Introduction
Anastrepha obliqua (McQuart) (Diptera: Tephritidae), commonly named ‘West Indian fruit fly’, is a native Neotropical species that oviposit and develops into a wide range of wild fruits and it is the most significant fruit fly pest of Mangifera indica L. (‘mango’) (Aluja and Birke, Reference Aluja and Birke1993; Aluja et al., Reference Aluja, Rull, Sivinski, Norrbom, Wharton, Macías-Ordóñez, Díaz-Fleischer and López2003; Mangan et al., Reference Mangan, Thomas, Moreno and Robacker2011). This pestiferous fruit fly is widespread in Mexico, Central and South America, and the West Indies (Ruiz-Arce et al., Reference Ruiz-Arce, Barr, Owen, Thomas and McPheron2012; Santos et al., Reference Santos, Silva and Miranda2020), and it is a serious threat to other mango-producing regions (Jiron, Reference Jiron1996; Montoya et al., Reference Montoya, Cancino, Zenil, Santiago, Gutierrez, Vreysen, Robinson and Hendrichs2007; Ruiz-Arce et al., Reference Ruiz-Arce, Barr, Owen, Thomas and McPheron2012). A. obliqua mass rearing has been successfully established at the Moscafrut Biofactory, located in Metapa de Dominguez, Chiapas, Southern Mexico, mainly to implement the sterile insect technique (Rull Gabayet et al., Reference Rull Gabayet, Reyes Flores, Enkerlin Hoeflich, McPheron and Steck1996; Orozco-Dávila et al., Reference Orozco-Dávila, Quintero, Hernández, Solís, Artiaga, Hernández, Ortega and Montoya2017). The production of millions of A. obliqua eggs, larvae, and pupae also represent an opportunity to develop parasitoid mass rearing, to apply an effective biological control of this pestiferous tephritid fruit fly (Ovruski et al., Reference Ovruski, Aluja, Sivinski and Wharton2000; Montoya et al., Reference Montoya, Liedo, Benrey, Barrera, Cancino, Sivinski and Aluja2000b; Artiaga-López et al., Reference Artiaga-López, Hernández, Domínguez-Gordillo, Moreno and Orozco-Dávila2002). Anastrepha obliqua is a host that can be used as a natural reservoir for different species of parasitoids, since its larval development occurs in various wild fruits, such as those of the genus Spondias (Aluja et al., Reference Aluja, Guillen, Liedo, Cabrera, de la Rosa, Celedonio and Mota1990; Sivinski et al., Reference Sivinski, Piñero and Aluja2000; Silva et al., Reference Silva, Santos, Silva, Vidal, Nink, Guimarães and Araujo2010). These wild fruits have important physical characteristics for parasitoid oviposition, such as small size, large seed, a narrow and soft pulp layer, and an extremely thin rind (Aluja and Birke, Reference Aluja and Birke1993; López et al., Reference López, Aluja and Sivinski1999; Murillo et al., Reference Murillo, Liedo, Nieto-López, Cabrera-Mireles, Barrera and Montoya2016; Montoya et al., Reference Montoya, López, Cruz, López, Cadena, Cancino and Liedo2017). However, A. obliqua has generated physiological defense strategies against parasitoid development that may be the result of a co-evolutionary process (Silva et al., Reference Silva, Boleli and Simões2002). These authors have reported a wide variety of hemocytes characterized as generators of resistance to parasitoid development inside host larva. Probably, due to this fact, some native and sympatric parasitoid species of Anastrepha spp., such as Doryctobracon crawfordi (Viereck) and Opius hirtus (Viereck), are not viable to develop within A. obliqua larvae, although they do so in others Anastrepha species (Poncio et al., Reference Poncio, Montoya, Cancino and Nava2016).
