Field Cage Assessment of the Neotropical-Native Parasitoid Ganaspis pelleranoi as a Biocontrol Agent of the Invasive Pest Ceratitis capitata

Abstract

Ceratitis capitata (Diptera: Tephritidae), or medfly, is an invasive pest widespread in Argentina, where standardized management methods, such as cultural and chemical controls, are commonly implemented. The success in controlling medfly populations depends on implementing preventive, sustainable, long-term, and eco-friendly eradication/control strategies across all invaded environments. One strategy may involve augmentative biological control using parasitoids adapted to local conditions, such as Ganaspis pelleranoi (Brèthes) (Hymenoptera: Figitidae), a Neotropical-native parasitoid that mostly forages on tephritid larvae in a broad range of fallen fruit. Two hypotheses were tested in the current study: (1) G. pelleranoi females are more efficient in controlling medfly larvae infesting different fruits as the density of released females progressively increases, and (2) such parasitoid-induced host mortality capacity remains when host density is increased. Parasitism (reproductive effects) and additional host mortality (non-reproductive effects) were the indicator variables of parasitoid-induced host ability. Trials were performed in field cages (semi-field conditions) using two medfly-multiplier host fruit species, namely sour orange and peach, and with variations in both parasitoid release and host larval densities. Three major findings were highlighted: (1) G. pelleranoi females successfully parasitized host larvae on peach and sour orange, regardless of their strongly differing physical features, although medfly larvae in peaches were significantly more susceptible to the parasitoid; (2) medfly mortality significantly increased in both peach and sour orange relative to the gradual increase in released G. pelleranoi females, regardless of the increase in host density offered to parasitoids; and (3) G. pelleranoi females induced a substantially high host die-off rate when the additional mortality was added to the analysis, which was not revealed when parasitism alone was regarded as a medfly mortality variable. Such outcomes may provide relevant information for implementing an augmentative biological control against medfly using indigenous parasitoid species within an eco-friendly fruit fly pest management approach.

1. Introduction

Ceratitis capitata (Wiedemann, 1824) (Diptera: Tephritidae), commonly known as the Mediterranean fruit fly or medfly, is one of the major invasive pests affecting a broad range of commercially valuable fruit and vegetable crops worldwide [1,2]. Endemic to sub-Saharan Africa, the medfly has spread to temperate, subtropical, and tropical regions worldwide mainly due to its broad thermal tolerance and polyphagous and multivoltine nature [3,4]. More than 300 fruit species, many of agricultural importance, have been reported as medfly hosts in the world [5,6,7,8]. Medfly can cause direct and indirect damage to the fruit industry. The former involves damage to the skin of the fruit due to the female’s egg-laying and rotting of the fruit pulp caused by the larvae feeding on it [9]. The latter involves quarantine restrictions on the movement of fresh fruit imposed by importing countries [10]. Therefore, the medfly affects both the country’s local economy and its international trade. The first medfly detections in the Neotropical region were in South American countries, such as Brazil in 1901 and Argentina in 1934; then, in 1955, it was recorded in Central American countries [11]. In South America, the medfly is widely distributed across agro-ecosystems because environmental fragmentation and degradation, along with the introduction and spread of exotic fruit species, enabled its persistence and dissemination [12]. A combination of the pest’s high dispersal capacity and human-mediated pathways led to the medfly spreading throughout all Argentinean fruit-growing regions, covering latitudes from 22° to 45° S, from the northern border with Bolivia to southern Patagonia. Consequently, the medfly became one of the most damaging pests affecting fruit and vegetable crops in Argentina [13]. Therefore, in the early 1990s, the Argentine government implemented the National Fruit Fly Control and Eradication Programme (Spanish acronym: PROCEM) [14]. Current medfly mitigation actions involve an area-wide integrated fruit fly management (AW-IFFM) approach, particularly adapted to the agro-ecological and geographical characteristics of each Argentinean fruit-growing region. Integrated strategies nowadays include the sterile insect technique (SIT), mass trapping, and cultural, chemical, and biological controls, as well as quarantine measures through phytosanitary barriers [13].

Biological control using parasitoids has become a relevant complementary tool with other eco-friendly techniques for fruit-fly-integrated management in Latin America [15,16,17,18], particularly with the sterile insect technique [19], mass trapping [20], and novel bait spray [21]. This is mainly because there has been a growing trend over recent years towards sustainable farming practices to minimize environmental impact, preserve natural resources, and ensure long-term agricultural productivity [22,23]. Thus, including indigenous parasitoids for fruit fly biological control strengthens that up-and-coming trend even more [24,25,26,27]. One of the rising tactics of biological control is the augmentative method, which involves releasing large numbers of mass-reared natural enemies to achieve early control of the target pest [28,29]. This biological control approach has been used against fruit fly pests in various countries whose fruit production has been affected by these pests [15,18,30,31,32,33]. In this regard, releases of the Southeast-Asian-native larval parasitoid Diachasmimorpha longicaudata (Ashmead, 1905) (Hymenoptera: Braconidae) were performed in medfly-infested commercial fruit crops of San Juan, a province located in the central-western fruit-growing region of Argentina. Post-release data assessment demonstrated the success of mass releases of this exotic parasitoid species in reducing medfly populations in irrigated multi-fruit orchards under a dry environment with wide thermal variations [13]. Those promising outcomes with the exotic parasitoid D. longicaudata for medfly control encouraged further assessments of the Neotropical-native parasitoid species. One of these indigenous species is the larval parasitoid Ganaspis pelleranoi (Brèthes, 1924) (Hymenoptera: Figitidae), a generalist fruit fly parasitoid belonging to a guild of synovigenic, solitary, koinobiont endoparasitoids that parasitize mid- and late-instars of tephritid flies [34]. This figitid can successfully be reared on the larvae of both C. capitata and Neotropical-native Anastrepha fraterculus (Wiedemann, 1830) (Diptera: Tephritidae) [34,35,36,37], another economically important tephritid fruit fly species in Argentina [14]. In addition, G. pelleranoi can also produce offspring using the larvae of the pest tephritid species Anastrepha ludens (Loew, 1873) (Diptera: Tephritidae) under laboratory conditions [24]. This parasitoid is naturally associated with wild medfly populations in warm–humid, temperate–humid, and temperate–dry environments from the northwestern fruit-growing region of Argentina [38]. Although G. pelleranoi has long been regarded as a potential candidate for fruit fly biological control in the Americas [15], field cage and particularly open-field assessments of its effectiveness as a biocontrol agent have not been conducted to date.

