Essential Oil of Rosmarinus officinalis Ecotypes and Their Major Compounds: Insecticidal and Histological Assessment Against Drosophila suzukii and Their Impact on a Nontarget Parasitoid

Abstract

Essential oils (EOs) produced by plants in the Lamiaceae family may provide new insecticidal molecules. Novel control compounds are needed to control Drosophila suzukii (Matsumura), a severe economic invasive pest of thin-skinned fruit crops. Thus, we characterized the main compounds of EOs from three rosemary Rosmarinus officinalis ecotypes (ECOs) and evaluated their toxicity to D. suzukii adults, deterrence of oviposition behavior, and histological alterations in larvae. Additionally, we analyzed the lethal and sublethal effect on the pupal parasitoid Trichopria anastrephae. The main compounds identified in the R. officinalis ECOs were α-pinene, camphor and 1,8-cineole. In bioassays via topical application or ingestion, ECOs and their major compounds showed high toxicity on D. suzukii adults and a lower concentration could kill 50% and 90% of flies compared to spinetoram. The dry residues of a-pinene, 1,8-cineole, and camphor provided a repellent effect by reducing D. suzukii oviposition by ~47% compared to untreated fruit. Histological sections of 3^rd instar larval D. suzukii posttreatment revealed damage to the fat body, Malpighian tubules, brain, salivary gland, and midgut, which contributed to high larval and pupal mortality. Survival and parasitism by adult T. anastrephae were not affected. Thus, R. officinalis EO and their compounds have potential for developing novel insecticides to manage D. suzukii.

Native to Asia, the spotted-wing drosophila Drosophila suzukii (Matsumura) (Diptera: Drosophilidae) is an invasive pest that has caused significant losses in several fruit-producing countries, mainly of thin-skinned fruits (De La Veja et al. 2020, Rota-Stabelli et al. 2020). Economic impacts of this pest have been documented in North America (Dreves 2011, Goodhue et al. 2011, Farnsworth et al. 2017), South America (Santos 2014), and Europe (De Ros et al. 2015). In the United States, D. suzukii has caused annual losses of approximately US$511 million in small fruit production (Goodhue et al. 2011). In Brazil, strawberry (Fragaria × ananassa Duchesne) producers have reported losses in production of up to 30% per year (Santos 2014, Andreazza et al. 2016), without accounting for the additional costs generated by the monitoring and control to prevent initial infestations (Dreves 2011, Goodhue et al. 2011, Lee et al. 2011).

The species represents a global phytosanitary threat (Hamby et al. 2016) due to its wide host range (Lee et al. 2015, Poyet et al. 2015), high dispersion capacity (Little et al. 2020), and rapid generation time (Sánchez-Ramos et al. 2019, Schlesener et al. 2020, Spitaler et al. 2020). Therefore, the application of organophosphate, pyrethroid, and spinosyn synthetics insecticides are adopted worldwide (Gress and Zalom 2019, Mermer et al. 2020, Santoiemma et al. 2020). However, their indiscriminate use can cause environmental contamination, toxicological risks, selection of insecticide-resistant populations (Gress and Zalom 2019, Van Timmeren et al. 2019), and adverse effects on beneficial fauna (Biondi et al. 2012, Roubos et al. 2014, Schlesener et al. 2019).

The pupal parasitoid Trichopria anastrephae Lima, 1940 (Hymenoptera: Diapriidae) is a natural enemy of D. suzukii that can be affected by the application of synthetic insecticides in small fruit crops. This species has been reported to parasitize D. suzukii pupae in Brazilian orchards (Wollmann et al. 2016, Andreazza et al. 2017). The occurrence of T. anastrephae in small fruit production areas, such as strawberries and Rubus spp., is indicative of their establishment in the field (Wollmann et al. 2016), which has led to an interest in mass rearing of the parasitoid in the laboratory (Krüger et al. 2019, Vieira et al. 2019). Considering the importance of T. anastrephae in the biological control of D. suzukii, it is necessary to search for products that do not affect enemies in the field, and that can be used in integrated pest management (IPM) (Bernardi et al. 2017).

