Introduction

Trichopria anastrephae Costa Lima, 1940 (Hymenoptera: Diapriidae) is a parasitoid species endemic to South America (Cruz et al. 2011). It is classified as an idiobiont pupal endoparasitoid and deposits eggs in the hemocoel of fruit fly puparia (Wang et al. 2018; Gonzalez-Cabrera et al. 2019). In Brazil, it has been found on strawberry fruits infested by Drosophila suzukii Matsumura, 1931 (Diptera: Drosophilidae) (Wollmann et al. 2016; Andreazza et al. 2017a), a pest insect considered of major agricultural importance in small fruit crops worldwide (Walsh et al. 2011; Calabria et al. 2012; Cini et al. 2012), and in Brazil as of the year 2014 (Deprá et al. 2014; Schlesener et al. 2015; Andreazza et al. 2017b; Dos Santos et al. 2017).

In D. suzukii puparia, females of the genus Trichopria have the ability to cause the mortality of 100 individuals per generation (Yi et al. 2020). Trichopria anastrephae has a life cycle (egg to adult) of approximately 21 days (Krüger et al. 2019; Vieira et al. 2020) and demonstrates a high capacity for interspecific competition with the pupal parasitoid Pachycrepoideus vindemmiae Rondani, 1875 (Hymenoptera: Pteromalidae), also on D. suzukii pupae (Oliveira et al. 2020).

Because D. suzukii larvae have a well-developed immune system, producing a physiological response by increasing the amount of hemocytes and encapsulating the immature stages of larval parasitoids, the use of species such as Asobara japonica Belokobylskij, 1998 (Hymenoptera: Braconidae), Ganaspis brasiliensis Ihering, 1905, and Leptopilina spp. (Hymenoptera: Figitidae) (Poyet et al. 2013; Iacovone et al. 2018; Wang et al. 2021) can become compromised, so the use and conservation of the parasitoid pupal T. anastrephae are even more important (Kacsoh and Schlenke 2012).

Although the parasitoid T. anastrephae is effective against D. suzukii (Krüger et al. 2019; Vieira et al. 2020), chemical control with insecticides broad spectrum, including organophosphates, spinosyns, pyrethroids, neonicotinoids, and avermectins is the most used approach for pest management worldwide (Haye et al. 2016; Andreazza et al. 2017b). In Brazil, spinetoram (spinosyns) is the only insecticide registered for the control of D. suzukii to date (Agrofit 2021). In view of this, the insecticides used are those recommended for other fly species, such as Anastrepha fraterculus (Wiedemann, 1830) and Ceratitis capitata (Wiedemann, 1824) (Diptera: Tephritidae) (Andreazza et al. 2017b; Schlesener et al. 2019; Morais et al. 2021).

While biological control is a promising alternative for D. suzukii management (Schetelig et al. 2018; Gonzalez-Cabrera et al. 2019; Krüger et al. 2019; Lee et al. 2019), the frequent application of insecticides each season can have lethal and/or sublethal effects on T. anastrephae and also impair the population density and biological performance of the parasitoid over generations (Costa et al. 2014; Beloti et al. 2015; Schlesener et al. 2019; Morales et al. 2020). Therefore, the objective of the present study was to evaluate the lethal (mortality) and transgenerational effects on parasitism, emergence, sex ratio, and survival in F0, F1, and F2 generations of five insecticides widely used in Brazilian fruit growing on T. anastrephae adults.

Material and Methods

Insects

Drosophila suzukii was reared on an artificial diet based on cornmeal, yeast, and sugar as proposed by Schlesener et al. (2017). Trichopria anastrephae parasitoids were reared and multiplied in D. suzukii puparia as per Vieira et al. (2020). Both were kept in a climate-controlled room with a temperature of 25 ± 2 °C, relative humidity of 70 ± 10%, and photophase of 12 h.

Insecticides

We used commercial formulations of five insecticides, representing the main chemical groups used to control arthropod pests of fruit trees grown in Brazil (Table 1). The applied doses followed the recommendations of the manufacturer’s package inserts and were diluted in distilled water (Table 1).

