Trichopria anastrephae: A Promising Neotropical-Native Parasitoid for Drosophila suzukii Control

Abstract

Drosophila suzukii (Matsumura) is an invasive pest mainly affecting berry and stone fruit crops worldwide. In Argentina, it inhabits fruit-growing regions. An eco-friendly management strategy involves biological control by using resident natural enemies, such as the Neotropical-native pupal parasitoid Trichopria anastrephae Lima (Ta). The study compared the host-killing capacity and the offspring reproductive success of two Ta lineages on the puparia of both D. suzukii (Ds) and D. melanogaster (Dm) in no-choice and choice tests under laboratory conditions. The host preference and host-switching behaviors were also assessed. One parasitoid lineage was reared on Ds (Ta_Ds), and the second on Dm (Ta_Dm). In no-choice tests, both Ta lineages performed similarly on both hosts regarding the percentage of killed hosts and parasitoid offspring survival. The host-killing ability of Ta_Dm was only significantly lower when Ds was offered as a host, relative to Dm. In choice tests, Ta attacked mainly Ds at a 4–9 times Ds to Dm ratio, but at a 1.5–2 times Ds to Dm ratio, the host-killing ability was similar between both drosophilids. At an equal host ratio or higher Dm ratios, Ta preferred the native host. However, it was determined that Ta has the potential to parasitize the recently-introduced pest.

1. Introduction

Invasions of alien species into new regions usually produce adverse economic, ecological, and social effects [1,2]. The globalized trade of agricultural commodities is among the leading causes of the significant expansion of crop pests over the last decades, threatening worldwide food security [3], which leads to rigorous governmental strategies to either avoid the introduction of invasive species or implement large-scale eradication programs whenever prevention fails [4,5]. In that regard, the distribution range of the spotted-wing drosophila (SWD), Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), a polyphagous fruit pest native to Southeast Asia, has rapidly expanded in the last decade and became a severe pest in Europe [6], the Americas [7], and throughout Asia [8]. Drosophila suzukii is currently a significant pest of berries and stone fruit production worldwide [9]. This invasive pest damages mainly soft-skinned fruit such as Vaccinium spp. (blueberry) (Ericaceae), Fragaria spp. (strawberry), Rubus spp. (raspberry and blackberry), and Prunus spp. (cherry, peach, apricot, and plum) (Rosaceae) [10]. In addition, numerous wild and ornamental non-crop fruits have been infested by D. suzukii worldwide [11,12,13,14]. Unlike other drosophilid flies, the D. suzukii female oviposits through the skin of fresh, healthy, and ripening fruits still on the plant due to its serrated and sclerotized ovipositor [15]. Consequently, the larvae cause unmarketable fruit, resulting in high losses to the fruit industry in many countries [16].

Drosophila suzukii invaded Argentina in 2014, spreading throughout fruit-growing regions with highly contrasting climatic conditions, from the humid subtropical rainforests of the north to the semi-desert highlands and lowlands of western and southern Argentina [17]. Such swift expansion could result from two invasion events originating from countries that recorded the pest previously, such as the USA and Brazil [18]. The high environmental adaptability and behavioral and physiological plasticity make D. suzukii particularly suited to changing habitats and climatic conditions [19].

Soft fruits, such as berries and cherries, are among the most critical groups of high commercial value fruits that have increased their regional expansion, production, marketing, and export in Argentina. Blueberry (Vaccinium corymbosum L.) exports in 2020 reached 11,000 t, accounting for USD 110 million in revenues [20]. Strawberries (Fragaria×ananassa Duch.), raspberries (Rubus idaeus L.), blackberries (R. fruticosus L. and R. ulmifolius Schott), and cherries (Prunus avium L.) are also highly produced in Argentina [17,21]. The high population growth, thermal plasticity, and broad host range make D. suzukii a significant risk to Argentina’s soft fruit industry. Although there are few published records of damage caused by this pest, the known data are highly worrisome [17].