In this context, the search for products that cause less adverse impacts on the environment and natural enemies is important for an IPM program (Isman and Grieneisen 2014, Amoabeng et al. 2019). The potential use of vegetable-based insecticides, especially essential oils (EOs) and their individual compounds, can generate alternatives for the control of D. suzukii (Isman and Grieneisen 2014, Bernardi et al. 2017, Amoabeng et al. 2019, Souza et al. 2020a). Also, products derived from plants have several chemical compounds in a single EO, which contributes to the increase in control efficiency and can prevent or delay the evolution of pest resistance by acting on different sites (Isman 2000).

Among the plants with potential insecticidal activity, plants in the Lamiaceae family stand out. Rosmarinus officinalis L., popularly known as rosemary, is harvested for EO extraction (Sayorwan et al. 2013). Chromatography analyses of the EOs present in this species have identified α-pinene, 1,8-cineole, borneol, bornyl acetate, camphor, and verbenone as the most abundant and frequent compounds (Isman et al. 2008, Sayorwan et al. 2013). However, R. officinalis, like any other plant, can change its chemical and genetic composition due to the different environmental pressures to which the plant is exposed (for example, herbivory, genotype, geographical origin, soil, and climate), resulting in ecotypes (ECOs) (Feitosa-Alcantara et al. 2017). Thus, when testing EOs, it is important to analyze the chemical variability of the oil and determine which compounds act as insecticides on the pest (Isman et al. 2008).

The present study aimed to 1) identify and quantify the EOs composition of three R. officinalis ECOs collected in different locations in the Neotropical region; 2) evaluate the EOs toxicity of the R. officinalis ECOs and their main compounds on D. suzukii adults; 3) investigate the effects of EOs dry residues and their main compounds on the oviposition behavior of D. suzukii; 4) assess the histological damage in D. suzukii larvae induced by R. officinalis EOs and its main compounds to demonstrate the target sites where the biopesticides acted; and 5) analyze the toxicity of R. officinalis EOs and their individual compounds to the parasitoid T. anastrephae.

Material and Methods

Rearing of Drosophila suzukii and Trichopria anastrephae

The 30th generation of D. suzukii was established from a population collected in an organic strawberry plantation in Curitiba, Paraná state, Brazil (31° 38′ 20′′ S, 52° 30′ 43′′ W) in 2018. Flies were reared in 300 ml glass flasks containing 12 ml of an artificial diet based on cornmeal, sugar, and yeast (Schlesener et al. 2018). The 25th generation of T. anastrephae was established from organic blackberry fruits infected by D. suzukii collected in Pelotas, Rio Grande do Sul state, Brazil (31°38′ 20″ S, 52° 30′ 43″ W) in 2018. Parasitoids were reared on D. suzukii pupae and fed with a 10% honey solution (Vieira et al. 2019). Both species were kept at 25 ± 1 °C, 70 ± 5% RH, and a 12:12 hr photoperiod (L:D).

Plant Material and Essential Oil Chemical Analysis

For the extraction of EOs, fresh leaves of R. officinalis collected in Pinhais, Paraná (25° 23′ 30″ S, 49° 07′ 30″ W), Taquarituba, São Paulo (23° 31′ 59″ S, 49° 1′ 40″ W), and Botucatu, São Paulo (22° 53 ′ 21.40″ S, 48° 28 ′ 12.23″ W) were subjected to hydrodistillation in a Clevenger-type apparatus for 4 hr. Anhydrous sodium sulfate was used to separate the hydrolate, and the samples were kept at −20°C before undergoing chemical analysis. The common compounds α-pinene, 1, 8-cineol, and camphor were found in proportions greater than 20% in the EOs of at least one of the EO samples analyzed from R. officinalis. Therefore, these compounds were thus selected for bioassays. Specifically, commercially available α-pinene (CAS: 7785-70-8), 1,8-cineole (CAS: 470-82-6), and camphor (CAS: 76-22-2) were obtained from Sigma–Aldrich Brazil (São Paulo, Brazil); all compounds were >99% pure.

