Introduction
Fruit flies (Diptera: Tephritidae) are considered a threat to fruit crops worldwide due to direct damage to fruit quality and indirect damage caused by quarantine barriers imposed by importing countries (Oliveira et al., Reference Oliveira, Fonseca, Alvarenga, Giustolin, Rabelo and Coutinho2014; Ruiz et al., Reference Ruiz, Juárez, Alzogaray, Arrighi, Arroyo, Gastaminza, Willink, Bardón and Vera2014). The most widely distributed and economically damaging fruit fly species in Brazil belong to the genera Ceratitis and Anastrepha (Oliveira et al., Reference Oliveira, Fonseca, Alvarenga, Giustolin, Rabelo and Coutinho2014). Infestation of fruit flies (Diptera: Tephritidae) is one of the factors that affects fruit health, the injuries caused by infestation of fruit flies may impair fruit quality for consumption and commercial sale (Louzeiro et al., Reference Louzeiro, Miguel Filho, Adalton and Flávio2020).
Damage caused by larvae undergoing their entire developmental cycle occurs inside the fruit’s pulp render fruits unmarketable (Ghabbari and Jemâa, Reference Ghabbari and Jemâa2024). Puncture and oviposition of fruit flies, and larvae feeding can lead to fruit drop (Umeh et al., Reference Umeh, Olaniyan, Ker and Andir2004; Ghabbari and Jemâa, Reference Ghabbari and Jemâa2024), accelerated ripening (Jayanthi et al., Reference Jayanthi, Kempraj, Ravindra and Verghese2015), changes in fruit peel colour (Jayanthi et al., Reference Jayanthi, Kempraj, Ravindra and Verghese2015), and pulp deterioration (Jayanthi et al., Reference Jayanthi, Kempraj, Ravindra and Verghese2015; Omoloye et al., Reference Omoloye, Oladapo, Ibitoye and Alabi2016). Mature attacked fruits may develop a water soaked appearance, causing a depression on the surface (Ghabbari and Jemâa, Reference Ghabbari and Jemâa2024).
In Brazil, the genus Ceratitis is represented only by the Mediterranean fly Ceratitis capitata (Wiedemann), which was introduced in 1901 in the state of São Paulo and is currently widely distributed in most Brazilian states. This species has been most frequently recorded in exotic fruits; however, since its introduction, it has shown great ability to adapt to several new exotic fruits and to both cultivated and non-cultivated native hosts (Carvalho et al., Reference Carvalho, Nascimento and Matrangolo1998; Zucchi and Moraes, Reference Zucchi and Moraes2012; Leite et al., Reference Leite, Castellani, Ribeiro, Costa, Bittencourt and Moreira2017; Sá et al., Reference Sá, Oliveira, Oliveira, Santos, Moreira and Castellani2019).
Their management relies primarily on chemical insecticides (Li et al., Reference Li, Cai, Qi, Lu and Li2024), mainly neurotoxic compounds (i.e. organophosphates, carbamates, and pyrethroids), applied through sprays or toxic baits (Raga and Sato, Reference Raga and Sato2016). However, despite advances in the techniques and products used in chemical control, consumers of fresh fruits have demanded residue-free or chemical-free fruits in the production chain. Furthermore, pesticide resistance has been reported in several fruit fly species (Li et al., Reference Li, Cai, Qi, Lu and Li2024). Thus, efforts have been made to gradually replace conventional chemical control with alternatives that have lower impact on the environment and consumers, such as biological control, which can also be used with other fruit fly management strategies (Montoya et al., Reference Montoya, Liedo, Benrey, Cancino, Barrera, Sivinski and Aluja2000).
Biological control is one of the most promising alternatives to chemical control in pest management, and one of the predominant approaches augmentative biological control insect pests (Hegazi et al., Reference Hegazi, Khafagi, Herz, Konstantopoulou, Hassan, Agamy and Shweil2012). Species of the family Braconidae (Opiinae) are the most abundant and diverse parasitoids that can be used in the control of fruit flies (Guimarães et al., Reference Guimarães, Gallardo, Diaz, Roberto and Zucchi2003; Garcia et al., Reference Garcia, Ovruski, Suárez, Cancino and Liburd2020). The exotic parasitoid Diachasmimorpha longicaudata (Ashmead) (Hymenoptera: Braconidae) parasitises at least 12 tephritid species and is one of the most widely distributed in the Americas (Stibick, Reference Stibick2004), and currently used in Fruit Fly Control and Eradication Program in Argentina (Suárez et al., Reference Suárez, Biancheri, Murúa, Bilbao, García, Cancino, Martín, Molina, Laría and Ovruski2019; Carta Gadea et al., Reference Carta Gadea, Suárez, Biancheri, Murúa, Molina, Laria and Ovruski2020).