Successful Integrated Pest Management (IPM) programs involve an understanding the basic ecology of the target species in both the agroecosystem and the surrounding non-crop areas [22]. Key aspects, such as natural mortality factors influencing pest population dynamics, are essential for implementing control tactics by using biocontrol agents [23,24], and taking into account the heterogeneity of agricultural landscapes [25]. In this regard, several resident parasitoid species have been associated with D. suzukii worldwide [10,26,27,28,29], but few can overcome its robust immune system [30,31]. Therefore, resident parasitoids with the highest chance of successfully developing on D. suzukii are pupal parasitoids [32,33,34]. Among these parasitoids, Trichopria anastrephae Lima (Diapriidae) and Pachycrepoideus vindemiae Rondani (Pteromalidae) were the most abundant species parasitizing D. suzukii in non-crop areas of the Argentinian northwestern fruit-growing region [35]. The diapriid T. anastrephae is a native species to the South American Neotropical region, and it was recorded for Brazil and Argentina from saprophytic drosophilids and D. suzukii [36]. It is an endoparasitoid because the egg is placed into the hemocoel of the host pupa [37]. In contrast, the ectoparasitoid P. vindemiae is a cosmopolitan species, highly polyphagous, attacking a wide variety of cyclorrhaphous dipterans [38].

Native parasitoids can display genetic variations for host choice and use [39], which is mainly associated with variation in both the physiological and behavioral components of host foraging [40] and through superparasitism [41]. Such novel settings may encourage native parasitoids to include the novel host in their host range through physiological compatibility [42], inducing host switching, i.e., parasitism on the most abundant host available in a choice setting [41,43,44]. Some generalist parasitoid species may display a host-switching behavior to increase their reproductive success [45]; therefore, host preferences may change according to the relative abundance of involved host species [43].

The aims of this study were to compare both the host-killing capacity and the offspring reproductive success of two population lineages of T. anastrephae on puparia of D. suzukii, a novel host, and D. melanogaster, a resident host, in no-choice and choice tests under laboratory conditions. Likewise, the host preference and host switching were compared in choice tests. We hypothesized that regardless of host origin, the two lineages of T. anastrephae, one of them laboratory-reared on D. suzukii and the other one on D. melanogaster, could both kill D. suzukii puparia and successfully develop in the new host. Secondly, the mortality of D. suzukii puparia caused by T. anastrephae would not be affected by the presence of D. melanogaster host puparia in the same habitat. The results are discussed in relation to the feasibility of using T. anastrephae in biological control programs against D. suzukii within an IPM strategy.