An aliquot of 1 μl of each of the EOs was analyzed using gas chromatography with detection by flame ionization (CG-DIC 2010 – Shimadzu). Mass spectrometry (CG-EM 2010 Plus – Shimadzu) in splitless mode was used for some samples as well. In both analyses, an RTX-5 chromatographic column was used, measuring 30 m × 0.25 mm d.i. × 0.25 μm of film thickness, obtained from J&W Scientific, Folsom, California, USA. The chromatographic analysis began by subjecting the compounds to a temperature of 50°C for one minute and heating them at a rate of 7°C min⁻¹ to 250°C, which was maintained for 10 min. The temperature of both the injector and detector was maintained at 250°C, with a pressure of 200 kPa for one minute in the injector. The quantification and identification of the EOs’ chemical constituents were performed by comparing their mass spectra with those of commercial libraries (McLafferty and Stauffer 2014) and their linear retention indexes (Van Den Dool and Kratz 1963) after the injection of a series of alkanes (C₈–C₂₆), as well as by comparing them with data from the literature (Adams 2007).

Toxicity Bioassay on Drosophila suzukii

The lethal and sublethal effects of the EOs of R. officinalis (ECO 1, ECO 2, and ECO 3), as well as α-pinene, 1,8-cineole, and camphor, were evaluated by the topical and ingestion methods following methodology from Bernardi et al. (2017) and Souza et al. (2020a, 2021). For the topical application, treatments were sprayed using a Potter Tower (Burkard Scientific, Uxbridge, UK), spraying 1 ml of solution per sample unit at a working pressure of 0.049 MPa. After spraying, the insects were placed inside plastic cages (1 liter) sealed with voile mesh and fed an artificial diet and distilled water throughout the evaluation period. In the ingestion bioassay, the treatments were supplied to the flies by a soaked cotton wick inside a 10 ml glass bottle. After 8 hr of exposure, the treatments were removed and replaced with an artificial diet and distilled water until the end of the evaluation period. All bioassays were performed under laboratory conditions of 25 ± 1°C, 70 ± 5% RH, and 12:12 hr (L: D) photoperiod in a completely randomized design.

Concentration–Response Curves Against Drosophila suzukii

The bioassays were carried out in two stages: 1) Pretests to estimate the lethal concentration to kill 50% (LC₅₀) and 90% (LC₉₀) of the flies exposed to the maximum 80 mg liter⁻¹ concentration in each treatment. Seven concentrations were obtained through serial dilutions in acetone: 2.5, 5.0, 7.5, 10, 20, 40, and 80 mg liter⁻¹. 2) Tests were carried out, with the ingestion bioassay using eight pairs of adults of approximately 72 hr-old D. suzukii per concentration and five replicates per treatment (n = 560). Meanwhile, the topical bioassay used four replicates of 20 flies (n = 80), which were also about 72 hr-old, exposed to each concentration (n = 80 per treatment, 560 total) for the topical bioassay. The spinetoram-based chemical insecticide, active ingredient (a.i.) Delegate 250 WG 7.5 g liter⁻¹, was the positive control at the dosage of 75 a.i. per liter of water. Water and acetone were used in the control treatments. The experimental design was completely randomized. Mortality was computed at 1 hr intervals for the first 24 hr posttreatment, and every 24 hr up to 120 hr posttreatment and was calculated using Abbott’s equation (1925).