Diachasmimorpha longicaudata is a solitary koinobiont endoparasitoid from the Indo-Australian region (Wharton and Gilstrap, Reference Wharton and Gilstrap1983) and is used in the biological control of tephritids worldwide (Garcia et al., Reference Garcia, Ovruski, Suárez, Cancino and Liburd2020). Diachasmimorpha longicaudata is important because of its ease of rearing in the laboratory and its ability to adapt quickly to different environments (Ovruski et al., Reference Ovruski, Bezdjian, Van Nieuwenhove, Albornoz-Medina and Schliserman2011). Furthermore, the species generalists are effective in tephritid control including species of the genera Anastrepha, Ceratitis, and Bactrocera (Paranhos et al., Reference Paranhos, Nava and Malavasi2019).
The success of biological control programs depends on basic research on natural enemies, which must be bred and released in large quantities in the field. In Argentina, the use of the parasitoid D. longicaudata has shown to be a great suppression strategy against medfly in fruit orchards (Sánchez et al., Reference Sánchez, Murúa, Suárez, Van Nieuwenhove, Taret, Pantano, Bilbao, Schliserman and Ovruski2016; Suárez et al., Reference Suárez, Biancheri, Murúa, Bilbao, García, Cancino, Martín, Molina, Laría and Ovruski2019, Reference Suárez, Biancheri, Murúa, Ordano, Wang, Cancino, Garcia, Sánchez, Beltrachini, Kulicheysky and Ovruski2023). In Guatemala, the release of D. longicaudata in coffee plantations to control C. capitata significantly reduced the pest population (Cancino et al., Reference Cancino, Ruiz, López, Aguilar, Galvez, Montoya and Liedo2019).
To ensure the successful mass production of D. longicaudata, high-quality host larvae are indispensable (Oliveira et al., Reference Oliveira, Fonseca, Alvarenga, Giustolin, Rabelo and Coutinho2014). In addition to the quality of the host larvae, the number of the larvae (Silva et al., Reference Silva, Bento and Zucchi2007; Cruz et al., Reference Cruz, Alvarenga, Oliveira, Conceição, Santos, Teresinha Augusta Giustolin and Souza2018; Altafini et al., Reference Altafini, Redaelli, Jahnke and Efron2019) and the duration of exposure to parasitism (Altafini et al., Reference Altafini, Redaelli, Jahnke and Efron2019) are important factors in successful breeding. Suárez et al. (Reference Suárez, Van Nieuwenhove, Murua, Bezdiian, Schliserman, Lara, Escobar and Ovruski2012) indicated that the use of larvae of Vienna-8 C. capitata strain at 40 min exposure time significantly increased overall D. longicaudata offspring production. However, Suárez et al. (Reference Suárez, Biancheri, Murúa, Bilbao, García, Cancino, Martín, Molina, Laría and Ovruski2019) and Carta Gadea et al. (Reference Carta Gadea, Suárez, Biancheri, Murúa, Molina, Laria and Ovruski2020) observed that an exposure time of 90 min favoured a greater emergence of D. longicaudata.
Therefore, studies such as the one, carried out in small-scale insect farming, can provide information on the behaviour of parasitoids in different species of tephritids (Rohr et al., Reference Rohr, Jahnke and Redaelli2019) and conditions for parasitism. In addition, they can potentially promote cost reduction efforts in the mass production of parasitoids for use in applied biological control (Camargos et al., Reference Camargos, Zamboni Costa and Miranda2017), among other topics that may help in the establishment of fruit fly management strategies. Therefore, the objectives of this study were to determine the exposure time, density, and ideal larval instar of C. capitata and to verify whether the D. longicaudata previous experience affects its interaction with the host insect and the influence of these factors on the parasitism rate.