Based on the G. pelleranoi bioecological features outlined above, a core research question arises: can G. pelleranoi effectively perform to reduce medfly population levels by increasing the number of released parasitoids? The parasitoid performance relates to the assessment of the host-killing effectiveness, i.e., the proportion of attacked hosts that die as a result of parasitization [39]. Thus, two conceptual hypotheses can be formulated in the face of this uncertainty: (1) G. pelleranoi females can find and kill medfly larvae infesting fruits of different physical features as the density of released parasitoid females in the tested patch increases, and (2) such medfly-killing capacity of G. pelleranoi females is sustained under variable host density conditions. Consequently, the overall aim of this study was to assess G. pelleranoi as a potential biological control agent of the exotic C. capitata through a gradual increase in the number of parasitoid females released under field cage (semi-field) conditions. In particular, three issues were tested: (1) the effect of increasing the density of released females on the parasitoid performance to reduce medfly emergence; (2) the parasitoid-induced medfly mortality based on the physical properties of two primary exotic host plants, namely Prunus persica (L.) Batsch (peach) (Rosaceae) and Citrus aurantium L. (sour orange) (Rutaceae); and (3) the potential effect of host larval density within both medium-sized (peach) and large-sized (orange) fruits on G. pelleranoi females’ performance. Both tested fruit species play an important role as reservoirs and multipliers of natural medfly populations in the Citrus-growing region of northern Argentina [40]. This study is framed within a renewed initiative to promote the use of a biological control to suppress fruit fly pest populations as an environmentally and human-health-friendly strategy. Therefore, the relevance of the findings is discussed in view of the potential use of this Neotropical-native figitid parasitoid species in augmentative [22,30,41] and conservative [25,42,43] fruit fly biological control programs scheduled or ongoing in Argentina and in the rest of the Latin American countries.

2. Materials and Methods

2.1. Source and Rearing of Insects

Both parasitoids and medflies were reared at the insectary of the Biological Control Department (BCD) from the Microbiological Industrial Processes and Biotechnology Pilot Plant (PROIMI, Spanish acronym). This scientific institution is located at 26°49′ S, 65°13′ W, at 452 m.a.s.l., in San Miguel de Tucumán, northwestern Argentina. The G. pelleranoi colony was initially established in November 2015 with individuals recovered from C. capitata puparia, which in turn were retrieved from feral peaches collected from the soil underneath the fruit tree. The fruit were taken from the Horco Molle Experimental Reserve (REHM, Spanish acronym), a protected wildlife area of the “Las Yungas” subtropical mountain rainforest belonging to the National University of Tucumán (UNT, Spanish acronym), located in Yerba Buena, Tucumán, at 26°55′ S and 65°05′ W, 600–800 m. The G. pelleranoi colony was held in 30 × 30 × 30 cm (length, width, and height) acrylic glass-structured, voile-fabric-covered cages at a density of 4000 parasitoid pairs per cage under 25 ± 1 °C; 75 ± 5% RH and a 12:12 (L:D) h photoperiod in a 6-m² room under a lighting condition of 1200 lux provided by six fairly distributed 36W fluorescent light tubes. Each cage was provided with water and honey every other day. Once a day, 12 × 1.5 cm (diameter × height) voile-cloth-screen-covered dishes holding ca. 1000 lab-reared third-instar medfly larvae were placed inside each parasitoid rearing cage. After host exposure, medfly larvae were placed in plastic containers with 2 mm of sterilized vermiculite (Intersum^®, Aislater S.R.L., Córdoba, Argentina) at the bottom as pupation medium. These containers were kept under lab conditions until both fly and parasitoid emergence. Medfly larvae were reared on an artificial diet consisting of wheat germ, sugar, brewer’s yeast, citric acid, agar-agar, vitamins, minerals, and preserving agents. Adult medflies were kept in the same cages and lab conditions as described above, and fed daily with a mixture of yeast hydrolysate enzymatic (MP Biomedicals, LLC, Solon, OH, USA), common refined sugar (Ledesma^®, Buenos Aires, Argentina), vitamins A and E, and water. The G. pelleranoi cohorts used in field cage trials were the 26th generation under artificial rearing.