Oviposition and Larvicidal Bioassay

Males and females of D. suzukii were separated after emergence to prevent mating before oviposition bioassays. When flies were 7 d old, they were placed in oviposition cages containing artificial fruits (Schlesener et al. 2018). These fruits had been previously treated with 1 ml of intact R. officinalis EOs and the main compounds α-pinene, 1,8-cineole, and camphor at the maximum concentration of 80 mg liter⁻¹. After 24 hr, the insects were removed, and eggs in each artificial fruit were counted using a stereoscopic microscope with 40X magnification. The experiment was completely randomized, including 30 replicates per treatment.

Larval toxicity tests with 3rd instar larval D. suzukii were performed following Souza et al. (2021) by setting groups of 20 individuals placed into glass vials of 1.3 cm diameter and 10 cm long. Each vial contained a 2.0 × 2.0 cm filter paper impregnated with 0.2 ml of solution for each treatment. Tests were randomized, with five replicates (n = 100) at the maximum concentration of 80 mg liter⁻¹. Larval mortality, pupation rate, and pupal mortality were assessed (Kumar et al. 2014, Singh and Kaur 2016) at 6, 24, and 48 hr posttreatments.

Histological Analysis

Third instar larval D. suzukii were exposed to the EOs of R. officinalis and their major compounds to a 80 mg liter⁻¹ concentration during 2 hr. Afterward, they were fixed in 10% buffered formalin for 30 min. To prepare slides, six longitudinal cuts were made in paraffin and sectioned into 4 μm thick sections and stained with Harris’ hematoxylin-eosin. The fat body, Malpighian tubules, brain, salivary gland, and midgut of treated larvae were observed and compared to the control negative larvae exposed to acetone. Each treatment included 30 larvae.

Toxicity Bioassay on Trichopria anastrephae

To analyze the toxicity of the treatments on the parasitoid T. anastrephae, the values for the LC₉₀ of the EOs of R. officinalis, as well as α-pinene, 1,8-cineole, camphor, and spinetoram, were determined previously upon 120 hr of exposure. Trichopria anastrephae adults were exposed to treatments via the topical application and ingestion bioassay method. Wasp mortality was evaluated at 120 hr posttreatment. To evaluate the sublethal effects of treatments on wasps, ten pupae of D. suzukii (24-hr-old pupae) were offered, per day, for seven days (beginning at 120 hr), to each surviving T. anastrephae female from the ingestion bioassay. Pupae of D. suzukii were exposed to the wasps on a wet hydrophilic cotton layer on an acrylic Petri dish. Pupae were removed daily, and placed in plastic cups (100 ml) sealed on top with voile until the fly or wasp emergence. During evaluation, the wasps were fed 80% honey/water (w v⁻¹). Parasitism (%) was determined by dividing the total number of wasp offspring per the total number of pupae offered, multiplied by 100.

Statistical Analysis

Generalized linear models (GLM) of the family of exponential distributions (Nelder and Wedderbum 1972) were used to analyze variables: eggs laid, proportion dead, pupation rate, and parasitism. When there were significant differences between treatments, multiple comparisons were performed (Tukey’s test, P < 0.05) using the glht function through the Multicomp package, with adjustment of P-values. All analyses were made using ‘R’ version 2.15.1 (R Development Core Team 2012). The LC₅₀ and LC₉₀ lethal concentrations and their 95% confidence intervals (CIs) were estimated using the Probit analysis (PROC PROBIT, SAS Institute 2000) to assess the toxicity against D. suzukii. A likelihood ratio test was used to test the hypothesis that the LC P values (LC at which a percent mortality P is attained) were equal. If the hypothesis was rejected, pairwise comparisons were performed, and significance was stated if CIs did not overlap. Finally, the average lethal time (LT₅₀) was estimated using Throne et al. (1995) for the Probit analysis of correlated data.