Taking into account the aforementioned evidence of high physiological resistance in A. obliqua, the expectations of its use as a host in parasitoid rearing would face a serious problem. The low level of parasitoid emerged from A. obliqua puparia under laboratory conditions is an indicator that weakens the objective of proposing it as a host for parasitoid mass rearing (Eben et al., Reference Eben, Benrey, Sivinski and Aluja2000; Cancino et al., Reference Cancino, Ruiz, Lopez and Sivinski2009). However, the use of nuclear techniques, such as radiation, in tephritid fruit fly hosts can be effective to reduce host immune resistance and analyze the viability for parasitoid mass rearing (Cancino et al., Reference Cancino, Ruiz, Viscarret, Sivinski and Hendrichs2012). It has been proved in both pestiferous fruit flies, Ceratitis capitata (Wied.) and Anastrepha ludens (Loew), that the application of radiation doses favors parasitoid development, and it can even be considered a direct relationship with the increase in radiation dose (Cancino et al., Reference Cancino, Ayala, Ovruski, Rios, López and Hendrichs2020; Suárez et al., Reference Suárez, Buonocore-Biancheri, Sanchez, Cancino, Murúa-Bruna, Bilbao, Molina, Laria and Ovruski-Alderete2020). Radiation dose between 20 and –30 Gy applied to A. obliqua larvae used as hosts for Diachasmimorpha longicaudata (Ashmead), a larval-pupal southeast Asian-native parasitoid, successfully allowed parasitoid development, and also prevented host emergence from non-parasitized larvae (Cancino et al., Reference Cancino, Ruiz, Lopez and Sivinski2009). However, the effect of higher radiation doses is unknown. In view of the above facts, it is undeniable that host radiation for parasitoid mass rearing is relevant and its use is intensifying. Therefore, assays were carried out to determine the best radiation dose (>20–30 Gy) to expose irradiated A. obliqua larvae of the most suitable age to D. crawfordi, Utetes anastrephae (Viereck), and D. longicaudata females to achieve maximum levels of parasitoid mass production with good quality individuals. So, mass production quality parameters such as larval host weight, host mortality, parasitoid emergence, and parasitoid offspring sex ratio were estimated. In addition, the number of scars in host puparia, the superparasitism, and the presence of melanization in parasitoid larvae were also assessed. The Neotropical D. crawfordi is not able to develop successfully in A. obliqua larvae in nature (Poncio et al., Reference Poncio, Montoya, Cancino and Nava2016), while the other native parasitoid U. anastrephae is closely associated with A. obliqua larvae in wild environments (Sivinski et al., Reference Sivinski, Aluja and López1997). On the contrary, the exotic D. longicaudata, which is a generalist parasitoid, successfully attacks larvae of different Anastrepha species (Montoya et al., Reference Montoya, López, Cruz, López, Cadena, Cancino and Liedo2017). For these reasons, it was hypothesized that increasing radiation dose under rearing conditions will reduce the A. obliqua larvae immune response, which will increase the parasitoid yield. The results are of great interest to use A. obliqua larvae for parasitoid mass production and its subsequent use in parasitoid mass releases under open-field conditions.
Materials and methods
Insect source
Samples of D. crawfordi (<350 generations), U. anastrephae (<300 generations), and D. longicaudata (<550 generations) were taken from parasitoid colonies kept under mass-rearing conditions at the Biological Control Department of the Moscafrut Plant. Adults parasitoids were provided with honey and water ad libitum and were kept at 24 ± 1°C; 65 ± 5% RH; and 12:12 h L:D. Ages from 5- to 8-day old A. obliqua larvae were used as parasitoid hosts. The age of the larva in days was determined according to the initial egg date sown in the diet. The host larvae were mass-reared on a corn-based diet containing corncob fractions (15%), torula yeast (5.83%), cornflour (8%), sugar (8.33%), guar gum (0.10%), sodium benzoate (0.23%), methyl p-hydroxybenzoate (0.11%), citric acid (0.63%), and water (61.77%) and kept under mass-production standard procedure at the Moscafrut Plant (Artiaga-López et al., Reference Artiaga-López, Hernández, Domínguez-Gordillo, Moreno and Orozco-Dávila2002). Batches of host larvae with fly emergence percentages <90 were discarded and not used in the tests.