2. Material and Methods

2.1. Insects Rearing

The colonies of D. suzukii, D. melanogaster, and T. anastrephae used in the assays were originated from puparia collected from fallen non-crop peaches (Prunus persica (L.) Batsch) in wilderness areas during summer (December) 2019 in Horco Molle (26°55′ S, 65°05′ W, 600–800 elevation), Tucumán province, northwestern Argentina. Drosophilid and parasitoid colonies were kept at 25 ± 1 °C, 75 ± 5% RH, and 12:12 (L:D) h photoperiod at the Pest Biological Control Department (DCBP, Spanish acronym) from the Biotechnology and Microbiological Industrial Processes Pilot Plant (PROIMI, Spanish acronym), in San Miguel de Tucumán, Tucumán, Argentina. Two T. anastrephae population lines were used in the trials. One of them was reared on D. suzukii puparia (Ta_Ds), whereas the second population line was reared on D. melanogaster puparia (Ta_Dm). Flies and parasitoids were held in cubical Plexiglas rearing cages (30 cm) with voile screen-covered sidewalls. Adult parasitoids were fed with honey every other day. Adult flies were fed daily with an artificial diet consisting of enzymatically hydrolyzed yeast (MP Biomedicals, LLC, Solon, OH, USA), corn gluten meal (Grupo Arcor S.A., La Reducción, Lules, Tucumán, Argentina), and standard white cane sugar (Ledesma SAAI, Libertador General San Martin, Jujuy, Argentina) in plastic Petri dishes (10 × 1.5 cm, diameter/deep) with a double layer of towel paper. Water was provided ad libitum by 200 mL plastic bottles with yellow absorbent cloth wicks. Four 140 mL disposable plastic cups, with 50 mL larval diet, were used as oviposition devices in each rearing cage. The larval diet was made of corn flour (32.5 g), brewer’s yeast (18.5 g), cane sugar (52.5 g), agar-agar (8 g), absolute ethyl alcohol 99.5% (15 mL), vitamin C (1 g), vitamin E (1 g), and Nipagin (methyl 4-hydroxybenzoate) (1.6 g). Agar-agar and corn flour were dissolved in 200 mL and 800 mL of water, respectively. Such ingredient proportions are enough for 500 g of diet. After 24 h, each oviposition device was removed, covered with a cotton voile cloth, and placed in an empty Plexiglass cage. Six days later, puparia were extracted from the larval diet, washed with a 10% sodium benzoate (Pura Química Laboratory, Córdoba, Argentina), and purified water solution. Then, puparia were placed in 500 mL hinged plastic cups with 5.5 cm² of vermiculite (Intersum^®, Aislater S.R.L., Córdoba, Argentina) previously sterilized on the bottom as a pupation substrate. These cups were placed in new adult-rearing cages. The procedure was carried out for each drosophilid species. One-day-old host puparia were exposed to parasitoid females for 48 h on 90 mm laboratory filter paper (Cytiva, Shanghai, China) moistened with distilled water inside plastic Petri dishes. The T. anastrephae population lines used in the trials were the 40th generation under artificial rearing.

2.2. Experimental Setup

No-choice and choice tests were conducted in an 8 m² room at the DCBP, under the aforementioned controlled laboratory conditions. The no-choice tests were performed to assess both the parasitoid’s host-killing capacity (parasitoid performance) and the ability to produce parasitoid surviving offspring (reproductive success) when D. suzukii and D. melanogaster puparia were singly exposed to parasitoid females from each population lineage. Experiments consisted of five naïve, i.e., never exposed to host puparia, 5-day-old, mated Ta_Ds or Ta_Dm females exposed to 50 D. suzukii or D. melanogaster puparia for two days. As previously described, one-day-old puparia of D. suzukii or D. melanogaster were provided to parasitoids. Cubical Plexiglas experimental cages (15 cm) with voile screen-covered sidewalls were used for each treatment. Control tests (no parasitoids) were performed simultaneously with treatments to check natural fly mortality and emergence rates. Once the 48 h exposition period ended, Petri dishes were removed from each experimental cage, and host puparia were placed in hinged lid plastic cups with sterilized vermiculite on the bottom. Puparia were kept in cups until adult flies or parasitoids emerged. Ten replicates per treatment and a control were performed. One week after parasitoid emergence, non-emerged host puparia were dissected to corroborate parasitism. A Leica^® EZ4D 40× stereomicroscope (Wetzlar, Germany) was used for dissections. The number and sex of the parasitoids, the number of flies, and the number of non-emerged puparia were recorded. Parasitoid performance was calculated using Abbott’s percent-corrected host mortality [46], which allows for calculating the parasitoid’s host-killing capacity through the relationship between host emergence rates from experimental treatment and control tests. Parasitoid reproductive success was based on the parasitoid offspring emergence, which was calculated as the total number of emerged parasitoids divided by the total number of exposed host puparia. The parasitoid offspring sex ratio was calculated as the percentage of live females recovered from the number of emerged parasitoids.