Results

In the chromatography analyses of the R. officinalis ECOs, 20 EOs peaks were identified (Fig. 1). The chromatography peaks were classified into hydrocarbon monoterpenes (20.9–47.3%), oxygenated monoterpenes (45.2–76.3%), hydrocarbon sesquiterpenes (0.7–2.0%), and ester (0.7–14.9%) (Table 1). ECOs 1, 2, and 3 showed α-pinene (9.3–20.1%), 1,8-cineole (13.0–49.1%) and camphor (12.1–20.5%) as common majority compounds. In the constitution of ECO 1 EO, 16 compounds were identified, accounting for 97.1% of its composition, with a predominance of α-pinene (20.1%), 1,8-cineol (13.0%), camphor (12, 1%), and bornyl acetate (14.9%) (Table 1). The chemical composition of ECO 2 includes 16 compounds, with α-pinene (19.5%), camphene (11.2%), 1,8-cineole (20.1%), and camphor (20.5%) representing the major compounds, totaling 98.7% of the oil identification (Table 1). The main compounds of the ECO 3 EOs were α-pinene (9.3%), 1,8-cineole (49.1%), and camphor (17.8%), among the 16 identified compounds out of a total of 98.6 % (Table 1).

In all ecotypes evaluated, their major compounds α-pinene, 1,8-cineole, and camphor, as well as spinetoram showed similar toxicity in the assays of ingestion with 65–80% mortality (F_{9, 32} = 17.84, P <0.0001) and topical application with 85–100% mortality (F_{9, 32} = 14.32, P <0.0001; Fig. 2). Based on the response concentration curves, ECO 1, ECO 2, ECO 3, and their compounds α-pinene, camphor, 1,8-cineole showed similar toxicity in the ingestion bioassay (LC₅₀ from 11.03 to 12.10 mg liter⁻¹; LC₉₀ from 19.48 to 22.24 mg liter⁻¹; Table 2). However, they were more toxic when compared to spinetoram (LC₅₀ = 63.10 and LC₉₀ = 86.17 mg liter⁻¹; Table 2). All treatments showed the same toxicity on D. suzukii adults in the topical application bioassay concerning the LC₅₀ values (LC₅₀ from 7.13 to 9.55 mg liter⁻¹; Table 2) and LC₉₀ (LC₉₀ from 17.12 to 22.34 mg liter⁻¹; Table 2). Finally, LT₅₀ values were concentration-dependent in both evaluated bioassays (Table 3).

When analyzing the deterrent effect on oviposition, D. suzukii females significantly reduced oviposition in ‘artificial fruits’ when exposed to the dry residues of the compounds α-pinene, camphor, and 1,8-cineole (Table 4). In contrast, ECO 1, ECO 2, ECO 3 did not have a detrimental effect on oviposition compared to the negative control (Table 4). Regarding the larvicidal effect, all ECOs and their compounds caused higher larval mortality than the control treatment, especially ECO 1, ECO 3, and the compounds camphor and 1,8-cineole, which caused larval mortality of ≅98% (Table 4). A similar effect was also observed in the biological parameters of pupation rate and pupal mortality (Table 4).

No change in the control group was found when assessing macroscopic damage or color change of the larval cuticles of D. suzukii (Fig. 3a–c). Motility and damage to cuticles were evident after 30 min of exposure to EOs. Diffuse oxidation (Fig. 3d–h), intense darkening in the head region (Fig. 3h and i), as well as edema (Fig. 3g) were observed in the cuticle of larvae treated with α-pinene, camphor, and 1,8-cineole. Besides, 2 hr posttreatment with EOs from ECO 1, ECO 2, and ECO 3, changes such as cuticle dehydration (Fig. 3j–l), degeneration of the respiratory spiracle (Fig. 3j and k), and wrinkling of the cuticle surface (Fig. 3j–l) were observed in the whole larva from the first to the sixth segment.