2.2. Study Timeframe, Experimental Location, and Environmental Conditions

Field cage trials were carried out in the BCD’s backyard between late November and mid-December in 2022, corresponding to late spring. The study was carried out at the time when the highest medfly population peak began to occur (late spring) in northwestern Argentina [40]. The PROIMI institution is located in the central piedmont sector of the Tucuman lowlands at the foot of the western mountains at altitudes ranging from 350 m to 500 m. The original native vegetation was characterized by a subtropical rainforest. However, residential neighborhoods and extensive citrus, soft fruits, sugar cane, and horticultural crops replaced it. The climate is humid and warm–temperate, with a rainy–warm season from October to April and a dry–cold season from May to September; its mean annual rainfall ranges between 1300 and 1600 mm, with a mean annual temperature of 18 °C [40]. Meteorological data, such as both mean temperature and relative humidity (RH), were provided by a digital weather station (LUFT^®, model WS80, Shenzhen, China) during the trials. This station was located outside the field cages but only 1 m away from them. The weekly temperature and RH average data recorded during the testing date are shown in Table 1.

Table 1. Minimum (=Min), maximum (=Max), and average temperature and relative humidity during field-cage trial dates between November and December 2022 (late spring).

Testing Dates	Temperature (°C)			Relative Humidity (%)
	Min.	Max.	Average	Min.	Max.	Average
November 18	17	25	21	45	55	50
November 21	15	37	26	55	70	62.5
November 24	15	30	22.5	65	77	71
November 28	21	35	28	70	75	72.5
December 1	23	46	34.5	70	78	74
December 4	21	38	29.5	75	79	77
December 8	24	29	26.5	62	75	68.5
December 8	24	29	26.5	62	75	68.5
December 12	20	30	25	65	76	70.5
December 15	25	30	27.5	58	76	67

2.3. Host Fruit Species Selection

Citrus aurantium and P. persica (unknown feral cultivars) were used during field cage trials. Such fruit species were selected because they are among the most widespread medfly wild host plants growing adjacent to commercial crops throughout the Citrus-producing areas of Argentina. Fruits were removed from the backyards of rural houses located within the REHM-UNT. Branches of all unsprayed fruit species trees, each containing 6–12 unripe fruits (small, green fruit), were covered with a cloth mesh to avoid early infestation by the medfly. Once the fruits were ripe, they were harvested and taken to the BCD’s laboratory. Fruit of similar size and ripeness degree per plant species were selected for use in the trials. Weight, rind thickness, and pulp depth were quantified for each fruit species. As such, those physical traits were 245.4 ± 3.5 gr, 7.4 ± 0.2 mm, 8.9 ± 0.5 cm for the sour orange (N = 100), and 49.6 ± 1.5 gr, 0.2 ± 0.1 mm, 2.5 ± 0.4 cm for the peach (N = 100).

2.4. Fruit Inoculation for Trials

Before the trials, the fruits were washed and sanitized with a sodium benzoate plus methylparaben solution. Then, the fruits were individually filled with 5-day-old (late second instar), lab-reared medfly larvae plus the diet on which they had been reared. Artificial infestation was performed by cutting the fruit in the middle using a sterilized scalpel. Then, a quarter of the pulp and liquid were removed from each fruit. The space shaped inside the fruit, ca. 50% of the fruit volume, was filled with the host larvae and their rearing diet. The peach stone was removed. Once the fruit was inoculated, both fruit sections, the small upper lid and the large lower section, were tightly fitted and linked together with a transparent silicone adhesive, Unipox^®-brand hot melt glue (Unipox^®, Lomas de Zamora, Buenos Aires, Argentina), which was melted with an electric silicone gun (Multitienda Mendoza, Mendoza, Argentina). Preliminary laboratory studies indicated that the use of the aforementioned silicone adhesive on the fruit did not affect the foraging behavior of the female parasitoids, as it did not interfere with the hosts’ detection, acceptance, and parasitization. Each inoculated fruit was an oviposition unit. After medfly larvae were put in each fruit, a 5 mm diameter sterilized metal rod was used to create eight equidistant equatorial punctures that simulated larval exit holes. These holes resemble cavities commonly found in fallen ripe fruit during surveys in natural environments [40]. The holes in the fruit were made because G. pelleranoi females could penetrate the fruit in search of larvae by biting through the epicarp and mesocarp but also through fissures or hollows already existing in the fallen fruit. The depth of such holes varied according to the rind thickness and pulp depth of each fruit species. In the sour orange, the holes were 6 cm deep, while in the peach, they were 2 cm deep. Puncturing occurred a few minutes before the trial started. The diet was added to the fruit in order to provide a feeding and acclimatization medium within the fruit for larvae previously reared on trays with an artificial diet, and to prevent the larvae from promptly exiting the fruit through the artificial punctures.

2.5. Experimental Setup

Field cage trials were performed to assess G. pelleranoi-induced medfly mortality in two key host fruit species under a natural free-foraging condition and the variations in both parasitoid female release and host larval densities. Trials were carried out inside 3.5 × 3.0 m cylindrical iron-framed, nylon-mesh-covered field cages. At 10 m above the roof of each field cage, both a 130 g/m² black shade cloth, UV-resistant net (AGROREDES^®, San Isidro, Buenos Aires, Argentina), to protect from direct sunlight, and a 100μ-translucent high-density plastic cover (AGROREDES^®), to prevent rain falling directly on the cage, were installed. Five 60 × 200 cm galvanized wire-structured, white voile-fabric-covered, cylindrical cages were placed inside every main nylon field cage. Each internal experimental cage had a 1.5 m long zipper in its middle part to enter. A 1 m high potted peach or orange tree was placed inside each experimental cage to provide both a more natural environment and a larger surface area for parasitoid shelter. According to the experimental design, four, six, or eight artificially inoculated and punctured peaches or sour oranges were placed on the floor of each experimental cage equidistant from one another. Each fruit was placed into a 5 × 5 × 0.5 cm white expanded polystyrene tray, which was filled with a 0.5 cm layer of sterilized 8 mm thick expanded vermiculite on the bottom as a pupation substrate. Fruits were placed in a circle around the potted tree. Treatments involved three densities of medfly larvae per experiment and four densities of G. pelleranoi females released per experimental cage. Host larval densities used were 200, 300, and 400 lab-reared medfly larvae per experiment, which involved four, six, and eight fruit per tested species. Each fruit was inoculated with 50 medfly larvae. The same larval density per fruit species was used to homogenize the number of host larvae. The densities of G. pelleranoi females used were 20, 40, 80, and 160 parasitoid females per experimental cage. Host–parasitoid ratios per treatment are shown in Table 2.