Parasitoid emergence and parasitoid offspring sex ratio
Tests were accomplished to achieve high parasitoid emergences with a female-biased sex ratio under an optimal radiation dose and a suitable host larval age for parasitoid mass rearing using A. obliqua as the host. Samples of 5-, 6-, 7, and 8-day-old A. obliqua larvae were taken from the mass-rearing larval diet. The diet was removed from the host larvae by washing them with fresh water. Five samples of 100 larvae for each age were individually placed in 7.5 × 4 cm2 (diameter by height) cylindrical plastic containers. Each sample of the larval host age was respectively exposed to gamma radiation at 40, 80, 120, and 160 Gy in a 127 Gamma Beam panoramic Irradiator (Nordion®, Ottawa, ON, Canada) with a cobalt 60 dry storing source at a rate of 4.60 Gy min−1 and 22°C. The irradiated larvae were placed again into containers with a rearing diet. Non-irradiated larvae were also included in the study as a control test (0 Gy). For each treatment, equivalent to a certain irradiation dose and larval host age, 100 A. obliqua larvae mixed with a rearing diet were individually exposed to 30 mated, 5–10-day-old D. crawfordi, U. anastrephae, and D. longicaudata females, all with previous oviposition experience. The larvae were exposed to parasitoids inside a 10 × 1.5 cm2 (diameter by height), the base of Petri dish which was covered with organdy screen, fastened with an elastic band. This was introduced into a cubical wooden structure cage (27 × 27 × 27 cm3) covered with a plastic screen. A ratio of 3.3:1, 100 hosts/30 parasitoid females ratio was used. The host exposure time was 60 min; after exposure, the host larvae were kept on the rearing diet according to their corresponding treatment in a 7.5 × 4 cm2 (diameter by height) cylindrical plastic container with a lid. When host larvae were aged 9-day-old, they were washed with fresh water only to eliminate the rearing diet. Subsequently, the parasitized larvae were returned to the container with vermiculite at the bottom ready for pupation. The treatments continued in the containers at 26°C and 60–80% RH for 15 days until U. anastrephae and D. longicaudata adult emergence, and for 20 days until D. crawfordi emergence. Each treatment was replicated ten times. For data assessment, the parasitoid emergences and the parasitoid offspring sex ratio were estimated. Parasitoid emergence was calculated as the number of emerged adults divided by the total number of offered pupae × 100, while offspring sex ratio was calculated as the fraction of daughter over son parasitoid offspring.
Effect of radiation doses on A. obliqua larvae
Larval host weight and host mortality were evaluated to know the effects of higher radiation doses on these two quality parameters. The larval host weight was determined from a 100-larva sample by treatment on a semi-analytical scale (PIONEER PA512C, OHAUS® Ohaus Corporation, Parsippany, NJ, USA). The host mortality was recorded 72 h after A. obliqua larvae exposed to parasitoids were placed inside pupation medium. Dead host larvae were counted and removed from each container. Each treatment was replicated ten times.
Scars, parasitoid first instar, and melanization levels
Tests were carried out to determine the effect of both radiation doses and larval host age on the number of scars in the host's puparia, superparasitism, and the level of melanization in parasitoid larvae. For this, 3–4-day-old pupae coming from 5-, 6-, 7-, and 8-day-old A. obliqua larvae exposed to parasitoids were dissected under a stereoscopic microscope (Discovery V8, Carl Zeiss® Gottingen, Germany). Ten pupae from each treatment were dissected on the base of a Petri dish by using dissecting needles. Before dissection, the number of oviposition scars on the surface of the puparium was counted, which represent oviposition attempts or oviposition performed on the host larva (Montoya et al., Reference Montoya, Liedo, Benrey, Barrera, Cancino and Aluja2000a). After that, each puparium was opened, and the first instar number of the corresponding parasitoid species was recorded. The presence of more than one parasitoid first instar larva within the host's body was considered a superparasitized host (Montoya et al., Reference Montoya, Cancino, Pérez-Lachaud and Liedo2011). The first instar of D. crawfordi, U. anastrephae, and D. longicaudata was determined as described by Miranda et al. (Reference Miranda, Sivinski, Rull, Cicero and Aluja2015), Aluja et al. (Reference Aluja, Ovruski, Sivinski, Córdoba-García, Schliserman, Núñez-Campero and Ordano2013), and Ibrahim et al. (Reference Ibrahim, Palacio and Androhani1994), respectively. The size and shape of both mandibles and cephalic heads were used as basic features to identify the first instar parasitoid. In addition, the degree of melanization was also evaluated for those encapsulated parasitoid larvae. A classification criterion was applied regarding the presence of melanization in parasitoid larvae, ranging from one to ten according to the increasing amount of melanin covering the parasitoid body, a qualitative indicator of host immunological reactions (Suárez et al., Reference Suárez, Buonocore-Biancheri, Sanchez, Cancino, Murúa-Bruna, Bilbao, Molina, Laria and Ovruski-Alderete2020). Each treatment was repeated ten times.
