Natural parasitism of Doryctobracon areolatus (Szépligeti) (Hymenoptera: Braconidae) on various development stages of Anastrepha obliqua (Macquart) (Diptera: Tephritidae) attacking Spondias mombin L. fruits was studied under field conditions. We collected 120 fruits of S. mombin from which we got 495 A. obliqua larvae of different instars. A total of 88% of these larvae were parasitized. Within the parasitized cohort, the first-instar of D. areolatus was frequently detected in all 3 larval stages (L1 = 94.3%, L2 = 98.1%, and L3 = 100%), and the rest (i.e., L1 = 5.7%, L2 = 1.8%) corresponded to the presence of eggs. In fruits with controlled infestation and cage-induced parasitism under field conditions, D. areolatus oviposited in mature eggs and recently hatched larvae of A. obliqua with comparable frequencies. Seven preimaginal stages of D. areolatus were observed during their development, which was completed in 27 days. It is concluded that D. areolatus has the capacity to oviposit in embryo eggs and neonate larvae of A. obliqua and that its first-instar larvae (with three distinct sizes) are capable of synchronizing their development with the development of the host larvae. This finding represents the first report of a native parasitoid attacking eggs or neonate larvae of a tephritid in the Neotropics. The implications of this finding are discussed within the context of the competitive interactions of this species with other parasitoid species under sympatric conditions, as well as the relevance for developing laboratory rearing methods for biological control purposes.

Egg parasitoid, laboratory breeding, interspecific competition, morphology, fruit flies, biological control

The native parasitoid guild that attacks fruit flies of the genus Anastrepha Schiner in the Neotropics is mainly composed of a group of solitary koinobiont endoparasitoids (primarily Braconidae and Figitidae) that oviposit in the host larvae and emerge from the pupae. The genus Doryctobracon constitutes 27% of the parasitoid species and shares a closely related evolutionary history with Anastrepha (López et al. 1999, Ovruski et al. 2000).

Another smaller group of Anastrepha parasitoids are pupal idiobionts that attack their hosts when they are in the soil and is represented by five species within the genera Coptera and Trichopria (Diapriidae) and three polyphagous species, Pachycrepoideus vindemiae (Rondani), Spalangia cameroni Perkins, and S. endius Walker (Pteromalidae) (Ovruski et al. 2000). The parasitoid guild of Anastrepha spp. in the Neotropics represents an important source of species with potential to be used in biological control programs against native tephritids (Aluja et al. 2003). However, native Anastrepha parasitoids attacking eggs or early larval developmental stages have not been reported. The only report of a parasitoid attacking Anastrepha eggs in the Americas correspond to Wharton et al. (1981), who found a small level of parasitism of the introduced Fopius arisanus (Sonan), apparently on A. striata (Schiner) eggs.

According to Aluja et al. (2009) species of the genera Doryctobracon, such as D. crawfordi (Viereck) and D. areolatus (Szépligeti), exhibit great potential as biological control agents and should be examined from the point of view of mass rearing projects. D. areolatus has been reported as a solitary koinobiont endoparasitoid that attacks third instar larvae of Anastrepha spp., both in native and exotic commercial fruits, with a wide distribution from Florida to Argentina (Hernández-Ortiz et al. 1994, López et al. 1999, Ovruski et al. 2000, Sivinski, et al. 2000, Aluja et al. 2003, 2009). This species frequently shows field dominance among concurrent parasitoid species attacking Anastrepha spp. (López et al. 1999, Sivinski et al. 1997, 2000; Aluja et al. 2003, Ovruski et al. 2004). The presence of diapause (Aluja et al. 1998, Ovruski et al. 2004) and an extrinsic capacity to find patches with low host density (Sivinski et al. 1998) allows an ample distribution in regions with low plant diversity (Eitam et al. 2004).

In the central region of Veracruz, Mexico, D. areolatus is the most abundant parasitoid species attacking Anastrepha obliqua (Macquart) in Spondias spp. (Cabrera et al. 2006), where it has frequently been observed at the beginning of the fruiting season. This suggests that this species could parasitize earlier developmental stages in relation to sympatric parasitoid species. Normally, parasitoids attacking eggs and first instar larvae of their hosts become more competitive than those attacking later stages (Wang and Messing 2002, Wang et al. 2003, Wang et al. 2008, Argov et al. 2011), showing important potential to be used as biocontrol agents. Therefore, our aims in this study were: 1) to determine if D. areolatus parasitize immature stages of A. obliqua and 2) to characterize its developmental stages during each phase of host development.

The presence of eggs and larvae of D. areolatus in the interior of eggs and recently hatched A. obliqua larvae represent a novel finding within the native parasitoid guild that attack fruit flies in the Neotropics, because there have been no previous reports of any native parasitoid covering this ecological niche (López et al. 1999, Ovruski et al. 2000).

Among the particular observations regarding this finding under forced conditions, it was notable that D. areolatus oviposit inside embryo-eggs of A. obliqua, depositing a flexible egg that can fold inside the interior of the host embryo, and that the first-instar larvae present a prolonged development with three distinct sizes that synchronize with the development of the host larva and pupa. The low number of embryo-eggs found with egg parasitoids could be explained by the short developmental time of eggs (less than 24 hours).

In Mexico, D. areolatus has been reported to be closely associated with A. obliqua in fruit hosts of the genus Spondias (López et al. 1999, Ovruski et al. 2000, Sivinski et al. 2000), which most likely is favored by the presence of semiochemicals that could allow it to attack early stages of A. obliqua (eggs and recently hatched larvae). This scenario seems similar to that of F. arisanus, which detects marking pheromones and kairomones that emanate from the eggs of its host or from the interaction of the fruit and the host egg (Rousse et al. 2005, 2007, Pérez et al. 2013).