Table 2. Host–parasitoid ratios (h/p) per treatment based on the three testing medfly larval densities and the four released Ganaspis pelleranoi female densities.

Host Larval Densities	Parasitoid Females Densities
	20	40	80	160
200	10 h/p	5 h/p	2.5 h/p	1.3 h/p
300	15 h/p	7.5 h/p	3.8 h/p	1.9 h/p
400	20 h/p	10 h/p	5 h/p	2.5 h/p

Naïve, mated, 3–4 day-old parasitoid females were used in all trials. Controls, with no released parasitoids, were carried out to determine natural medfly emergence from both inoculated peach and sour orange. All four treatments involving different parasitoid release densities and the respective controls were carried out in the same main nylon field cage. Parasitoid females were released on the floor of the experimental cage around the potted tree and 2 cm away from the fruits. Parasitoids were allowed to forage on fruit for 48 h starting at 10:00 a.m. After the tests were finished, both the fruit and the vermiculite from the trays were removed and taken to the BCD’s laboratory. There, the fruits were dissected to remove living medfly larvae, and the vermiculite was sieved to recover puparia. Larvae and puparia from the same fruit and from each tray were placed into 10 × 7 cm plastic cups with sterilized vermiculite on the bottom as a pupation substrate, and with a voile-covered top. The cups were kept under lab conditions until the flies and parasitoids emerged. Numbers of emerged parasitoids, adult flies, and non-hatched puparia were recorded. Once the parasitoid emergence period was over, the non-hatched puparia were dissected to detect the presence or absence of immature parasitoid stages or non-emerged adult parasitoids. Dead puparia due to pathogens or unknown causes were also reported. All this information was used to calculate parasitism and estimate the additional host mortality (=AM) as indicators of the parasitoid-induced host mortality ability. Each treatment combination (fruit × host density × parasitoid density) was replicated 10 times, with a total of 240 experimental units. Controls (no parasitoid release) were also replicated 10 times. For each replicate, new inoculated fruits, pupation substrate, and parasitoid females were always used.

2.6. Data Analysis

The R-4.5.1 software was used for statistical analysis [44]. Statistical significance was set at α = 0.05. The parasitism was calculated using the formula: 100 × (number of emerged parasitoids + number of non-emerged parasitoids)/(total number of exposed hosts). The parasitism was analyzed using generalized linear mixed models (GLMMs) with a binomial error distribution and logit link function. Fruit type (peach and orange), host density (200, 300, and 400 hosts per cage), parasitoid release density (20, 40, 80, and 160 released parasitoids per cage), and their interactions were included as fixed effects. Data from controls were excluded from models to avoid complete separation arising from structural zeros (i.e., no parasitism occurs in the absence of parasitoids). The AM was also analyzed using GLMMs with a binomial error distribution and logit link function. Fruit type, host density, parasitoid release density (0, 20, 40, 80, and 160 released parasitoids per cage), and their interactions were included as fixed effects, while the experimental period and a random effect at the observation level were included as random intercepts. Controls were included in the AM models. The AM was derived on the response scale using model-based predictions from the fitted GLMM. Particularly, the AM was calculated as the difference between the predicted probability of host mortality in the treatment cages (presence of parasitoids) and the predicted probability of host mortality in the control cages (no parasitoids), for each fruit type × host density combination:

$$\mathsf{\Delta }AM=p \left(\mathrm{dead} \mathrm{pupae}\right|\mathrm{parasitoid} \mathrm{release} \mathrm{density}>0) - p (\mathrm{dead} \mathrm{pupae}\mid \mathrm{control})$$

where $p(\cdot )$ denotes the marginal probability of host mortality predicted by the GLMM on the response scale. For reporting purposes, the AM was expressed in percentage points as follows:

$$\mathsf{\Delta }AM(\mathrm{\%})=100\times \mathsf{\Delta }AM$$

The experimental period (a 2-day exposure time) was included as a randomized blocking factor. The residual overdispersion was accounted for by including a random effect at the observation level, consistent with standard practice for binomial GLMMs. The models were fitted using the glmmTMB package [45]. In order to assess whether host exposure to parasitoids caused additional mortality relative to baseline levels, hypothesis tests comparing the treatment group to the control group were conducted. For this purpose, estimated marginal means (EMMs) based on GLMMs of the dead host pupae were used. For each combination of fruit type × host density, each parasitoid release density was compared with the control. These comparisons tested the null hypothesis H₀: ΔAM = 0 against the alternative H₁: ΔAM ≠ 0, that is, whether exposure to parasitoids increased host mortality, in addition to parasitism, relative to controls. Tests were performed on the model’s linear predictor (logit) scale, reporting estimates, standard errors, z-statistics, and p-values. The p-values were adjusted using Sidak’s method for multiple comparisons within each fruit type × host density combination. EMMs and treatment vs. control contrasts were computed using the emmeans package [46]. Pairwise contrasts between different parasitoid density levels were then performed. Similarly, to compare the two fruit types within each combination of host and parasitoid density, EMMs were computed for fruit type within each treatment level, followed by pairwise contrasts. Contrasts were evaluated on the log-odds scale using Tukey’s adjusted method to control the family-wise error rate for multiple comparisons. Results are reported as odds ratios (ORs) with their corresponding standard errors, t-ratios, and p-values, providing a measure of effect size for each comparison. Data cleaning, transformation, and reshaping were performed with the tidyverse function, particularly the dplyr and tidyr packages [47]. Graphical representations were produced using the ggplot2 package [48]. In order to compare the host mortality recorded from parasitism alone with the combined effect of parasitism and the AM, a percentage-based metric was developed. This metric, hereafter referred to as parasitism + AM, was calculated by adding the percentage of parasitism to the corresponding AM, which was estimated for each treatment combination. The parasitism + AM metric was used as a descriptive index to combine successful parasitism and the mortality associated with the action of parasitoids into a single measure of the overall impact on the host. This index was used only for graphical comparisons between treatments and experimental runs and was not subjected to further inferential testing.

3. Results

3.1. Fruit Effect on Parasitism and Parasitism + AM

Parasitism fluctuated considerably across treatments, ranging from a minimum of 12.2% ± 0.8% (SE) to a maximum of 41.3% ± 1.1% (SE), and fruit type significantly affected this outcome. Parasitism odds were significantly lower in the orange than in the peach under the GLMM model’s reference conditions (β = −0.270, SE = 0.104, z = −2.596, p = 0.009), revealing a considerably lower incidence of parasitism in the orange relative to the peach. Pairwise fruit contrasts within each host density × parasitoid release density combination showed that the peach frequently had higher parasitism odds than the orange. This contrast was particularly significant in nine of the 12 treatment combinations. The three non-significant parasitism contrasts occurred at the intermediate host density (300 hosts per cage) with 20, 40, and 80 parasitoids released per cage (p = 0.5070, p = 0.3031, and p = 0.0572, respectively) (Figure 1A, Table 3).

Figure 1. (A) Parasitism (%) and (B) parasitism plus additional mortality (%) in peach (green) and orange (blue) at different parasitoid release densities (20–160 parasitoids per cage) and host densities (200, 300, and 400 hosts per cage); n = 10 for each trial. Large points represent treatment means, which are interconnected by trend lines across parasitoid release densities; error bars indicate ± 95% confidence intervals. Small points represent individual replicates (horizontally jittered).

Table 3. Fruit type (peach vs. orange) contrasts for parasitism and parasitism + additional mortality (AM) by host and parasitoid release densities. Odds ratios (ORs) are expressed as peach/orange; OR > 1 indicates higher odds in peach. Parasitism contrasts are derived directly from the parasitism GLMM. Contrasts for parasitism + AM are reported as approximate, as this composite metric was constructed on the response scale and not modeled as a direct binomial response. The p-values indicate statistical significance at α = 0.05. An asterisk (*) means non-significant.

Host/Parasitoid Density	Parasitism		Parasitism + AM
	OR (Peach/Orange)	p-Value	OR (Approx.) (Peach/Orange)	p-Value (Approx.)
200:20	1.310	0.0094	2.463	<0.0001
300:20	1.054	0.5070 *	1.603	<0.0001
400:20	1.218	0.0099	1.566	<0.0001
200:40	1.783	<0.0001	2.698	<0.0001
300:40	0.925	0.3031 *	1.359	0.0032
400:40	1.798	<0.0001	2.326	<0.0001
200:80	1.283	0.0061	1.989	<0.0001
300:80	1.151	0.0572 *	1.861	<0.0001
400:80	1.503	<0.0001	2.441	<0.0001
200:160	1.551	<0.0001	2.261	<0.0001
300:160	1.443	<0.0001	2.291	<0.0001
400:160	1.956	<0.0001	3.389	<0.0001

Additional mortality (AM) varied across treatments, ranging from a minimum of 0.5% ± 1.4% (SE) to a maximum of 21.8% ± 1.3% (SE), and fruit type significantly influenced this component. Parasitism + AM odds were significantly higher in peach than in orange under the GLMM model’s reference conditions (β = 0.491, SE = 0.086, z = 5.720, p < 0.001) (Figure 1B), revealing a considerably greater total host mortality in peach when accounting for non-parasitic effects. Pairwise fruit contrasts within each host density × parasitoid release density combination showed that peach consistently had higher parasitism + AM odds than orange. Unlike the analysis based on parasitism alone, in which three non-significant contrasts were found, the inclusion of AM data resulted in statistically significant contrasts across all 12 treatment combinations (Table 3). This highlighted the enhanced discriminatory power of the composite metric.