Data analysis
A generalized linear model with a normal distribution for the analysis of parameters was used. The analysis was performed under a bifactorial design, where the radiation dose and the larval host age were used as fixed factors. Each species of the parasitoid was analyzed individually. Mean comparisons were analyzed by Tukey's honestly significant difference (HSD) test at P = 0.05. The means of emergence and sex ratio in D. crawfordi were compared applying the Bonferroni adjustment to avoid statistical problems with zero values. The qualitative data of melanization were analyzed by a non-parametric robust analysis of variance (ANOVA). The comparison of the means was performed with the least significant difference test. JMP software, version 11 (SAS Institute, 2013) (JMP®SAS Institute, Inc.) and R version 3.6.1 (R Core Team, 2020) were used for the different analyses.
Results
Parasitoid emergence and parasitoid offspring sex ratio
Both radiation doses and larval host age, and their interaction had a significant effect on the parasitoid emergence in the three parasitoid species (table 1). The younger host larvae (5-, 6-, and 7-day-old) irradiated at higher doses (120–160 Gy) produced significantly the highest percentages of parasitoid emergence (fig. 1; table 2). There was a significant effect of radiation doses on the sex ratio in D. crawfordi, due to considerable difference with the control (0 Gy) (table 1), since the parasitoid emergence only occurred from irradiated host larvae (table 2). The highest proportion of D. crawfordi female offspring was generated in irradiated youngest larvae (5-day-old) (table 2). The sex ratio in both U. anastrephae and D. longicaudata was not significantly affected by the variation in radiation dose, but larval host age had a significant effect (table 1). The highest proportion of U. anastrephae female offspring was produced in 6-day-old larvae, while in D. longicaudata was slightly higher in older larvae (table 2). However, the interaction between both radiation doses and larval host age on sex ratio was not significant in the three parasitoid species (table 1).
Figure 1. Percentages of parasitoid emergence (±SE) in 5-, 6-, 7-, and 8-day-old A. obliqua host larva irradiated at different doses and parasitized by D. crawfordi, U. anastrephae, and D. longicaudata.
Table 1. Summary of two-way generalized linear models on the effect of radiation dose (RD) and larval host age (LHA), and their interactions on quality control parameters in D. crawfordi, U. anastrephae, and D. longicaudata reared on A. obliqua larvae
*Significant variables.
a Generalized linear model and multiple means analysis by Tukey's HSD test (α = 0.05).
b Non-parametric robust ANOVA and multiple means analysis by minimal difference significant (P = 0.05).
Table 2. Parasitoid emergence and offspring sex ratio (mean ± SE) recorded from 5-, 6-, 7-, and 8-day-old larvae of A. obliqua irradiated at different gamma-radiation doses and exposed to D. crawfordi, U. anastrephae, and D. longicaudata females
The same lowercase letters indicate no significant differences among columns and the same uppercase letters indicate no significant differences among rows (Tukey's HSD test, P = 0.05). Only different uppercase letters indicate significant differences among columns and rows by factor interactions. The means of parasitoid emergence and sex ratio in D. crawfordi were compared using the Bonferroni adjustment (α/n).
Effect of radiation doses on A. obliqua larvae
Significant effects and interaction of radiation doses with age were found on the weight of larval host exposed to U. anastrephae (table 1). In both D. crawfordi and D. longicaudata, there was no interaction between dose and age in the average weight and mortality of exposed larvae. However, a significant weight difference in larval host age was found in the three evaluated parasitoid species (table 1). Host weight increased significantly in host larvae aged 6–8-day-old (table 3). Interestingly, a significant effect of larval host age on host mortality was found in both D. crawfordi and D. longicaudata, but not in U. anastrephae (table 1). Mortality was significantly higher in younger host larvae (table 3).