Materials and methods
Diachasmimorpha longicaudata and Ceratitis capitata rearing
Two bioassays were conducted involving the parasitoid D. longicaudata and its host C. capitata. The parasitoids were obtained from the breeding stock established in 2012 at the Fruit Fly Laboratory of the State University of Southwest Bahia (Vitória da Conquista, Bahia, Brazil) using pupae hosts of C. capitata tsl Vienna-8 parasitised by D. longicaudata from the Brazilian Agricultural Research Corporation (Embrapa), Cassava, and Fruticulture Unit (Cruz das Almas, BA, Brazil). The rearing conditions were 25 ± 2°C, 65 ± 10% relative humidity, and a 12 h light/dark cycle, according to the methodology described by Carvalho and Nascimento (Reference Carvalho and Nascimento2002). Parasitism units were prepared by wrapping groups of third-instar larva of C. capitata with voile fabric, secured with elastic material. The larvae were periodically exposed to the parasitoids for 1 h and then placed in 500 mL plastic containers with a thin layer of vermiculite and covered with paper towels until the adults emerged. The emerged parasitoids were placed in acrylic cages (30 × 30 × 30 cm) and fed water and a diet based on honey and agar.
The C. capitata larvae used in the rearing of D. longicaudata and in the bioassays were obtained from a population maintained in the same laboratory for 16 years and from Embrapa Cassava and Fruticulture Unit, which adds periodic collections of wild individuals every year. The adults were kept in acrylic cages (30 × 30 × 30 cm) and were fed daily with a sugar-based diet and Biones® at a 3:1 ratio and water. The eggs were collected daily, disinfected, and placed in plastic containers with an artificial diet (based on brewer’s yeast, soy extract, oat, sugar, sodium benzoate, nipagin, citric acid, and water) for the larvae. The containers were closed and kept at a temperature of 25 ± 2°C until the larvae reached the adult stage. The adults were then released into the cages, where the cycle was restarted.
In all bioassays, previously mated 8-day-old D. longicaudata females were used (Rohr et al., Reference Rohr, Jahnke and Redaelli2019). To prevent the presence of males from influencing the behaviour of the females during the evaluation of parasitism in the bioassays, the females were kept in with males for 2 days to ensure mating and removed after this period (Oliveira et al., Reference Oliveira, Fonseca, Alvarenga, Giustolin, Rabelo and Coutinho2014). Similarly, for all bioassays, the host was offered to the female parasitoid in parasitism units of a specified number of C. capitata larvae with a small portion of the larval diet, wrapped in voile fabric and hung from the upper part of the cage. The inclusion of the larval diet in the parasitism unit was necessary to prevent dehydration of the larvae, as larvae without food either die of lack of moisture or enter the pupal stage early.
Density and larval instar of Ceratitis capitata on parasitism by Diachasmimorpha longicaudata
In the first bioassay, the effect of the density and larval instar of C. capitata on parasitism by D. longicaudata was evaluated with eight treatments and eight replicates in a completely randomised 2 × 4 factorial design, with two instars (second 4–5 days and third 6–7 days) and four larval densities (5, 10, 15, and 20), for a total of 64 groups. For each group, a plastic container (cage) with a capacity of 1 L held a parasitism unit consisting of C. capitata larvae of instars and densities corresponding to each treatment affixed to the upper part of the cage.
One female parasitoid, without previous experience of oviposition, was released into each cage and allowed to remain for 2 h. After the exposure period, the larvae were removed from the parasitism unit and allowed to pupate in plastic containers (100 mL) with a thin layer of vermiculite, closed with perforated lids, at a temperature of 25 ± 2°C, a relative humidity of 65 ± 10%, and a light/dark cycle of 12 h.
Time exposure of Ceratitis capitata larva to Diachasmimorpha longicaudata
The second bioassay was performed to determine the appropriate duration of exposure of C. capitata larva to the parasitoid. Two parts were performed: one involving female with previous experience of oviposition and the other involving females with no experience of oviposition. Both (with or without experience of oviposition) were conducted in accordance with a completely randomised design with five treatments consisting of exposure times of 30, 60, 120, 240, and 480 min, and eight replicates, totalling 40 groups. For each group, a plastic container (cage) with a capacity of 1 L held a parasitism unit consisting of 20 third-instar larvae of C. capitata and a small amount of the larval diet wrapped in voile fabric, which was suspended from the upper part of the cage. A total of 800 larvae were used in the bioassay.
Previous experience of oviposition was obtained by introducing female parasitoids to parasitism units composed of third-instar larvae of C. capitata for 3 h, 24 h before the beginning of the bioassays.
One mated female parasitoid (with or without experience in ovipositing) was released into each cage for the designated time period. Subsequently, the larvae were allowed to pupate in plastic containers (100 mL) containing a thin layer of vermiculite, closed with perforated lids, at a temperature of 25 ± 2°C, a relative humidity of 65 ± 10%, and a light/dark period of 12 h.