Table 1 Commercial insecticides used to assess lethal and sublethal toxicity in Trichopria anastrephae
Full size table

Bioassay of toxicity and transgenerational effects

First, 24-h-old D. suzukii pupae were separated and deposited on Petri dishes (8-cm diameter) (approximately 150 pupae/treatment). Subsequently, insecticides diluted in distilled water (treatments) were applied by spraying via a calibrated Potter tower to deposit a volume of 1.25 ± 0.25 mg cm−2 according to the protocols established by the IOBC (Hassan et al. 2000). Distilled water was used as a negative control. The pupae were kept for one hour on filter paper at a temperature of 25 ± 2 °C and relative humidity of 70 ± 10% to allow evaporation of excess spray. After that time, 15 pupae exposed to the treatments were offered for parasitism to each pair of T. anastrephae (aged 24 h) in plastic cages made from acrylic tubes (2.5-cm diameter × 4.5-cm height) closed at the top with voile fabric (Vieira et al. 2020). The adults of T. anastrephae were fed with pure honey droplets and the pupae were exposed to parasitism for 24 h. After this period, pupae were removed and packed in new acrylic tubes (until emergence, approximately 18 days under bioassay conditions) and T. anastrephae pairs were kept in 2.5 cm × 8.5 cm flat-bottomed glass tubes closed at the top with voile tissue. The experimental design was entirely randomized, with six treatments and 10 replicates (acrylic tubes) per treatment, with each replicate consisting of a pair of T. anastrephae. The biological parameters evaluated were mortality of T. anastrephae after 24 h in contact with pupae contaminated by the treatments, pupal parasitism defined by counting emerged parasitoids and opening puparia without emergence to verify the presence of D. suzukii or T. anastrephae, the emergence of parasitoids and sex ratio [Σ♀/Σ (♀ and ♂)].

From the total number of emerged individuals from each treatment (F1 generation), a minimum of five and a maximum of 15 pairs of T. anastrephae were separated and placed in acrylic cages, as mentioned above, and offered 15 pupae of D. suzukii (24 h of age) for a period of 24 h. After this time, the parasitoids were removed and placed in new cages (acrylic tubes) and fed with droplets of pure honey to evaluate the survival of males and females. Pupae were placed in an acclimatized room with a temperature of 25 ± 2 °C to evaluate parasitism, emergence, and the sex ratio of F2 generation T. anastrephae.

IOBC classification

To determine toxicity classes, the reduction (R) in parasitism (F0 and F1) and emergence (F1 and F2) was calculated using the equation R = 100 − [(insecticide treatment value/control value) × 100], according to Hassan (1994). Then, insecticides were grouped into toxicity classes according to the calculated values based on the criteria established by the International Organization for Biological and Integrated Control (IOBC) (Hassan et al. 2000). The toxicity classes were class 1 = harmless (R < 30%); 2 = slightly harmful (R = 30–79%); 3 = moderately harmful (R = 80–99%); and 4 = harmful (R > 99%).

Data analysis

The data obtained were tested for normality by the Shapiro–Wilk test and for homogeneity of variance by the Bartlett test. When these assumptions were not met, the data were subjected to non-parametric Kruskal–Wallis analysis of variance, and means were compared using the Dunn test with Bonferroni correction at 5% error probability. For the survival curve, data on the longevity of individuals were used to estimate survival curves using the Kaplan–Meier estimator and comparing the survival curves by the log-rank test through the program SigmaPlot (v.12.5, Systat Software Inc., California, USA). Statistical analyses were performed with R version 4.0.0 software (R Development Core Team 2020).

Results

Toxicity on T. anastrephae and on biological parameters in F 0

When adult parasitoids were exposed to D. suzukii pupae treated with insecticides, significant mortality values were observed among the treatments evaluated (Kw = 44.51; df = 5, 54; p-value < 0.001) (Table 2). The insecticide malathion showed the highest acute toxicity, causing 100% mortality of T. anastrephae adults (Table 2). By contrast, the insecticides deltamethrin and spinetoram caused 60 and 50% mortality of T. anastrephae, respectively (Table 2), whereas thiamethoxam and abamectin were statistically similar to the control treatment (Table 2). Regarding parasitism rate, significant differences (Kw = 47.62; df = 5, 54; p-value < 0.001) were found in all treatments compared to the control. The lowest parasitism rates were seen for the insecticides malathion (P = 0.60 parasitized pupae) and deltamethrin (P = 1.20 parasitized pupae) (Table 2). These produced the greatest reductions in parasitism, and thus, malathion (PR = 95.27%) and deltamethrin (PR = 90.50%) were classified as moderately harmful (class 3) (Table 2). Conversely, the insecticides thiamethoxam (RP = 55.11%), spinetoram (RP = 48.81%), and abamectin (RP = 32.28%) were classified as slightly harmful (Class 2) (Table 2).