The dual-choice tests were performed to assess the parasitoid’s host-killing capacity and reproductive success, and also the host species preference and host switching behavior when different proportions of D. suzukii and D. melanogaster puparia were simultaneously offered to parasitoid females from both population lineages. Experiments consisted of five naïve 5-day-old, mated Ta_Ds or Ta_Dm females exposed to 50 host puparia for 48 h, but at nine different ratios of D. suzukii and D. melanogaster, as follows: 45:5, 40:10, 35:15, 30:20, 25:25, 20:30, 15:35, 10:40, and 5:45. Puparia of both D. suzukii and D. melanogaster were placed together in the same standard-size plastic petri dish. Trials were carried out using the same procedure as described above. Drosophila suzukii puparia can be easily distinguished from the D. melanogaster puparia by the external shape of the anterior spiracles, which have two tubes with plumose-shaped tips on the top [33]. The number and sex of the parasitoids and the number of flies and non-emerged puparia were recorded. Treatments and controls were replicated 10 times. Parasitoid performance and reproductive success (parasitoid emergence rate) were determined. The host species preference was calculated using the formula E₁/E₂ = C × N₁/N₂, as described by [47], where C, a constant, is the preference index for a host species in equal abundance based on the proportion of attacked hosts, which in the current study was 25:25. N₁ and N₂ are the numbers of two host species, i.e., N₁ = D. suzukii and N₂ = D. melanogaster, offered to the parasitoid. E₁ and E₂ are the observed numbers of the two host species parasitized, i.e., host parasitism. When the C value is =0 or <1, the preferred host species is #2; when the C value is >1, the preferred host species is #1, but when the C value is =1, there is no preference [48]. Parasitism was calculated as the emerged parasitoids plus non-emerged parasitoids derived from host puparia dissection, divided by the number of offered host puparia. The host-switching behavior was tested to establish whether parasitism was low when a host was uncommon [44]. The switching trials were assessed using the formula P₁ = C × F₁/(1 − F₁ + C × F₁), as described by [49], where F₁ is the proportion of the host species #1, i.e., D. suzukii, in a particular trial. The P₁ parameter is the expected ratio of the host species #1 among all parasitized hosts, and C is the constant described above. When host switching occurs, the observed parasitized ratio (E₁/E₂) is higher than expected at a given N₁/N₂ value [50].

2.3. Statistical Analysis

In order to compare the parasitoid’s host-killing capacity, the reproductive success, the parasitoid offspring sex ratio, and the host switch index for both Ta_Ds and Ta_Dm population lineages, Kruskal–Wallis’ rank sum tests were performed, with Dunn’s post hoc pairwise comparison tests to look for differences between factor levels using the Bonferroni–Holm method for adjustment. Mann–Whitney–Wilcoxon tests were performed with a Bonferroni–Holm adjustment method to compare the host preference index between Ta_Ds and Ta_Dm population lineages. The R-4.3.2 statistical software was used for analysis [51]. The box plots were plotted to show the resulting data, which involve median (horizontal line inside the box), mean (X inside the box), interquartile range Q1–Q3 (bottom and top ends of the box), range (minimum: Q0, maximum: Q4; both ends of the whisker on the vertical line outside the box), and raw data dispersal (colored circles). Letters that display the significant difference in figures were included with the R-library ‘rcompanion’ function.

3. Results

3.1. Parasitoid Performance and Reproductive Success

There were significant but small differences when the host-killing effectiveness of T. anastrephae was evaluated under no-choice conditions (H_{(3, n = 40)} = 13.384, p = 0.004). The performance of Ta_Dm when D. suzukii was offered as a host was significantly lower than that of Ta_Dm when D. melanogaster was the host (Figure 1A). There were no significant differences between T. anastrephae population lineages regarding their performance parasitizing either D. suzukii or D. melanogaster (Figure 1A). The host-killing capacity of T. anastrephae was >80% in all four treatments. There were no significant differences between the reproductive success of the two T. anastrephae population lineages or when both fly species were exposed to parasitoid females (H_{(3, n = 40)} = 3.811, p = 0.283). The mean offspring survival in both parasitoid lineages varied between 50% and 70% (Figure 1B).