Table 3. Larval host weight and host mortality (mean ± SE) tested in 5-, 6-, 7-, and 8-day-old larvae of A. obliqua irradiated at different gamma-radiation doses exposed to D. crawfordi, U. anastrephae, and D. longicaudata females
The same lowercase letters indicate no significant differences among columns and the same uppercase letters indicate no significant differences among rows. Only different uppercase letters indicate significant differences among columns and rows by factor interactions (Tukey's HSD test, P = 0.05).
Scars, parasitoid first instar, and melanization levels
There were no D. crawfordi adults that emerged from non-irradiated A. obliqua larvae, although these host larvae were parasitized. This was checked by the scars in the host's puparia and by the parasitoid's first instar larvae inside hosts. There was no significant relationship between radiation dose, larval host's age, and their interaction as a function of both the number of scars and D. crawfordi first instar (table 1). Superparasitism in A. obliqua larvae by D. crawfordi was infrequent. The means did not exceed 1.3 first instar parasitoid larvae per dissected host puparium (table 4). Significant effects of radiation doses, larval host's age, and their interaction were found on the number of scars in the host's puparia parasitized by U. anastrephae (table 1). A significantly higher number of scars was found with low radiation doses (0–80 Gy) in host puparia from 8-day-old larvae (table 4). There was no significant effect of increasing radiation doses and their interaction with the larval host's age on the presence of parasitoid first instars larvae per host larvae (table 1). However, the number of parasitoid first instar was significantly influenced by the larval host's age (table 1). The highest superparasitism level was recorded in 7-day-old A. obliqua larvae, while the lowest superparasitism value was recorded in 5-day-old host larvae (table 4). The superparasitism caused by U. anastrephae was quite low (table 4). Similarly, increasing radiation doses did not significantly influence the number of scars caused by D. longicaudata (table 1). Nevertheless, a significant decrease in the number of scars was found in 8-day-old host larvae compared to 5-day-old larvae (table 4). Significant effects of radiation dose and larval host age were found on the number of the parasitoid in the first instar, but the interaction between both fixed factors was not significant (table 1). An increase in the first instar per host puparium occurred at the highest radiation dose evaluated (160 Gy). A greater number of first instar parasitoid larvae was found in host puparia from 5- and 7-day-old larvae (table 3). Unlike the other two Neotropical species, superparasitism was more frequent in D. longicaudata (table 4).
Table 4. Scars on host puparia and first instar larvae of parasitoid (mean ± SE) found in host puparia coming from 5-, 6-, 7-, and 8-day-old A. obliqua larvae irradiated at different gamma-radiation doses and exposed to D. crawfordi, U. anastrephae, and D. longicaudata females
The same lowercase letters indicate no significant differences among columns and the same uppercase letters indicate no significant differences among rows. Only different uppercase letters indicate significant differences among columns and rows by factor interactions (Tukey's HSD test, P = 0.05).
The melanization decreased in the younger larvae at higher doses of radiation when the larva was parasitized by D. crawfordi. The age of larva and doses of radiation had a similar influence on the percentage of melanization obtained in parasitized larvae by both U. anastrephae and D. longicaudata, although there was no interaction (table 1). Non-irradiated and the oldest larva had the highest percentages of melanization. The decrease of melanization was not clear in older larvae when it was parasitized by U. anastrephae (fig. 2).
Figure 2. Percentages of melanization (±SE) in 5-, 6-, 7-, and 8-day-old A. obliqua host larva irradiated at different doses and parasitized by D. crawfordi, U. anastrephae, and D. longicaudata.
Discussion
An optimal host radiation dose is essential to improve the standard operational capacity of the fruit fly parasitoid mass production in augmentative biological control programs. In this regard, the use of well-implemented radiation provides multiple practical benefits, such as fly emergence inhibitions, good quality of host larvae used for parasitoid rearing, and an increased parasitoid adult emergence (Cancino et al., Reference Cancino, Ruiz, Viscarret, Sivinski and Hendrichs2012). The trials of this study showed that younger A. obliqua larvae (5–6-day-old) irradiated at higher radiation doses (120 and 160 Gy) are mainly suitable to produce D. crawfordi, U. anastrephae, and D. longicaudata under mass-rearing conditions. Predictably, high gamma-radiation doses did not affect the host larva quality or health. Only the weight of host larvae used for U. anastrephae had an interaction between the age and the radiation dose, which could be part of the close coevolutive relationship between the host–parasitoid (Marsaro Júnior et al., Reference Marsaro Júnior, Adaime, Ronchi-Teles, Lima and Pereira2011; Jesus-Barros et al., Reference Jesus-Barros, Adaime, Oliveira, Silva, Costa-Neto and Souza-Filho2012; Murillo et al., Reference Murillo, Cabrera-Mireles, Barrera, Liedo and Montoya2015). However, in general, the relevant quality parameters in radiated A. obliqua larvae, such as larval host weight and host mortality (Orozco-Dávila et al., Reference Orozco-Dávila, Artiaga-López, Hernández, Domínguez and Hernández2014), were not affected regarding the non-irradiated larvae. Similarly, the normal development of the parasitoid within the host was not affected by the use of higher radiation doses.