In both bioassays, the numbers of flies and emerged parasitoids and the parasitism rate and sex ratio were quantified according to Carvalho (Reference Carvalho2003):
The parasitism rate was determined by the following equation:
\begin{align*}{\text{Parasitism}} &(\% ) = \\
&\left[ {\frac{{{\text{Number of parasitoids}}}}{{{\text{Number of parasitoids + Number of flies}}}}} \right]{\text{ }} \times {\text{ 100 }}\end{align*}The sex ratio of the parasitoids was determined by the following equation:
\begin{equation*}{\text{Sex ratio = }}\frac{{{\text{Number of females}}}}{{{\text{Number of females + Number of males }}}}\end{equation*}Data analysis
For the bioassay that evaluated the effect of the density and larval instar of C. capitata on parasitism by D. longicaudata, the data were analysed using a linear mixed effects model (LMER) in R version 3.5.4. To fit the data to a normal distribution, the number of flies was transformed into 
$\sqrt {{\text{(x)}}} $. In the regression of the relationship of parasitism by D. longicaudata as a function of larval density in LMER, the models were fitted using parameter estimation via the restricted maximum likelihood, and the coefficient of determination (R 2) was estimated according to the method described by Nakagawa and Schielzeth (Reference Nakagawa and Schielzeth2013) for linear mixed-effect models.
The data obtained from the bioassay evaluating the exposure time of females of D. longicaudata with and without previous experience of oviposition were analysed using a generalised linear model (GLM) with the ‘lmer’ function of the ‘lme4’ package (Bates et al., Reference Bates, Mächler, Bolker and Walker2015), executed in R version 3.5.2 (R Development Core Team. R, 2019). The Poisson distribution was used for the number of flies, and the normal (Gaussian) distribution was used for the sex ratio and parasitism rate. The means were compared using the ‘lsmeans’ package (Lenth, Reference Lenth2016). The relationship between parasitism D. longicaudata and the exposure time of the larvae was tested using the curve-fitting regression analysis procedure with the 2D Table Curve (Systat, San Jose, CA, USA). The fitted model was chosen based on the principle of parsimony, with high F and R 2 values.
Results
Density and larval instar of Ceratitis capitata on parasitism by Diachasmimorpha longicaudata
The larval density of C. capitata in the parasitism units of third-instar larva had significant effects on the number of emerged flies (R 2 = 0.99, P = 0.01), on the number of parasitoids (R 2 = 0.99, P < 0.0001), and on the parasitism rate (R 2 = 0.81, P = 0.03), while the density of second-instar larva affected only the number of parasitoids (R 2 = 0.87, P < 0.02) (fig. 1A–C). Fly emergence followed a quadratic pattern, with the lowest (5 larvae) and highest (20 larvae) densidites resulting in higher fly emergence (fig. 2A). The effect on the number of parasitoids was linear and positive for both larval instars, with parasitoid emergence increasing as a function of the increase in larval density (fig. 2B). The effect of larval density on the parasitism rate followed a quadratic model that was significant only for 3rd instar larva, with increases occurring as a function of larval density tending to stabilise between 15 and 20 larvae (fig. 1C).
Numerical relationship between the larval density of Ceratitis capitata (5, 10, 15, and 20) in the parasitism units and the number of emerged flies (A), number of parasitoids (B), and parasitism rate (C) of D. Longicaudata in second- and third-instar larvae.
Number of Ceratitis capitata fruit flies that emerged (A), number of parasitoids (B), and parasitism rate (C) of D. longicaudata in relation to the second- and third-instar larvae. The bars represent the mean (±SD). Bars followed by the same letter are not significantly different at p < 0.05 according to the Tukey test (adjusted model: LMER, confidence level adjustment by the Šidák method).
Regardless of larval density, the third instar had the highest number of emerged parasitoids (F = 6.82, P = 0.01), but there were no significant differences between instars in terms of parasitoid number or parasitism rate (F = 0.82, P > 0.05; F = 1.54, P > 0.05, respectively) (fig. 2A–C). Sex ratio values above 0.5 were obtained only when 10, 15, and 20 third-instar larva were used (table 1).