There were significant differences between choice treatments when comparing the host-killing capacity of the Ta_Dm lineage (H_{(17, n = 180)} = 118.248, p < 0.001). The killing capacity of the parasitoid on D. suzukii was significantly higher than the D. melanogaster-killing parasitoid ability when only the ratio of D. suzukii puparia was from 4- to 9-fold higher than that from D. melanogaster (Figure 2A). However, when the D. suzukii puparia ratio was 1.5- and 2-fold higher than that of D. melanogaster, the parasitoid’s host-killing capacity was similar for both drosophilid hosts; although when the D. suzukii:D. melanogaster ratio was =1 or <1, females of the Ta_Dm lineage killed significantly more puparia of D. melanogaster than D. suzukii puparia (Figure 2A). The reproductive success of the Ta_Dm lineage differed significantly between treatments (H_{(17, n = 180)} = 126.723, p < 0.001). The parasitoid’s surviving offspring sourced from D. suzukii puparia was higher than that coming from D. melanogaster at 2–9 D. suzukii puparia per D. melanogaster puparium ratio (Figure 2B). When the D. suzukii puparia ratio was 1.5-times higher than D. melanogaster there were no significant differences between the Ta_Dm surviving offspring from both host species. In contrast, when the D. suzukii:D. melanogaster ratio was =1 or <1, the parasitoid offspring that emerged from D. suzukii puparia was significantly lower than that from D. melanogaster puparia (Figure 2B).

Similarly to the results with the Ta_Dm lineage, there were significant differences between choice treatments when comparing the performance of the Ta_Ds lineage (H_{(17, n = 180)} = 141.553, p < 0.001). The Ta_Ds female had a significantly higher capacity to kill D. suzukii puparia when its proportion was 4- and 9-fold higher than for D. melanogaster (Figure 3A). The Ta_Ds lineage performance was similar for the two hosts at ratios of 1.5- and 2-fold more D. suzukii puparia than D. melanogaster, but at D. suzukii:D. melanogaster puparia ratios =1 or <1 there was significantly higher mortality of D. melanogaster puparia (Figure 3A). The reproductive success of the Ta_Ds lineage differed significantly between treatments (H_{(17, n = 180)} = 133.622, p < 0.001), and also exhibited a similar trend to that of the Ta_Dm lineage concerning tested D. suzukii:D. melanogaster puparia ratios. When D. suzukii puparia ratios were 2-, 4- and 9-fold higher than that from D. melanogaster puparia, there was a significantly higher emergence of parasitoid offspring from D. suzukii puparia (Figure 3B). When the D. suzukii puparia ratio was 1.5-fold higher than D. melanogaster there was similar Ta_Ds offspring emergence from both drosophilid hosts. In contrast, at D. suzukii:D. melanogaster puparia ratios =1 or <1, the Ta_Ds offspring from D. suzukii puparia was significantly lower than that from D. melanogaster puparia (Figure 3B).

The offspring sex ratio of the two T. anastrephae lineages was similar in both choice and non-choice tests (

Table 1. Female offspring percentage (sex ratio) recorded from both Trichopria anastrephae lineages, one reared on Drosophila suzukii (Ta_Ds) and the other one on Drosophila melanogaster (Ta_Dm), at different ratios of D. suzukii (Ds) over D. melanogaster (Dm).