Increased parasitoid emergence could be achieved with radiation doses higher than 160 Gy. However, it is advisable to assess the effects of radiation exposure time and crowding effects on the host larvae. Particularly, parasitoid emergences in the three evaluated parasitoid species were enhanced and the sex ratio was slightly female-biased. The sex ratio was related to the host age in a different way for each species. More female offspring were recovered from young host larvae in the two native braconid species (5-day-old for D. crawfordi and 6-day-old for U. anastrephae). Koinobiont parasitoids usually deposit female eggs on young hosts for two reasons: to avoid a stronger antagonistic reaction from older hosts and to provide suitable nutrition to parasitoid larval instars inside the host. This could be considered an adaptive strategy (Vinson and Iwantsch, Reference Vinson and Iwantsch1980; Hu and Vinson, Reference Hu and Vinson2000; Kaeslin et al., Reference Kaeslin, Reinhard, Bühler, Roth, Wilhelm and Lanzrein2010), displayed by both Neotropical braconids. Even though D. longicaudata is a kind of koinobiont, it could have another strategy, taking into account its status as an exotic species that established a new trophic association with A. obliqua. Therefore, D. longicaudata could attack older larvae by avoiding host immune reactions through oviposition of eggs with entomopoxvirus (Lawrence, Reference Lawrence2005).
Interestingly, the results showed that D. crawfordi has been able to survive and develop successfully inside irradiated A. obliqua larvae, even at 40 Gy. Lower radiation doses (<20 Gy) were not effective. However, this Neotropical parasitoid was unable to develop into non-irradiated A. obliqua larvae even though they have been parasitized. Parasitoid emergence and biased female sex ratio were linked to radiation doses, which could be used as indicators of a decrease in antagonistic reactions of A. obliqua larvae (Strand and Pech, Reference Strand and Pech1995; Reed et al., Reference Reed, Luhring, StaVord, Hansen, Millar, Hanks and Paine2007; Xu et al., Reference Xu, Yang, Lin, Zang, Tian and Ruan2016). In that sense, it is important to note that the host's immune system is weakened by radiation (Hendrichs et al., Reference Hendrichs, Bloem, Hoch, Carpenter, Greany and Robinson2009), which reduces its ability to suppress parasitoid development (Al khalaf and Abdel Baki, Reference Al khalaf and Abdel Baki2013; Sang et al., Reference Sang, Yu, He, Ma, Zhu, Zhu, Wang and Lei2016). This fact was verified with D. crawfordi adult emergence from irradiated A. obliqua larvae since as expressed by Poncio et al. (Reference Poncio, Montoya, Cancino and Nava2016) A. obliqua is not a suitable host for D. crawfordi in nature despite the sympatric coexistence between them. Furthermore, the larval host's age is another critical factor, which adversely affects parasitoid emergence. The host's immune system usually strengthens as the larvae grow older, providing greater protection against parasitoid development (Hegazi and Khafagi, Reference Hegazi and Khafagi2008; Beckage, Reference Beckage, Resh and Cardé2009) and in turn, a remarkable reduction in host mortality in Anastrepha species mass rearing (Orozco-Dávila et al., Reference Orozco-Dávila, Quintero, Hernández, Solís, Artiaga, Hernández, Ortega and Montoya2017). Younger host larvae are usually more sensitive to handling under rearing conditions, and also are more vulnerable to the parasitoid attack due to the early stage of development of the host's immune system (Sisterson and Averill, Reference Sisterson and Averill2003; Ideo et al., Reference Ideo, Watada, Mitsui and Kimura2008). Data from trials of the present study verified this assertion, being that both the mortality and the parasitism percentages were appreciably higher in the youngest host larvae compared to the older larvae.