The finding of the parasitism of D. areolatus on eggs and recently hatched larvae of A. obliqua sheds light on two relevant aspects of its role as a natural enemy and biological control agent of fruit flies: 1) its competition and coexistence with other opiine parasitoids, highlighting the exotic species Diachasmimorpha longicaudata (Ashmead) and the native species U. anastrephae (García-Medel et al. 2007, Sivinski et al. 1998, Paranhos et al. 2013, Aluja et al. 2013), and 2) the promising development of its mass rearing under laboratory conditions (Eitam et al. 2003, Aluja et al. 2009).

It has been argued that D. areolatus is an inferior competitor compared to D. longicaudata (Sivinski et al. 1998, Eitam et al. 2004) and to U. anastrephae (Aluja et al. 2013). It has also been suggested that D. longicaudata, given its larger ovipositor, could cause a local extinction by displacing D. areolatus when deprived of free space left by its competitors, as it has been suggested to explain the reduction of the dispersion range in Florida, USA, in the presence of D. longicaudata (Sivinski et al. 1998). For U. anastrephae, it has been suggested that the historic sympatry of D. areolatus and U. anastrephae depends on the ability of D. areolatus to avoid competition with the intrinsically superior competitor by exploiting hosts in larger fruits that are out of the reach of the smaller ovipositor of U. anastrephae (Sivinski et al. 1997, Aluja et al. 2013). However, our new findings suggest that both hypotheses can be reformulated in relation to the biology and oviposition behavior of D. areolatus.

An early action of D. areolatus against immature A. obliqua could represent an ecological advantage that prevents its displacement or local extinction by other competitors such as U. anastrephae and D. longicaudata, since these latter species invariably will attack mature larval stages that could already be parasitized by D. areolatus. According to Wang et al. (2003, 2008), in F. arisanus this earlier attack increases the probability to suppress the invasive larva through starvation or suffocation mechanisms. Field observations seem to support this assertion. Even though D. longicaudata has become established in numerous sites in Mexico where it has been released, its presence in Spondias species is inferior to that of D. areolatus (López et al.1999, Sivinski et al. 2000, Montoya et al. unpublished data). Our data showed that this species also competes successfully against U. anastrephae by parasitizing eggs and recently hatched larvae, which enables D. areolatus to become the dominant parasitoid species in these hosts.

Laboratory studies have reported that D. areolatus is an inferior competitor relative to D. longicaudata and U. anastrephae (Paranhos et al. 2013, Aluja et al. 2013) because larvae of D. longicaudata and U. anastrephae kill larvae of D. areolatus during competition through sequential exposures. However, these studies were conducted using mature host larvae (3rd instar), which, according to our results, presents a disadvantage to D. areolatus.

One difficulty in rearing D. areolatus has apparently been the oviposition stimuli in oviposition units (artificial devices with third-instar host larvae mixed with food) (Eitam et al. 2003), which has not been a problem in the case of D. longicaudata because this species detects its host by larval vibrations when feeding or moving (Lawrence 1981). However, in light of our new findings, new perspectives on laboratory rearing of this species are realized. The use of late-stage eggs or neonate larvae, similar to Fopius arisanus rearing (Harris et al. 1991, Zenil et al. 2004, Rousse et al. 2005, Montoya et al. 2009), should be tested.

Unsuccessful attempts have been made to rear D. areolatus using fruits with third instar host larvae (Eitam et al. 2003). This could be because D. areolatus females need different stimuli, such as chemical signals emitted by host (eggs or young larvae) interacting with fruit volatiles, as has been demonstrated for F. arisanus (Rousse et al. 2007, Pérez et al. 2013).

The preimaginal development of D. areolatus in A. obliqua required approximately 27 days, and 7 preimaginal stages were classified: egg, three larval stages, prepupa, pupa, and pharate adult. The morphological observations of the preimaginal stages of D. areolatus are in agreement with what has been reported for F. arisanus and D. longicaudata (Rousse et al. 2005, Carabajal-Paladino et al. 2010). However, the development of D. areolatus is more akin to F. arisanus because both exhibit a facultative synchronization between their larval development and that of its host, which is reflected in the long period of time for the first-instar larva, which finally changes when its host reaches the prepupal stage (Rousse et al. 2005). This type of development allows the second or third instars of F. arisanus (and probably D. areolatus) to occupy most of the available space inside the pupa, which facilitate the elimination of competing larvae (Wang et al. 2003, 2008).

Our study shows that D. areolatus can parasitize A. obliqua eggs and recently hatched larvae, giving an advantage over other parasitoids that attack the later-stage larvae. This finding represents a novel report regarding the oviposition behavior of this species, suggesting that it may occupy an ecological niche that was previously thought empty in the Americas. These findings also open new perspectives for the biological control of fruit flies. If mass rearing methods are developed, this will allow release of the most dominant fruit fly parasitoid species in the Neotropics.

We thankfully acknowledge Ana Lilia Montero (ECOSUR), Gerardo de Jesús Alonso, and Guillermo Jaime (ITUG) for their assistance in fieldwork and Maria Guadalupe Nieto (ECOSUR) for her assistance in laboratory. This study was funded by the SAGARPA-CONACYT Sectorial Fund project 163431, and it was part of Félix D. Murillo Ph. D. thesis at ECOSUR with a fellowship from CONACYT.