Parasitoid Lineages/Host Species	Parasitoid Females Offspring Percentage (Median and Range); (Treatments: Drosophila suzukii:Drosophila melanogaster Puparia Ratios)
	No-Choice Tests	Choice Tests
	50:0/0:50	45:5	40:10	35:15	30:20	25:25	20:30	15:35	10:40	5:45
TaDs − Ds	47 (44−62) a	57 (43−67) a	57 (42−67) a	51 (38−77) a	60 (46−80) ab	60 (50−100) ac	65 (25−67) a	67 (33–100) a	50 (0–100) a	100 (0–100) a
TaDm − Ds	56 (46–61) a	58 (46−70) a	58 (46−70) a	62 (46−73) a	65 (46−80) a	67 (40−80) a	58 (40–80) a	67 (50–67) a	50 (33–100) a	25 (0–100) a
TaDs − Dm	52 (45–60) a	0 (0−100) a	58 (0−100) a	58 (0−80) a	55 (0−72) ab	60 (0−67) ac	59 (0–67) a	59 (54–72) a	56 (40–75) a	59 (47–64) a
TaDm − Dm	53 (43–61) a	58 (0−100) a	50 (0−100) a	50 (0−80) a	53 (0–67) b	50 (0−75) bc	51 (0–66) a	64 (59–75) a	50 (37–65) a	58 (48–70) a
Statistical results:
H=	3.91	2.86	1.10	2.13	7.76	9.46	2.26	5.41	1.52	1.51
df=	3	3	3	3	3	3	3	3	3	3
n=	40	40	40	40	40	40	40	40	40	40
p=	0.270	0.410	0.780	0.550	0.050 *	0.020 *	0.520	0.140	0.680	0.680

Different letters in the same column represent significant differences at p > 0.05 (Dunn’s test). * Significant variation.

). Trichopria anastrephae mainly exhibited a female-biased sex ratio (1.1–1.8:1 females per male), and there was no significant difference in offspring sex ratio when choice tests were compared with each other (H_{(8, n = 360)} = 12.760, p = 0.120).

3.2. Host Preference and Switching Indexes

In the treatment with an equal proportion of D. suzukii and D. melanogaster, the parasitism ratio on both drosophilid hosts ranged from 0.55 (0.50–0.61, 0.40–0.67) (Median, IQR (25th–75th percentiles), and minimum and maximum data (range)) for Ta_Dm lineage to 0.56 (0.54–0.63, 0.50–0.69) for Ta_Ds lineage. The “C” index was <1, indicating a preference of T. anastrephae for D. melanogaster. Statistical comparison between both parasitoid lineages indicated no significant differences (U = 116.0, n = 10, p = 0.427).

In treatments with unequal proportions of D. suzukii and D. melanogaster, the observed parasitism was significantly higher than expected at 45:5 and 40:10 D. suzukii:D. melanogaster ratios for both parasitoid lineage (

Table 2. Host switching behavior by two Trichopria anastrephae lineages, one reared on Drosophila suzukii (Ta_Ds) and the other one on Drosophila melanogaster (Ta_Dm), to different ratios of D. suzukii over D. melanogaster.

Observed (Obs)/ Expected (Exp) Parasitism	Switching Tests: Drosophila suzukii/Drosophila melanogaster Puparia Ratios (Median and Range)
	45:5	40:10	35:15	30:20	20:30	15:35	10:40	5:45
TaDs − Obs	2.5 (1.3–4.3) a	1.6 (0.9–1.8) a	1.3 (1.0−2.0) a	0.8 (0.6–1.2) b	0.6 (0.4–0.8) a	0.5 (0.2–0.7) a	0.3 (0.2–0.8) a	0.3 (0.2–0.7) a
TaDm − Obs	1.8 (1.2–3.8) a	2.1 (1.0–3.3) a	1.3 (0.9−2.7) a	0.9 (0.8–1.3) b	0.6 (0.4–0.8) a	0.5 (0.3–0.7) a	0.4 (0.2–0.8) a	0.3 (0.3–0.6) a
TaDs − Exp	−1.7 (−1.5–−3.5) b	−3.0 (−2.0–−6.3) b	−7.0 (−6.0−35.0) a	2.5 (1.9–3.0) a	0.5 (0.5–0.6) a	0.3 (0.2–0.3) b	0.2 (0.1–0.2) b	0.1 (0.06–0.1) b
TaDm − Exp	−1.6 (−0.8–−3.1) b	−2.7 (−1.1–−5.0) b	−2.5 (−2.3−35.0) a	2.6 (2.1–6.0) a	0.5 (0.4–0.6) a	0.3 (0.2–0.3) b	0.2 (0.1–0.2) b	0.1 (0.05–0.1) b
Statistical results:
H=	29.676	29.657	1.443	29.519	2.043	20.544	18.177	29.796
df=	3	3	3	3	3	3	3	3
n=	40	40	40	40	40	40	40	40
p=	<0.001 *	<0.001 *	=0.695	<0.001 *	=0.564	<0.001 *	<0.001 *	<0.001 *
Obs/Exp results:	Obs > Exp	Obs > Exp	Obs = Exp	Obs < Exp	Obs = Exp	Obs > Exp	Obs > Exp	Obs > Exp