Histological sections of D. suzukii larvae showed several changes 2 hr posttreatments with R. officinalis ECOs and their individual compounds in the cuticle (Fig. 4A), fat body (Fig. 4B), Malpighian tubules (Fig. 4C), and brain (Fig. 4D); no changes were observed in the control larvae (Fig. 4I). Cuticular projection was reported for ECO 1 and ECO 2 (Fig. 4A II and III), while rarefaction of epidermal cells (Fig. 4A IV) and cuticle detachment (Fig. 4A IV) were observed after contact with ECO 3. The fat body of D. suzukii showed granules of the visceral layer (Fig. 4B II), irregular trophocytes with condensation of chromatin (Fig. 4B III), and intense cytoplasmic vacuolization (Fig. 4B III and IV) were observed in ECO 1, ECO 2, and ECO 3. For these treatments, Malpighian tubules were also observed with the pyknotic nuclei in the epithelium (Fig. 4C II and III) and disintegration of the brush border (Fig. 4C IV). On the other hand, the brain showed degeneration and vacuolization in the cortical layer (Fig. 4D II and III), as well as cytoplasmic granulation (Fig. 4D III and IV).

Likewise, the exocrine system of D. suzukii larvae showed several changes 6 hr posttreatment with α-pinene (Fig. 5A II, III, and IV). Deformation of the lumen, vacuolization of the secretory ducts (Fig. 5 A II), and the condensation of the nuclei with polytene chromosomes were observed in the salivary gland (Fig. 5 A III and IV). On the other hand, the midgut of D. suzukii larvae showed marked necrosis of the intestinal tract, characterized by condensation of chromatin, pyknotic nuclei, and loss of architecture after exposure with camphor (Fig. 5 B II, III, and IV). The hindgut of the individuals in the groups treated with 1,8-cineole showed a very similar morphology to that of the individuals in the control group; however, they had pyknotic nuclei (Fig. 5 C II, III, andIV).

In the lethal toxicity bioassay on T. anastrephae adults, ECO 1, 2, and 3 and their major compounds α-pinene, camphor, 1,8-cineole resulted in mortality of less than 20% of adults in the topical application and ingestion bioassay (Table 5). These values were significantly lower when compared to spinetoram (mortality of 64.2 by topical, 46.1% by ingestion, Table 5). In contrast, all treatments evaluated did not have a sublethal effect in daily parasitism over seven days (Table 5).

Discussion

In this study, qualitative and quantitative differences in the EOs chemical composition were identified in the three R. officinalis ECOs collected in the Neotropical region. The major components consisted of terpenes, namely α-pinene, 1,8-cineole, and camphor. These findings corroborate previous studies on ecotypes of R. officinalis collected in Spain (Arnold et al. 1997), Canada (Isman et al. 2008), Italy (Beretta et al. 2011), and India (Kiran and Prakash 2015). However, a variation in the composition of these compounds was observed, with rates ranging from 9.3 to 20.1% (α-pinene), 13.0 to 49.1% (1,8-cineole), and 12.1 to 20.5% (camphor). This variation in the chemical composition of the main compounds within the same species can be attributed to environmental, climatic and genetic factors, geographic location, and extraction method (Isman et al. 2008, Feitosa-Alcantara et al. 2017, Trombin-Souza et al. 2017).

The bioactivity of R. officinalis EOs has been documented (Andrade et al. 2018), along with their properties as insecticides (Isman et al. 2008, Kiran and Prakash 2015), fumigants (Papachristos and Stampoulos 2004, Kiran and Prakash 2015), repellents (Isman et al. 2008), larvicides (Duarte et al. 2015; Tak and Isman 2015, 2017), and acaricides (Choi et al. 2004, Miresmailli and Isman 2006). We found that R. officinalis ECOs and their major compounds (α-pinene, 1,8-cineole, and camphor) were highly toxic to D. suzukii adults by ingestion and topical application, similar to the action of the commercial insecticide based on spinetoram. The performance of these EOs was likely due to their lipophilic constitution and the low molecular weight of their compounds, which favor the EO diffusion through the cell membrane by regulating neurotransmitters such as gamma-aminobutyric acid (GABA), octopamine, tyramine, or acetylcholinesterase (AChE) (Jankowska et al. 2017). In the present study, D. suzukii showed evidence of neurological intoxication since the flies exhibited signs of tremors and hyperextension of the legs and abdomen, followed by paralysis and consequent death after ingestion or contact with the products (Enan 2005, Blenau et al. 2012).