3.2. Parasitism and AM Responses to Host and Parasitoid Release Densities

Parasitism increased with higher parasitoid release densities in both fruit species. Relative to the release density of 20 parasitoids per cage (reference), positive and significant effects were found for densities of 40 (β = 0.364, SE = 0.096, z = 3.795, p < 0.001), 80 (β = 0.531, SE = 0.094, z = 5.625, p < 0.001), and 160 (β = 1.030, SE = 0.092, z = 11.249, p < 0.001). Host density also influenced parasitism. Compared to 200 hosts per cage (reference), densities of 300 (β = 0.398, SE = 0.090, z = 4.415, p < 0.001) and 400 hosts (β = 0.213, SE = 0.088, z = 2.409, p = 0.016) showed higher parasitism odds. Significant interaction terms indicated that the magnitude of parasitoid release effects varied by fruit type and host density. The specific differences between treatments are illustrated in each panel of Figure 1A. The number of dead host pupae increased with parasitoid release density, even after accounting for baseline mortality found in controls. In the GLMM fitted to host mortality data (dead host pupae), all parasitoid release densities showed strong positive effects relative to controls: 20 (β = 0.672, SE = 0.084, z = 7.966, p < 0.001), 40 (β = 0.738, SE = 0.084, z = 8.796, p < 0.001), 80 (β = 0.805, SE = 0.084, z = 9.638, p < 0.001), and 160 (β = 0.992, SE = 0.083, z = 12.024, p < 0.001). On the response scale, AM (expressed as percentage points, pp) was higher in peach than in orange and tended to increase with parasitoid release density (Table 4). In peach, AM ranged from 10.0% to 21.8%, whereas in orange ranged from 0.5% to 9.5%. The AM effect increased with higher parasitoid release densities. These patterns varied across host densities, consistent with fruit- and host-dependent modulation of parasitoid-associated additional mortality.

Table 4. Significance of additional mortality (ΔAM) by fruit type, host density, and parasitoid release density. Results from treatment versus control contrasts derived from estimated marginal means (EMMs) of the GLMM fitted to host mortality data. p-values were adjusted using Sidák’s method for multiple comparisons within each fruit type × host density combination. Significance codes: *** p < 0.001, ** p < 0.01, * p < 0.05, ns = non-significant (p ≥ 0.05).

Fruit Species	Host/Parasitoid Density	ΔAM (%)	SE (%)	p-Value	Level of Significance
Citrus aurantium L.:	200/20	0.46	1.43	0.996	ns
	200/40	2.61	1.46	0.2548	ns
	200/80	3.51	1.47	0.0616	ns
	200/160	7.06	1.5	<0.0001	***
	300/20	3.84	1.26	0.0077	**
	300/40	2.57	1.24	0.1357	ns
	300/80	3.13	1.25	0.0426	*
	300/160	9.54	1.3	<0.0001	***
	400/20	4.48	1.16	0.0003	***
	400/40	5.8	1.17	<0.0001	***
	400/80	3.04	1.15	0.0267	*
	400/160	6.4	1.17	<0.0001	***
Prunus persica (L.) Batsch:	200/20	10.9	1.36	<0.0001	***
	200/40	12.2	1.38	<0.0001	***
	200/80	13.55	1.39	<0.0001	***
	200/160	17.55	1.43	<0.0001	***
	300/20	12.64	1.2	<0.0001	***
	300/40	10.77	1.19	<0.0001	***
	300/80	14.84	1.22	<0.0001	***
	300/160	21.84	1.26	<0.0001	***
	400/20	9.86	1.11	<0.0001	***
	400/40	13.81	1.14	<0.0001	***
	400/80	15.23	1.15	<0.0001	***
	400/160	21.09	1.17	<0.0001	***

Treatment versus control contrasts derived from the estimated marginal means (EMMs) of the GLMM fitted to host mortality data (dead host pupae) indicated that AM was significantly higher than zero at all combinations of parasitoid and host densities in peach (all p < 0.001) (Table 4). In contrast, in orange, AM was significantly higher than zero only at intermediate and high parasitoid release densities, with non-significant effects observed at 20 parasitoids across all host densities and at 40 parasitoids at 200 and 300 hosts (Table 4). Detailed results, including estimates, standard errors, and z-ratios, are provided in File S1.

3.3. Parasitism vs. Parasitism + AM

Host mortality attributed to the composite metric (parasitism + AM) was consistently higher than that caused by parasitism alone across treatment combinations (Figure 2). Given the descriptive nature of this composite metric, no inferential statistics were calculated directly on this index. Instead, patterns were evaluated through graphical inspection. However, the AM values were significantly higher than zero in most treatment combinations (Section 3.2, Table 4), implying that parasitism + AM outperformed parasitism alone, as the latter variable did not account for the host’s additional mortality component. The difference between the two metrics increased with parasitoid release density and was particularly evident in peach at high release densities. In orange, the difference between the two variables was smaller at low parasitoid release densities and more pronounced at high release densities. These patterns were consistent across all experimental runs, indicating a standardized trend in both fruit species (Figure 2). At the highest parasitoid release density (160 parasitoids per cage), parasitism + AM caused mean host mortalities ranging from 51.3% to 62.4% in peach across all host densities, whereas parasitism alone accounted for 33.8% to 41.3%. In orange, parasitism + AM caused mortality rates ranging from 31.8% to 38.9%, compared with 24.7% to 29.3% for parasitism alone (Table 3, File S2). The composite metric substantially enhanced host mortality differences between fruit species under high parasitoid pressure (File S2).

Figure 2. Boxplots show the distribution of parasitism (%) (green boxes) and parasitism plus additional mortality (%) (pink boxes) across parasitoid release densities for each host density (rows) and fruit type (columns). Boxes represent the interquartile range with the median indicated by the central line; whiskers extend to 1.5× the interquartile range, and error bars indicate ± 95% confidence intervals. Each box is based on n = 10 replicates per combination of fruit, host density, and parasitoid density. Jittered points correspond to individual experimental cages, highlighting the variability within the treatment.