Mean sex ratio of Diachasmimorpha longicaudata as a function of density and larval instar of Ceratitis capitata
Time exposure of Ceratitis capitata larva to Diachasmimorpha longicaudata
The exposure time of C. capitata larva in the parasitism units to female parasitoid D. longicaudata with and without experience of oviposition significantly affected the number of flies that emerged (R 2 = 0.77, F = 10.14, P = 0.04; R 2 = 0.96, F = 94.65, P < 0.01, respectively) and the parasitism rate (R 2 = 0.79, F = 11.54, P = 0.04; R 2 = 0.96, F = 77.93, P < 0.01, respectively), following a negative linear model for the number of flies and a positive linear model for the parasitism rate (fig. 3A and B). The highest parasitism rates were obtained at an exposure time of 480 min, resulting in 96.75% and 90.75% parasitism when females with and without experience in oviposition, respectively (fig. 3B).
Relationships between the duration of exposure to Ceratitis capitata larvae and the number of flies emerged (A), the parasitism rate (B), and the sexual ratio (C) of D. longicaudata.
The sex ratio of the parasitoids was affected by the exposure time of C. capitata larvae in the parasitism units only if the female parasitoid lacked previous experience of oviposition (R 2 = 0.93, DF = 1.0, F = 42.45, P < 0.01). At exposure times of 30, 60, 120, and 240 min, more males than females emerged; that is, the sex ratio was lower than 0.5. With 480 min of exposure, the sex ratio of the parasitoids was greater than 0.6 (fig. 4C); that is, more female parasitoids than males emerged.
Number of Ceratitis capitata flies that emerged (A), parasitism rate (%) (B), and sex ratio (C) of parasitism by D. longicaudata with and without previous oviposition experience. The bars correspond to the mean (±SD). Bars followed by the same letter are not significantly different at p < 0.05 according to the Tukey test (adjusted model: GLM, linkage function: logit, confidence level adjustment by the Šidák method).
Previous oviposition experience in D. longicaudata females did not influence the evaluated variables, as no differences were observed in the number of emerged flies, parasitism rate, or sex ratio between experienced and inexperienced parasitoid females.
Discussion
The larval density in the parasitism units and the instar (second or third) of the host larvae influenced the number of parasitoids, the number of emerged flies and the parasitism rate. At 20, third-instar larvae of C. capitata, there was a higher rate of parasitism (above 90.0%), which is an important aspect of the mass breeding of these insects. These results contrast with those obtained by Cruz et al. (Reference Cruz, Alvarenga, Oliveira, Conceição, Santos, Teresinha Augusta Giustolin and Souza2018) because, in the same situation, with 1 female of D. longicaudata and 20 host larvae of the same insect species as in the present study, they found a parasitism rate of 26.82%. The authors varied the number of female parasitoids (1–15) and the number of host larva (10–50) and concluded that an equal number of females parasitoids and host larva resulted in better production efficiency of female parasitoids. A likely explanation for the differences between the results mentioned may be related to the exposure time of the host larvae to the female parasitoids, which in the experiment was between 120 and 240, with did the number of parasitoids increase exponentially, while exposure used in the study by Cruz et al. (Reference Cruz, Alvarenga, Oliveira, Conceição, Santos, Teresinha Augusta Giustolin and Souza2018) was of 60 min. However, Suárez et al. (Reference Suárez, Biancheri, Murúa, Bilbao, García, Cancino, Martín, Molina, Laría and Ovruski2019) and Carta Gadea et al. (Reference Carta Gadea, Suárez, Biancheri, Murúa, Molina, Laria and Ovruski2020) found that within 120 min there was a high proportion of emergence of D. longicaudata adults from C. capitata Vienna-8 larvae. The number emerged flies and parasitism rate varied as expected: as the parasitism rate and number of parasitoids increase, the number of emerged flies decreases.
According to previous studies, the parasitoid D. longicaudata usually parasitises second- and third-instar tephritid larvae (Sivinski et al., Reference Sivinski, Vulinec and Aluja2001; Sime et al., Reference Sime, Daane, Nadel, Funk, Messing, Andrews, Marshall, Johnson and Pickett2006). In the present study, a larger number of parasitoids were produced in third-instar larvae, as reported in some previous studies (Carvalho, Reference Carvalho2005; Ovruski et al., Reference Ovruski, Bezdjian, Van Nieuwenhove, Albornoz-Medina and Schliserman2011; Montoya et al., Reference Montoya, López, Cruz, Cadena, Cancino and Liedo2017). The larval instars of C. capitata (first, second, and third) tested by Rohr et al. (Reference Rohr, Jahnke and Redaelli2019), with no free choice, did not affect the average number of emerged parasitoids, whereas in the free-choice bioassay, the average number of emerged parasitoids was greater for second- and third-instar larvae.