Table 2 Mortality, parasitism rate, parasitism reduction, and IOBC classification for F0 generation of Trichopria anastephae when subjected to different insecticides
Full size table

Transgenerational effects in the F1 generation

The emergence rate of the F1 generation of T. anastrephae was significantly affected by the tested insecticides (Kw = 46.29; df = 5, 54; p-value < 0.001). The control treatment had a mean of 12.20 emerged parasitoids (Table 3), while the lowest emergence rates were caused by the insecticides malathion, with 0.50 parasitoids (SR = 95.90%), and deltamethrin with 1.20 parasitoids (SR = 90.16%) (Table 3), which were thus classified as moderately harmful (class 3). By contrast, the insecticides thiamethoxam (RE = 53.27%) and spinetoram (RE = 48.36%) were classified as slightly harmful (class 2) and abamectin (RE = 29.50%) as harmless (class 1) (Table 3). No significant differences were observed in the sex ratio among the treatments (Kw = 9.12; df = 5, 54; p-value = 0.10 (Table 3) or with respect to the parasitism rates of the F1 generation (Kw = 9.94; df = 4, 60; p-value = 0.04) (Table 3). Based on the percentage reduction of F1 parasitism, the insecticides were classified as harmless (class 1) (Table 3).

Table 3 Emergence rate and parasitism, emergence reduction and parasitism, sex ratio, and IOBC classification for F1 generation of Trichopria anastephae when subjected to different insecticides
Full size table

The survival curves of males and females of the F1 generation of T. anastrephae showed a significant difference between treatments (males: χ2 = 77.4; df = 4; p-value < 0.001, females: χ2 = 77.9, df = 4, p-value < 0.001) (Fig. 1). The insecticides deltamethrin (10.80 days) and thiamethoxam (13.06 days) caused the greatest reductions in male insect survival, relative to the control treatment (29.73 days) (Fig. 1A). The same pattern was observed for T. anastrephae females (Fig. 1B). Abamectin did not affect the longevity of males and females of T. anastrephae compared to the control treatment (Fig. 1A and B).

Fig. 1
figure 1

Survival curve of adults of the F1 generation of Trichopria anastrephae, A males and B females, when subjected to different insecticides. The curves were generated using Kaplan–Meier estimators and compared using the log-rank test (P < 0.05)

Full size image

Transgenerational effects in the F2 generation

No significant differences were observed in the emergence rate of T. anastrephae in the F2 generation (Kw = 9.77; df = 4, 60; p-value = 0.04) between the treatments evaluated (Table 4). In view of this, all the insecticides evaluated were classified as innocuous (class 1). Similarly, no significant differences were observed in the sex ratio of T. anastrephae of the F2 generation (Kw = 1.49; df = 4, 60; p-value = 0.83) (Table 4).

Table 4 Emergence rate, emergence reduction, IOBC rating and sex ratio for F2 generation of Trichopria anastrephae when subjected to different insecticides
Full size table

Discussion

Chemical control involving insecticides is an important part of pest arthropod management in Brazilian fruit production. However, the ecological services provided by natural enemies are now receiving due recognition as more sustainable practices are sought. Therefore, studies aiming to evaluate the compatibility between insecticides and natural enemies, within the precepts of IPM, are indispensable (Roubos et al. 2014). In this study, all insecticides tested have neurotoxic effects, and given that there are similarities between the modes of nerve impulse transmission between different animal phyla, they are usually classified as less selective to non-target organisms (Amarasekare et al. 2016; Fontes et al. 2018). The fact was verified about T. anastrephae adults in F0 generation, with emphasis on the organophosphate insecticide.

The highest mortality caused by malathion (F0 generation of T. anastrephae) may have occurred by contact or inhalation during the period of exposure to parasitism, and the lethal impact of this organophosphate insecticide may be attributed to the rapid transformation of oxygenase enzymes, thus inhibiting the action of acetylcholinesterase and other enzyme systems that perform detoxification (Büyükgüzel 2006). These results are similar to those found when T. anastrephae adults were exposed to dry insecticide residues via the tarsal contact method (Schlesener et al. 2019) and for the parasitoid Palmistichus elaeisis Delvare & LaSalle, 1993 (Hymenoptera: Eulophidae) exposed to the insecticide malathion (100% mortality) (Cruz et al. 2017).