4. Discussion

Parasitoid species selected as potentially effective fruit fly biocontrol agents entail a prior and exhaustive assessment of their host foraging and killing abilities in different host fruits and surrounding environments [26,49]. Understanding those ecological aspects is strategically essential for developing and implementing a biological control program using indigenous parasitoids [50]. In this framework, the current study mainly reports the ability of the Neotropical-native parasitoid G. pelleranoi to kill the exotic C. capitata infesting two substantially different medfly-multiplying fruit species, through simulated augmentative releases under field cage conditions. Consequently, results of this study highlight valuable biological information as follows: (1) G. pelleranoi females performed satisfactorily once released under non-controlled environmental conditions as they successfully parasitized host larvae on peach and sour orange, regardless of their strongly differing physical features; (2) both host and parasitoid densities influenced the ability of G. pelleranoi females to kill medfly larvae, and the fruit type modulated this effect; and (3) G. pelleranoi females induced a substantially high host die-off rate when AM was added to the analysis, which was not revealed when parasitism alone was regarded as a medfly mortality variable.

The first finding showed G. pelleranoi as a suitable parasitoid to successfully attack medfly larvae in fruits with highly contrasting physical attributes, as is the case between the peach and the orange. Individual fruit physical features, such as rind thickness and pulp depth, can mostly influence fruit fly parasitism rates [51,52]. The peach has physical traits that facilitate natural parasitism in tephritid larvae lodged internally in the fruit, such as small size, thin skin, and shallow pulp [40]. The highest fruit fly parasitism levels have usually been linked to fruit with more advantageous features, enabling parasitoid females to find and parasitize host larvae [22,53,54]. In contrast, the sour orange, with its large size and a rind 37 times thicker and a pulp 4 times deeper than those of the peach, sharply restricts natural parasitism [40]. Fruit flies can escape parasitism when they infest large fruit because fly larvae can feed at a greater depth in the fruit pulp, which reduces host accessibility to parasitoids [55,56]. This occurs mainly with braconid larval parasitoids, such as Doryctobracon spp., Utetes spp., and Opius spp., whose females lay eggs on the tephritid larvae feeding inside fruits by drilling with their ovipositor through the pericarp from the exterior [53]. Thus, the physical barrier imposed by the fruit on the female parasitoid is one of the main hurdles to achieving the successful release of fruit fly parasitoids [57]. As such, a large fruit can provide shelter, as it reduces or prevents attacks by parasitoids [58]. However, the results of the current study revealed that releases of the figitid G. pelleranoi induced up to 50% of pest mortality in the sour orange. This information supports previous field data recorded from fruit surveys in wilderness areas of northwestern Argentina, in which G. pelleranoi was recovered from non-crop Citrus spp. highly infested by C. capitata, although natural parasitism remained below 10% [40]. The host-searching behavior likely played a role in enabling G. pelleranoi females to detect medfly larvae and oviposit in fruit with unsuitable features for natural parasitism by braconid wasps. Ganaspis pelleranoi females mainly forage host larvae in fallen fruit and mostly find them by entering the fruit through fissures in the rind, by holes produced by its jaws, or even by attacking the fly larva when it exits the fruit to pupate [59,60]. These host foraging and oviposition strategies exhibited by this figitid species appear to be an important advantage over the foraging behavior of braconids when the infested fruit is a Citrus species. In addition, the lack of development of Neotropical-native braconid larvae in C. capitata, probably caused by encapsulation or antibiosis [40], would be overcome by G. pelleranoi, as it successfully develops inside medfly larvae.

The second finding highlighted that increased medfly mortality in both tested fruit species occurred with augmented numbers of released female parasitoids, even at higher host densities. In this regard, when comparing the maximum parasitoid release density, i.e., 160 parasitoids per experimental cage, in both testing fruit species and in the three host larval densities, G. pelleranoi was highly effective in killing medfly larvae. Therefore, it was proven that the progressive increase in the number of released parasitoid females significantly improved the effectiveness of G. pelleranoi in reducing medfly emergence in the fruits of the two tested species. Although the results recorded in field cages are still preliminary, they suggest that augmentative releases of G. pelleranoi may substantially suppress medfly populations in the citrus-growing region of northwestern Argentina, characterized by subtropical weather conditions. The findings of the current study is consistent with previous field cage trials conducted with two population lines of the exotic braconid parasitoid D. longicaudata [61]. In those trials, the authors found significant reductions in C. capitata emergence in infested commercial peaches at the highest D. longicaudata release rates, e.g., 160 and 320 parasitoid females. In the current study, the maximum release density did not exceed 160 parasitoids; it cannot be regarded as an optimal release rate based on the host densities tested. The optimal release rate of predators and parasitoids for effective pest control depends on the species assessed, although exceeding the optimal rate does not improve control of the target pest [62]. Factors that can affect the relative impact of open-field release rates include the timing and method of biological control application, the reproductive capacity and foraging ability of natural enemies, the host density and availability, community interactions, environmental conditions [62,63,64], and traits of fruit species hosting the pest [53,57]. However, two components in the host–parasitoid interaction are relevant in determining the parasitoid release rate: the intrinsic growth rate of the host population relative to the maximum growth rate of the parasitoid [65]. Although the latter is lower than the growth rate of the host, a threshold still exists for the number of parasitoids that can be released to control the host population growth [65]. This is the case of G. pelleranoi, whose intrinsic increment rate when using different fruit fly species as hosts, e.g., Anastrepha ludens [24], A. fraterculus [34], and C. capitata [35], was significantly lower than the mean intrinsic growth rate of the invasive medfly [66]. However, this does not preclude the potential use of G. pelleranoi as a medfly biological control agent through augmentative release, as suggested by the current study. Interestingly, the performance of G. pelleranoi females was conditioned by the tested fruit species. The peach evidently exerted a significant influence on the parasitoid-induced host mortality capacity. Medfly mortality was particularly higher in the peach than in the sour orange on 85% of the different combined densities of released parasitoids and exposed host larvae. Medfly mortality rates were particularly higher in those host densities involving 300 to 400 medfly larvae exposed to G. pelleranoi females, but always with a preference for infested peaches over sour oranges. Apparently, increasing the number of fruits in the experimental cages, to increase the host density offered to parasitoids, enabled females to increase their host foraging possibilities and decrease intraspecific interference. Previous lab studies showed that G. pelleranoi females had a higher propensity to forage non-parasitized host larvae rather than medfly larvae previously parasitized by conspecific females [67]. Thus, the ability of G. pelleranoi females to discriminate previously parasitized hosts suggests low levels of conspecific competition, which may lead females to search for hosts in alternative microenvironments, e.g., in other fallen or unfallen fruits [67].