A recent study with pepper (Piper spp.) EOs on D. suzukii adults used different exposure routes, and better results were obtained with topical application (Souza et al. 2020a). Similar effects were observed in this study as the EOs of the R. officinalis ECOs and their major compounds killed the D. suzukii faster when administered topically (LT₅₀ 7.10–0.41 hr) compared to the ingestion method (LT₅₀ 48.1–0.55 hr). These results may be linked to the fact that these products penetrate directly into their hemolymph in a single topical application of the EOs on D. suzukii dorsal regions. Another fact is that the products remain in the digestive tract of insects during the feeding period (8 hr), requiring a longer time to metabolize and/or excrete substances (Souza et al. 2020a). Although, we have not quantified the consumption of EOs by flies, we hypothesize that EOs may not have a good taste, which decreases the ingestion and makes this method less toxic to insects.

In addition to causing high toxicity in D. suzukii adults, the compounds α-pinene, 1,8-cineole, and camphor showed deterrent effects on oviposition, reducing the number of eggs laid per fruit by up to 41.7% when these compounds were applied, a fact not observed for ECOs 1, 2, and 3. Finding products that affect the oviposition behavior is important for managing the pest since the damage is caused by the rupture of fruit skins, particularly for thin-skinned fruit (Goodhue et al. 2011, Lee et al. 2011, Andreazza et al. 2016, Farnsworth et al. 2017). Therefore, deterrent products can reduce skin rupture and, consequently, reduce infestation by pathogens that accelerate the fruit deterioration processes (Mitsui et al. 2006, Bernardi et al. 2017, Souza et al. 2020b). Furthermore, the deterrents reduce pest population density in the orchard (Ioannou et al. 2012, Bernardi et al. 2017, Geisler et al. 2019). Besides, we found that R. officinalis ECOs and their major compounds had larvicidal effects, did not inhibit parasitism rate, and did not cause parasitoid mortality of D. suzukii. Regarding EO penetration into the larvae, it is worth mentioning that the cuticle is a biphasic structure with lipophilic and hydrophilic layers (Yu 2008). This suggests that the larvicidal effect reported by us may be related to the EOs polarity (lipophilic substances), which allow oils to penetrate the larvae cuticle, interfering with their physiological functions (Chaaban et al. 2019) and directly hindering their development (Kumar et al. 2014, Chaaban et al. 2019).

In addition to causing direct mortality to D. suzukii larvae, we observed macroscopic damage caused by the EOs of R. officinalis ECOs and their compounds in larval D. suzukii, including edema, diffuse darkening, and wrinkling on the cuticle surface. Similar observations have been found for larval Aedes aegypti L. (Diptera: Culicidae) (Oliveira et al. 2013, Chantawee and Soonwera 2018), Cochliomyia macellaria (F.) (Diptera: Calliphoridae) (Chaaban et al. 2019), and Lucila sericata (Meigen) (Diptera: Calliphoridae) (Shalaby et al. 2016, Bedini et al. 2019). Moreover, when analyzing histological lesions in D. suzukii, including rarefaction of the epidermal cells and the destruction of the cuticular layer, we found that they were higher when using EOs from intact ECOs. The interactions of the chemical compounds existing in R. officinalis EOs can probably contribute to increased penetration and cuticular damage. This fact was observed in Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae) when the binary mixture of 1,8-cineole and camphor was applied to this insect (Tak and Isman 2015). Another study on T. ni showed an increase of up to 19 times in the EOs penetration in the larval integument when camphor and 1,8-cineole were administered together, suggesting greater penetration as the main synergy mechanism (Tak and Isman 2017).