In addition to providing high toxicity on T. anastrephae adults, in the F0 generation, all insecticides significantly affected the rate of parasitism of T. anastrephae. This may be attributed to the fact that neurotoxic insecticides affect the neurosecretory system of arthropods, and since reproduction is regulated by hormones, the insecticides may have caused the hormonal imbalance in the insects, interfering in parasitism of the species (Maddrell and Reynolds 1972). Similar results were reported by Fontes et al. (2018) for Trichogramma achaeae Nagaraja and Nagarkatti, 1971 (Hymenoptera: Trichogrammatidae) for the insecticides abamectin and thiamethoxam, when sprayed on host eggs. The results for deltamethrin may be directly related to the high toxicity, penetration capacity, and fast action of the product since they act as modulators of sodium channels, causing paralysis and physiological and behavioral changes. The repellent action of this chemical may also have contributed to the reduction of parasitism of T. anastrephae (Bos and Masson 1983; Costa et al. 2020). Consequently, the emergence rate of the F1 generation was also significantly affected, such that malathion and deltamethrin reduced emergence by more than 90%.

Despite their negative effects on the emergence of F1 generation adults, the insecticides did not affect the sex ratio of T. anastrephae. Several studies have shown that agrochemicals products can also cause changes in the sex ratio of beneficial insects. For instance, the organophosphorus insecticide chlorpyrifos modified the sex ratio of the offspring of several Hymenoptera parasitoid (Delpuech and Meyet 2003), whereas imidacloprid significantly changed the sex ratio of the progeny of Encarsia inaron Walker (Hymenoptera: Aphelinidae) by increasing the number of male offspring (Sohrabi et al. 2012). However, the mechanisms underlying the change in the sex ratio of beneficial arthropods caused by insecticides have not been evaluated yet. In the present study, the proportion of the sex ratio of T. anastrephae has not changed. This lack of effect on the sex ratio emphasizes the potential of this species as a biological control agent for D. suzukii. By contrast, negative effects, as reported by Costa et al. (2014) for Trichogramma galloi Zucchi, 1988, would reduce the proportion of females, making it impossible for them to perform their ecological role.

The insecticides deltamethrin and thiamethoxam caused the greatest reductions in the survival of T. anastrephae. The active ingredient thiamethoxam, belonging to the neonicotinoid group, is a competitive agonist of nicotinic acetylcholine receptors, being able to induce continuous excitation in neuronal membranes, which results in discharges, paralysis, and the depletion of cellular energy. Pazini et al. (2019) demonstrated similar results regarding the survival of the F1 generation of Telenomus podisi Ashmead, 1893 (Hymenoptera: Platygastridae) when eggs of Euschistus heros Fabricius, 1798 (Hemiptera: Pentatomidae) were exposed to thiamethoxam prior to parasitism by F0. As well as, the effect of deltamethrin on the nervous system of T. anastrephae affected adult emergence and subsequent longevity of insects (Garcia et al. 2006). Therefore, the use of deltamethrin-based insecticides in pest management will provide difficulties for the parasitoid to find the host and negatively affect the parasitism of D. suzukii.

The F2 generation of T. anastrephae was not affected by any of the treatments, so all insecticides were classified as innocuous at this stage. Similar results were found by Beloti et al. (2015) for Tamarixia radiata Waterston, 1922 (Hymenoptera: Eulophidae) when treatments were sprayed on orange tree discs and the F0 parasitoids remained in contact for 24 h. Likewise, Paiva et al. (2018) reported that when treatments were sprayed on eggs of Ephestia kuehniella Zeller, 1879 (Lepidoptera: Pyralidae) prior to parasitism by the F0 generation of Trichogramma pretiosum Riley, 1879 (Hymenoptera: Trichogrammatidae), there were no transgenerational effects on the F2 generation. However, it is worth noting that the present study was conducted under laboratory conditions, so the parasitoid was exposed to the worst possible conditions. Thus, studies in semi-field and field situations should be conducted since the effects of the environment, such as sunlight (Paiva et al. 2018), can accelerate the degradation of the chemicals and lead to lower acute and sublethal toxicity than in the laboratory setting.

The findings in this study will contribute to the development of IPM programs in which the integration of chemical and biological control is sought of D. suzukii; this is because we have the constant occurrence of T. anastrephae, a recurrent species in small fruit crops infested by these flies in Brazil (Wollmann et al. 2016, Bernardi et al. 2017). Although the insecticides evaluated in this study (abamectin, deltamethrin, malathion, spinetoram, and thiamethoxam) do not show transgenerational effects on T. anastrephae, the use of these products in the management of D. suzukii and other arthropod pests that infest the strawberry crop must be used with care, so as not to harm the natural biological control of D. suzukii or in a possible mass release program of this parasitoid in the field (Krüger et al. 2019; Vieira et al. 2020). Thus, in areas with the occurrence of T. anastrephae, producers should use alternative products, such as plant extracts and essential oils or products based on azadirachtin (De souza et al. 2021), as they do not present toxicity to the parasitoid and are toxic for D. suzukii adults.