Superparasitism in A. obliqua larvae in both D. crawfordi and U. anastrephae was scarce; apparently, superparasitism is not a very usual strategy in Neotropical Anastrepha parasitoid species (Ayala et al., Reference Ayala, Pérez-Lachaud, Toledo, Liedo and Montoya2018). Although the number of oviposition scars recorded in the host puparia parasitized by U. anastrephae was higher at low doses in 7–8-day-old larvae, there was no consistency with the parasitoid first instar number. The first instar larva recorded was often in 7-day-old host larva. There is not sufficient information about the foraging and host selection in U. anastrephae. The low superparasitism level in the native U. anastrephae may indicate that it is not necessarily a survival mechanism, as is the case of introduced parasitoid species (Vinson and Iwantsch, Reference Vinson and Iwantsch1980; Kraaijeveld et al., Reference Kraaijeveld, Van Alphen and Godfray2011). Utetes anastrephae has a sympatric co-evolutionary relationship with A. obliqua, which would justify the low superparasitism in larvae of this tephritid. In contrast, superparasitism in D. longicaudata was a lot more frequent and constant than in both Neotropical parasitoid species, but with a greater preference in A. obliqua larvae irradiated at high doses. This introduced braconid parasitoid has adapted to Anastrepha spp. larvae in nature (Montoya et al., Reference Montoya, López, Cruz, López, Cadena, Cancino and Liedo2017), and optimizes their resources (host larvae) by using superparasitism as an effective survival mechanism (Montoya et al., Reference Montoya, Liedo, Benrey, Barrera, Cancino, Sivinski and Aluja2000b, Reference Montoya, Pérez-Lachaud and Liedo2012; González et al., Reference González, Montoya, Perez-Lachaud, Cancino and Liedo2007). Superparasitize host larvae increased the probability of survival of one D. longicaudata individual per host after high intraspecific competitive activity in the first instar (Montoya et al., Reference Montoya, Cancino, Pérez-Lachaud and Liedo2011).
The presence of melanin in parasitized host larvae may be a practical qualitative indicator of the host immune reactions (Nappi and Vass, Reference Nappi and Vass1993; Suárez et al., Reference Suárez, Buonocore-Biancheri, Sanchez, Cancino, Murúa-Bruna, Bilbao, Molina, Laria and Ovruski-Alderete2020). Melanin is the result of enzymatic activity from phenoloxidase as the host's cellular and humoral reaction to parasitization (Boman and Hultmark, Reference Boman and Hultmark1987; Nappi and Ottaviani, Reference Nappi and Ottaviani2000; Liu et al., Reference Liu, Jiravanichpaisal, Cerenius, Lee, Söderhäll and Söderhäll2007). The gradual melanin level depletion is a relevant indicator of the adverse effect caused by increased radiation. Radiation may induce physiological changes involving reduced phenoloxidase action in tephritid larvae. This was reported in larvae of Anastrepha suspensa (Loew) (Nation et al., Reference Nation, Smittle and Milne1995), C. capitata (Mansour and Franz, Reference Mansour and Franz1996), and Bactrocera dorsalis (Hendel) (Chang et al., Reference Chang, Goodman, Ringbauer, Geib and Stanley2016). Nevertheless, the qualitative determination of melanization is not feasible in all Anastrepha species, because observations in A. ludens larvae did not provide evidence of melanin in both D. crawfordi and U. anastrephae eggs and larvae as a signal of the host's immunological reaction (Cancino et al., Reference Cancino, Ayala, Ovruski, Rios, López and Hendrichs2020). The immunological reactions of A. obliqua larvae could be related to a more forceful way to parasitism, considering that larval development occurs mainly inside small host fruits, which have soft pulp, thin skin, and large seeds, such as native Spondias (Anacardiaceae) species. These host fruit species have highly favorable conditions for parasitoids to find and oviposit A. obliqua larvae (Ovruski et al., Reference Ovruski, Aluja, Sivinski and Wharton2000; Sivinski et al., Reference Sivinski, Piñero and Aluja2000). Finally, the host's immune response capacity may decline due to increased radiation, which is of considerable significance to be applied as an alternative rearing procedure in different parasitoid–host relationships that are not viable under lab conditions (Hoffmann et al., Reference Hoffmann, Ode, Walker, Gardner, van Nouhuys and Shelton2001; Muhammad et al., Reference Muhammad, Ahmad, Rashidi and Ahmad2013; Hasan et al., Reference Hasan, Yeasmin, Athanassiou, Bari and Islam2019). Similarly, lab-reared parasitoid production levels are low due to the immunological action of the artificially reared host, which could be avoided using radiation (Consoli et al., Reference Consoli, Parra and Vinson2000; Hasan et al., Reference Hasan, Yeasmin, Athanassiou, Bari and Islam2019).
