Brazil, only S. endius (Aguiar-Menezes et al., 2003;Silva et al., 2003;Uchôa-Fernandes et al., 2003;Nicácio et al., 2011) andC. haywardi (Aguiar-Menezes et al., 2003) were not collected during the present study (Table 2).
Pachycrepoideus vindemmiae was the most common species in this survey, possibly due to its generalist behaviour (Marchiori et al., 2013;Zhao et al., 2013), which favors its presence in various environments and regions. In several parts of the world, records indicate that P. vindemmiae parasitizes species of fruit flies of economic importance in the genera Anastrepha, Ceratitis, Bactrocera, and Rhagoletis (Noyes, 2014). In some arid regions such as Tunisia and Benin, P. vindemmiae has been recorded parasitizing pupae of C. capitata (Harbi et al., 2015) and Bactrocera invadens Drew, Tsuruta & White (Vayssières et al., 2011).
In Africa, there are reports of S. simplex parasitizing pupae of various Ceratitis species (Vayssières et al., 2002). In the Americas, S. simplex has been observed to parasitize Anastrepha species, such as A. ludens in Mexico, Anastrepha suspensa (Loew) in the United States and Anastrepha acidusa (Walker) in Puerto Rico (Gibson, 2009). In Brazil, S. simplex was only obtained from pupae of Anastrepha serpentina (Wiedemann) in the state of São Paulo (Fernandes et al., 2013). Thus, this is the first record of S. simplex in association with pupae of C. capitata in Brazil (Table 2).
The species S. leiopleura was described based on specimens collected in North America and Mexico (Gibson, 2009). Recently this species was reported in Brazil in the state of Minas Gerais. However, this report was based on specimens collected in traps, so it was not possible to know its host (Juliato et al., 2017). In this study, S. leiopleura was collected from C. capitata pupae in Rio Grande Norte, so this is the first record of the association of S. leiopleura with a host of the Tephritidae family in Brazil.
Spalangia impunctata is possibly a circumtropical species (Gibson, 2009). This species was described based on specimens obtained from Drosophila Fallén pupae in India (Sureshan and Farsana, 2014). As reported, this is the first record of S. impunctata in Brazil. The specimens of S. impunctata were obtained from pupae of C. capitata in Rio Grande do Norte. This shows that Brazilian semiarid regions may contain species not yet reported in Brazil.
Trichopria anastrephae was described based on specimens obtained from pupae of Anastrepha sp. and A. serpentina in Rio de Janeiro, Brazil (Lima, 1940). Of the 12 Trichopria species cataloged in Brazil, T. anastrephae was the only one reported to parasitize Anastrepha Una/BA Anastrepha spp. Souza-Filho et al. (2007) Divinópolis/MG Anastrepha spp. Silva et al. (2003) Not mentioned/RJ Anastrepha sp. and A. serpentina Lima (1940) * First reports in Brazil; ** New distribution reports; *** New reports of association with fruit flies in Brazil. (Santis, 1980;Margaria, 2019). Trichopria anastrephae has been observed parasitizing fruit flies (Anastrepha) in the states of Rio de Janeiro (Lima, 1940;Aguiar-Menezes et al., 2001), Minas Gerais (Silva et al., 2003), Rio Grande do Sul (Garcia and Corseuil, 2004;Cruz et al., 2011) and Bahia (Souza-Filho et al., 2007). Therefore, this is the first record of T. anastrephae in a semiarid environment, and it is also the first time that T. anastrephae has been observed parasitizing C. capitata pupae in Brazil (Table 2). In Brazil, the few reports of fruit fly pupae parasitoids known are concentrated in the midwestern and southern regions, which indicate that surveys of these parasitoids have been neglected in various regions of the country. In this study, six species of parasitoids are recorded for the first time, obtained from pupae (sentinels) of C. capitata distributed in a semiarid environment in northeastern Brazil. This information extends reports of pupae parasitoids associated with fruit flies in Brazil and records the presence of S. impunctata in the country for the first time. In addition, reports were made from pupae of C. capitata, which is an important exotic species that is spreading in Brazilian semiarid regions.
Therefore, the information obtained shows that if surveys are expanded, new reports of parasitoids associated with fruit fly pupae may be recorded in various regions of the country. Thus, further studies on these pupal parasitoids should be performed, aiming at their use in the management of C. capitata in fruit orchards located in semiarid regions of Brazil.

Figure 1
States with recordings of pupal parasitoids associated with fruit flies (Anastrepha spp. and / or Ceratitis capitata) in Brazil.