However, according to Paranhos et al. (Reference Paranhos, Walder and Alvarenga2007), under laboratory conditions, the parasitism rate can be influenced by several intrinsic factors (age and quality of the parasitoids produced; sex ratio of parental males and females) and external factors (host:parasitoid ratio, species of fruit host of the fruit flies, age of the host larvae). Exposing C. capitata larvae to the female parasitoid for 480 min (8 h) promoted higher rates of parasitism (above 90.0%), regardless of the condition of the female (with or without previous experience). This result can indicate that increasing the contact time between parasitoid females and host larvae enables higher parasitism. This observation is highly relevant, especially in the mass production of parasitoids for release to control fruit flies.
For females without previous experience of oviposition, the sex ratio was also linearly related to the parasitism rate, indicating that increasing exposure time increases the oviposition of fertilised eggs, resulting in females that are larger and probably more vigorous, favouring female progeny; in contrast, unfertilised eggs, which give rise to males, were laid in second-instar larvae, which are smaller and less vigorous (Oliveira et al., Reference Oliveira, Fonseca, Alvarenga, Giustolin, Rabelo and Coutinho2014). The increase in the sex ratio with increasing exposure time of the larvae to the parasitoid was expected since leaving the female parasitoid confined for a longer time with the host larvae might have allowed oviposition in previously parasitised, which would explain the high sex ratio.
Montoya et al. (Reference Montoya, Cacino, Perez-Lachaud and Liedo2011, Reference Montoya, Cancino and Ruiz2012) reported that the sex ratio was correlated with the intensity of superparasitism, and the male-to-female ratio was significantly affected by the intensity of parasitism.
Superparasitism is common in this species: D. longicaudata females not only can identify parasitised hosts but also preferentially superparasitise previously parasitised hosts, although negative effects on progeny demography have not yet been reported (Montoya et al., Reference Montoya, Cancino and Ruiz2012). This result is relevant considering that females lay eggs, promoting the biological control of fruit flies. In the past, superparasitism was considered an unwanted trait and was attributed to the failure to identify an already parasitised host. However, superparasitism is currently understood as an adaptive advantage in which the female increases the chances of offspring survival by laying more fertilised eggs inside the host (González et al., Reference González, Montoya, Pérez-Lachaud, Cancino and Liedo2010). Altafini et al. (Reference Altafini, Redaelli and Janke2013) suggested that the highest superparasitism rates observed were influenced by the size of the host; thus, Anastrepha fraterculus larvae, which are larger than C. capitata larvae, had higher superparasitism rates. However, the hypothesis that the occurrence of superparasitism was related to the sex ratio could not be confirmed in the present study, since the intensity of parasitism in the previous study was assessed by evaluating the oviposition scars left by the females, which were not examined in this study.
The data obtained indicate that providing D. longicaudata females with 180 min of oviposition experience, acquired 24 h before the bioassays, was not enough to affect the results because the differences between the values of flies, parasitism, and sex ratio were not significant (fig. 4). A number of factors affect the behaviour of parasitoids, and this behaviour may be innate or may be modified by experience, resulting in learning (Matthews and Matthews, Reference Matthews and Matthews2010).
An alternative hypothesis is that both categories of females (with and without previous experience of oviposition) respond primarily to the signal of larval food that was common to all parasitism units, resulting in similar parasitism rates. Thus, research that addresses the importance of these tritrophic relationships and the need for more information will contribute to a better understanding of the ecological and evolutionary interactions between plants, herbivores, and natural enemies, which may be highly important in the development of conservationist agriculture, in which insect pests are controlled with reduced impact on the environment (Gonçalves, Reference Gonçalves2015). However, female parasitoids of this species can retain learning about certain volatiles, such as vanilla essential oil, for up to 24 h (Zadra et al., Reference Zadra, Sant’Ana, Radaelli and Tognon2018).
In the present study, previous experience with oviposition did not affect the variables, probably indicating that parasitisation behaviour is innate, i.e. it may be triggered by a signal regardless of experience, which may enable the elimination of one step in mass breeding. In general, the results of the present study indicate that to increase the parasitism rate of D. longicaudata, third-instar larva of C. capitata must be grouped into parasitism units of 20 larvae and exposed to a single female of the parasitoid for 8 h, or to conditions temperature of 25º ± 2°C and the age of the third-instar larva at 7–8 days old.
Acknowledgements
We thank the Foundation for Research Support of the State of Bahia (FAPESB) and the CAPES Foundation (Brazilian Ministry of Education; Finance Code 001) for their financial support.