Different letters in the same column represent significant differences at p > 0.05 (Dunn’s test). * Significant variation.

), showing that host switching occurred in both host proportions, in which T. anastrephae preferentially attacked D. melanogaster. The observed and expected parasitism were not significantly different at 35:15 and 20:30 D. suzukii:D. melanogaster ratios for either parasitoid lineage (Table 2), which means that T. anastrephae attacked indifferently one or another host. The expected parasitism was significantly higher than observed at the 30:20 D. suzukii:D. melanogaster ratio for both parasitoid lineages (Table 2). This suggests that the host switch did not occur because T. anastrephae mainly attacked D. melanogaster, despite a higher proportion of D. suzukii puparia. The observed parasitism was significantly higher than expected at 15:35, 10:40, and 5:45 D. suzukii:D. melanogaster ratios for both parasitoid lineages (Table 2). These findings indicate that the host switch occurred because T. anastrephae primarily attacked D. melanogaster over D. suzukii.

4. Discussion

The current study provides information on the D. suzukii-killing capacity of T. anastrephae for two parasitoid lineages faced with D. suzukii and the native host D. melanogaster, and the subsequent parasitoid reproductive success in terms of the offspring’s survival. Based on these parameters, the study reports host preference and host-switching behavior at various ratios of D. suzukii and D. melanogaster.

The results show that regardless of the population lineage, T. anastrephae performed similarly well on D. suzukii and D. melanogaster with regard to the percentage of killed hosts and offspring survival when there was no choice between hosts. The high mortality of D. suzukii recorded in these tests supports previous studies on parasitism levels of a T. anastrephae Brazilian population line on D. suzukii under laboratory conditions [37,52,53,54,55] and in greenhouse trials [56]. Similarly, Trichopria drosophilae Perkins, another resident pupal parasitoid found parasitizing D. suzukii in North America [57,58], Central America [59], Europe [60,61,62], and Asia [63] is also able to induce high mortality of D. suzukii puparia.

The results also revealed that both lineages of T. anastrephae mainly focused on attacking D. suzukii puparia at very high proportions of this host relative to D. melanogaster in choice situations. However, at higher proportions but no more than twice as many D. suzukii over D. melanogaster, T. anastrephae females attacked both hosts equally. At equal proportions of both hosts, T. anastrephae focused its attack on D. melanogaster, but the parasitoid intensified its parasitism on D. melanogaster at higher proportions of native host over the exotic host in choice tests. In contrast to the present study, the cosmopolitan T. drosophilae showed a slight preference to parasitize D. suzukii puparia than Drosophila immigrans Sturtevant or D. melanogaster puparia in laboratory studies when the same host-to-host ratio was assessed [63]. However, the percentages of T. drosophilae male and female offspring recorded from D. suzukii puparia individually compared with those emerged from D. immigrans were similar [63]. These results suggest that T. drosophilae, like T. anastrephae, is naturally associated with saprophytic drosophilids. However, the larger size of the D. suzukii puparium compared to that of D. melanogaster appears to be a factor driving the host preference of T. drosophilae for D. suzukii [57,58,63,64,65]. That would not be the case with T. anastrephae; despite the small size of D. melanogaster puparium, the parasitoid prefers it in an equal host-choice condition. Other biological factors may be influencing host preference by T. anastrephae. Probably, olfactory cues associated with the native host puparium play a relevant role in host searching behavior of T. anastrephae, in addition to the host-habitat (fruit) olfactory stimuli, which have a significant influence in the long-range host location [56].