The third finding revealed that the composite metric (parasitism + AM) was a better predictor variable of the true host mortality inflicted by the parasitoid than the parasitism rate. Therefore, evaluating parasitism alone may not accurately represent how many hosts are successfully suppressed by G. pelleranoi, and so the effectiveness of this figitid parasitoid as a medfly control agent can be underestimated. This is because the parasitoid-induced host mortality is usually based on evidence of host death followed by successful parasitoid offspring development, i.e., a reproductive mortality [68]. Therefore, the induced host mortality without visible evidence of parasitism, i.e., non-reproductive effects, is usually overlooked [68,69]. These non-reproductive impacts, i.e., when the host is not involved in reproductive processes of the parasitoid, can kill the host or induce behavioral, reproductive, and developmental disorders, reducing host fitness [70]. The hidden host mortality can be caused by several factors [68,70], including both stinging activity in the host without oviposition and superparasitism [71]. Both events may increase host mortality rates when fruit fly larvae are attacked by parasitoids [71,72,73,74]. Females of the braconid D. longicaudata can cause serious damage to Anastrepha spp. larvae due to multiple punctures as a result of probing with the ovipositor [71] or an overly high number of successful ovipositions, causing a superparasitized host [72,75]. Both situations can often kill the tephritid fly larva [71,72]. As found in the current study, the figitid Aganaspis daci (Weld, 1951) (Hymenoptera: Figitidae), a promising candidate for the biological control of C. capitata in the Mediterranean region [73,74], can also induce non-reproductive mortality in medfly larvae [73], potentially caused by superparasitism [76]. Interestingly, the combined induced mortality and parasitism rate caused by A. daci on medfly averaged at 87% under field conditions in the Spanish Mediterranean region [74]. Based on these data, the effectiveness of G. pelleranoi in controlling medfly under semi-field conditions was lower than that of A. daci. The figitid G. pelleranoi induced an average mortality rate of around 57% in medfly at the maximum parasitoid release density.

Superparasitism was detected in some non-hatched medfly puparia through dissection, but this parameter was not quantified, so no conclusions could be drawn about whether superparasitism by G. pelleranoi was a factor influencing the high corrected host mortality rates recorded in the current study. However, a recent study [67] found that self-superparasitism was higher than conspecific superparasitism in G. pelleranoi, and females could repeatedly both probe with their ovipositor and oviposit in the same medfly larva. Therefore, excessive probing activity by G. pelleranoi females could cause non-reproductive mortality in the host larva. Furthermore, combining AM with parasitism and analyzing them as a composite indicator reinforced the effect of fruit type on the impact of G. pelleranoi on host mortality. In this regard, the differences between peach and sour orange relating to host mortality rate were even greater. The integration of the AM variable produced a consistently detectable difference in odds between peach and orange across all experimental conditions. Consequently, the AM, which involved non-reproductive effects of the parasitoid on the host, has important practical implications for the evaluation of G. pelleranoi as a biocontrol agent of natural medfly regulation. Faced with this evidence, the resident parasitoid G. pelleranoi should be taken into account when analyzing different biological control strategies, such as conservative and/or augmentative approaches.

5. Conclusions

The results supported the preliminary hypotheses, as two main findings stood out. Firstly, G. pelleranoi females performed satisfactorily once released under non-controlled environmental conditions and successfully killed a high proportion of medfly larvae in the two tested fruit species, characterized by significant physical differences. Secondly, the parasitoid-induced medfly mortality significantly increased in both peach and sour orange relative to the gradual increase in released G. pelleranoi females, regardless of the increase in host density offered to parasitoids. Although such outcomes were limited to two medfly host fruit species confined to field cages, it served as a precedent for developing and testing the usefulness of the native parasitoid G. pelleranoi through open-field releases to control natural medfly populations within an eco-friendly pest management approach. Consistent with this approach, the ability of G. pelleranoi females to successfully attack medfly larvae in large exotic fruits, such as Citrus spp., may have significant practical implications, for instance, the implementation of augmentative biological control involving the mass rearing and release of G. pelleranoi in Citrus-producing areas or in other high-value fruit crops for Argentina’s fruit production, and in countries affected by C. capitata. However, successful implementation of augmentative releases requires precise timing, production at a mass-rearing facility —such as the San Juan biofactory in central-western Argentina—and, most importantly, the integration of habitat management to support the release of the parasitoid.