In summary, these findings identified both suitable radiation doses and host's ages of A. obliqua larvae under mass-rearing conditions, which enable its subsequent use in D. longicaudata, U. anastrephae, and D. crawfordi rearing to substantially improve parasitoid mass production. In this regard, it is noteworthy that the main objective is focused on augmentative biological control through parasitoid mass releases. However, and from a practical viewpoint related to open-field parasitoid releases against A. obliqua wild populations, only two parasitoid species of the three species studied, U. anastrephae and D. longicaudata, may be massively released in marginal areas surrounding commercial orchards, such as backyard orchards and wild vegetation areas. This is because D. crawfordi has a very low natural emergence rate from A. obliqua puparia (Sivinski et al., Reference Sivinski, Aluja and López1997). Authors such as Córdova-García (Reference Córdova-García2008) and Poncio et al. (Reference Poncio, Montoya, Cancino and Nava2016) have reported that D. crawfordi larva development is harshly affected by the immunological defenses of A. obliqua larva under natural and lab conditions. Doryctobracon crawfordi would be more associated with A. ludens in nature (Miranda et al., Reference Miranda, Sivinski, Rull, Cicero and Aluja2015; Montoya et al., Reference Montoya, López, Cruz, López, Cadena, Cancino and Liedo2017). In contrast, U. anastrephae is closely associated with A. obliqua in the Neotropics (Sivinski et al., Reference Sivinski, Aluja and López1997, Reference Sivinski, Piñero and Aluja2000; López et al., Reference López, Aluja and Sivinski1999), and it has a coevolutionary process since its short ovipositor is adapted to parasitize A. obliqua larvae in small fruits (Sivinski et al., Reference Sivinski, Vulinec and Aluja2001). As mentioned by Aluja et al. (Reference Aluja, Sivinski, Ovruski, Guillén, López, Cancino, Torres-Anaya, Gallegos-Chan and Ruiz2009, Reference Aluja, Sivinski, Van Driesche, Anzures-Dadda and Guillen2014), native vegetation can be managed to conserve and multiply native Anastrepha parasitoids in rural areas where farmers cannot apply expensive pest control and management procedures. Thus, massive releases of species such as U. anastrephae may facilitate such measures. On the other hand, the exotic D. longicaudata is considered a new successful association with Anastrepha (Schiner) in the Neotropics (Ovruski et al., Reference Ovruski, Aluja, Sivinski and Wharton2000; Cancino et al., Reference Cancino, Ruiz, Gómez and Toledo2002; Montoya et al., Reference Montoya, López, Cruz, López, Cadena, Cancino and Liedo2017; Garcia et al., Reference Garcia, Ovruski, Suárez, Cancino and Liburd2020). It has previously been used against A. obliqua in mango orchards in Chiapas, Mexico, through augmentative releases, and their permanent establishment in this region was confirmed (Montoya et al., Reference Montoya, Liedo, Benrey, Barrera, Cancino, Sivinski and Aluja2000b). Consequently, it is a valuable parasitoid species for use in Anastrepha biological control (Montoya et al., Reference Montoya, López, Cruz, López, Cadena, Cancino and Liedo2017).
Acknowledgements
Financial support was provided by the International Atomic Energy Agency (IAEA) – Research Contract No. 20562 ‘Use of Radiation to Reduce Host Antagonism Reaction to Fruit Fly Parasitosis Attacks’. We appreciate a lot the technical soport received from the staff of the Departamento de Control Biológico and the Departamento de Cría Masiva de A. obliqua of Moscafrut Program.