We used visual observations to record the damage to the fat body, Malpighian tubules, brain, salivary gland, midgut and hindgut, essential structures for insect homeostasis. Larvae of D. suzukii exposed to EOs showed changes in the fat body, including cytoplasmic vacuolization, irregular morphology of the trophocytes, and granules with a pyknotic nucleus. These changes can occur in a possible attempt to detoxify cells to excrete toxic substances (Cruz et al. 2010, Cousin et al. 2013). Despite the high plasticity of adipocyte cells in the fat body, excessive lipid deposition can cause fat body hypertrophy (Arrese and Soulages 2010), as observed for Culex quinquefasciatus (Say, 1823) (Diptera: Culicidae) (Castro et al. 2016) and Spodoptera frugiperda (Smith, 1797) (Lepidoptera: Noctuidae) (Silva et al. 2018, Dutra et al. 2019). Besides, we noticed the disintegration of the brush border in the Malpighi tubules, as well as the presence of pyknotic nuclei in the epithelium. Our results corroborate with those of previous studies on C. macellaria (Chaaban et al. 2019) and Culex pipiens pallens Coquillett (Liu et al. 2020), which state that morphological changes and chromatin condensation result in the death of the insect by apoptosis, that is, programmed cell death. The changes induced by intact EOs in D. suzukii brain have also been demonstrated by chromatin condensation, vacuolar degeneration, and cytoplasmic granulation, providing additional support to the neurotoxic effect of ECO 1, ECO 2, and ECO 3. Besides, a study on the histopathological brain damage caused by EOs concluded that the mode of action is similar, regardless of the target insect’s taxonomic classification (Chaaban et al. 2019).

In addition to the effects described above, cytoplasmic vacuolization of the salivary gland of 3rd instar larval D. suzukii, followed by compacting and fragmentation of the chromatin, was evident. These findings probably show the low transcriptional activity due to chromatin modifications, suggesting the advanced process of cell death (Silva-Zacarin et al. 2008), as observed for Euschistus heros (F.) (Heteroptera: Pentatomidae) (Cossolin et al. 2019). We also found that the region of the mid and hindgut of the 3rd instar larval D. suzukii are highly affected by camphor and 1,8-cineole, including loss of digestive tract architecture, condensation of chromatin, and presence of pyknotic nuclei in epithelial cells. Morphological alterations and pyknotic nuclei are frequently described in degenerating organs of dipterans (Castro et al. 2016, Chantawee and Soonwera 2018, Chaaban et al. 2019, Liu et al. 2020).

Another factor that can positively contribute to the management of D. suzukii populations using R. officinalis ECOs and their major compounds was the low mortality caused to T. anastrephae adults in the topical application and ingestion bioassay. In addition to providing low mortality, they did not cause sublethal effects on pupal parasitism over time, becoming evident that they are an alternative for D. suzukii IPMs (Desneux et al. 2007; Biondi et al. 2012, 2013; Guedes et al. 2016). Considering the high toxicity to D. suzukii adults, deterrence of oviposition and compatibility with T. anastrephae adults, R. officinalis EOs or their major compounds α-pinene, 1,8-cineole, and camphor can be used for the formulation of bioinsecticides. This study may contribute to the development of new products for D. suzukii IPM programs since R. officinalis is abundantly cultivated in the Neotropical region.

Acknowledgments

We would like to thank the National Council for the Improvement of Higher Education (CAPES) for granting a scholarship to M.T.S., D.C.O., M.C.M., D.J.M., and V.S.R. Thanks also to the National Council for Scientific and Technological Development (CNPq) for the financial support provided to M.T.S., D.B., and P.H.G.Z., and the Araucária Foundation of Scientific and Technological Development Support for the M.A.C.Z. scholarship.

References Cited