The preference for D. melanogaster displayed by the T. anastrephae in the current studies matches with field survey data recorded from non-crop fruits in wild environments from northwestern Argentina [26,27]. Those wilderness areas involved patches of secondary structure Yungas subtropical rainforest with a mix of feral exotic fruits plus native fruit species. Field studies found that T. anastrephae parasitized more resident saprophytic drosophilid puparia than D. suzukii. However, it is worth noting that an extremely high abundance of saprophytic drosophilids over D. suzukii was always present in the fallen fruits surveyed [27]. Therefore, that a disproportional natural relationship between D. melanogaster and D. suzukii puparia likely influenced the host preference displayed by T. anastrephae, as evidenced by the results of the current tests. There was only host switching to D. suzukii when the host ratio was 80–90% D. suzukii over D. melanogaster. These findings are supported by differences in the evolutionary history between T. anastrephae and both hosts because it is a Neotropical-native parasitoid species [66] coevolved in sympatry with saprophytic drosophilid species, such as those of the D. melanogaster group [26]. This may account for the close trophic association between T. anastrephae and non-pest saprophytic drosophilids, whereas a new trophic association was recently established with D. suzukii. Although T. anastrephae quickly adapted to D. suzukii, the prevalence of puparia from native drosophilid hosts inside fallen fruit in natural environments may reduce the effectiveness of this resident parasitoid on the exotic pest [26]. However, the results of the current study show that although T. anastrephae preferred D. melanogaster in a choice condition at equal host ratios, T. anastrephae was an effective natural enemy as it achieved mortality rates on D. suzukii between 40% and 42%. Even at host ratios where D. suzukii was disadvantaged, close to 50% of the D. suzukii puparia were parasitized by T. anastrephae, and the parasitoid offspring was always female-biased. Such findings are significant because they highlight T. anastrephae as an important D. suzukii mortality agent. In both Drosophila species studied, the host mortality caused by T. anastrephae was high. An additional mortality factor might be due to superparasitism, particularly when considering the relationship between the low density of exposed hosts and the long host exposure time in the tests. For the current study, verifying whether the host mortality was also caused by superparasitism was not achievable. However, a second step of the study has been planned to ascertain whether superparasitism influenced high host mortality levels reported herein.

Recent studies [35] showed that T. anastrephae can also forage D. suzukii puparia outside the fruit pulp, where saprophytic dipteran puparia do not prevail. Thus, T. anastrephae can parasitize D. suzukii puparia in less competitive microhabitats, such as the ground beneath fallen feral peaches and guavas in non-crop areas [35]. Previous studies have shown that most D. suzukii larvae pupate on the ground beneath fallen fruit [57,58,59,60,61,62,63,64,65,66,67,68], and puparia can be attacked by pupal parasitoids [56,69]. These earlier published data, plus information from the current study, reveal that T. anastrephae may exert mortality over D. suzukii puparia isolated on the ground. Augmentative releases [60] or conservation biological control [70] are strategies that can be focused from a D. suzukii area-wide integrated management approach [36,71]. Sustainable pest management must focus on landscape biodiversity conservation and enhanced natural control [72].

5. Conclusions

The results suggest that T. anastrephae was effective in killing D. suzukii based on its performance relative to the pest and producing reproductively successful offspring. Given the preference of T. anastrephae for resident saprophytic drosophilids, the concurrent presence of those hosts in equal or higher ratios to that of D. suzukii in the same microhabitat influenced on the performance of the parasitoid. However, the mortality of D. suzukii by T. anastrephae under such conditions was significant. Optimizing important biological traits can be explored through selective rearing in studies based on the experimental adaptation of resident parasitoids on D. suzukii. This would provide a more specific biological control agent targeted at the pest.