Toxicity and repellent activity of a carlina oxide nanoemulsion toward the South American tomato pinworm, Tuta absoluta

doi:10.21203/rs.3.rs-3671767/v1

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Toxicity and repellent activity of a carlina oxide nanoemulsion toward the South American tomato pinworm, Tuta absoluta

https://doi.org/10.21203/rs.3.rs-3671767/v1

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Plant essential oil (EO)-based insecticides represent a promising tool for IPM, though their formulation is limited by poor physio-chemical properties. EO encapsulation into stable formulations, like nano emulsions (NEs), could boost EO efficacy and stability. Carlina acaulis roots contain an EO recently studied for its excellent insecticidal activities, and chiefly composed by carlina oxide (>97%). Herein, we developed two carlina oxide NEs (0.25% and 0.5% w/w) through ultrasounds exposure and characterized them by dynamic light scattering. The NE insecticidal and repellent activities were investigated against T. absoluta eggs and larvae, and adults, respectively. 0.25% and 0.5% NEs showed a monomodal size distribution with a Z-average size of 113.87±1.32 nm and 127.27±0.47 nm, respectively. The polydispersity indexes measured at 0.15±0.01 and 0.16±0.01 indicate a low grade of polydispersity. The 0.25% NE showed significant contact toxicity on T. absoluta eggs, with a maximum egg hatching inhibition of 85.7% 11 days post-treatment. The highest larvicidal effect was observed in translaminar toxicity tests, with complete mortality after 24 h. The NE did not achieve significant oviposition deterrence. Overall, the tested NE showed promising effectiveness as ovicide and larvicide on T. absoluta, highlighting the need of further research shedding light on its modes of action, as well as to evaluate lethal and sublethal effects on tomato biological control agents and pollinators.

Carlina acaulis

Gelechiidae

eco-friendly botanical insecticide

nanopesticide

phytotoxicity

Nanoemulsion (NE) can boost the stability and the efficacy of insecticidal essential oils.
Here we developed carlina oxide NEs for insecticidal applications against Tuta absoluta.
0.25% (w/w) carlina oxide NE was stable for 3 months and slightly phytotoxic to tomato.
The NE was toxic towards T. absoluta eggs and larvae.
No oviposition deterrence effects were detected on adults.

Nanotechnology is a rapidly growing field that holds great promise and is being widely utilized in various applied sciences (Yousef et al. 2023). Its employment in agriculture is significantly increased over the last years (Pavoni et al. 2019; Kavallieratos et al. 2022; Yousef et al. 2023). Thanks to the nanometric size of the dispersed phase, nano-systems facilitate the delivery of active compounds (e.g., insecticides) more efficiently and with minor health and environmental impact (An et al. 2022; Bamisaye et al. 2023, Geng et al. 2017; Schulz 2004). In addition, they provide a viable way to use plant-borne biocides with limited physicochemical qualities, high volatility, and rapid environmental degradation for managing pests of economic importance (Pavela et al. 2021a; Pavoni et al. 2020). Colloidal dispersions, characterized by the diffusion of a core phase within a continuous medium, have garnered significant attention as a highly adaptable nano-system (Pavoni et al. 2019). Nanoemulsions (NEs) are a type of self-emulsifying colloidal systems. They are composed of two immiscible liquid phases and possess the unique ability to effectively transport lipophilic or poorly water-soluble chemicals, such as essential oils (EOs) (Pavela et al. 2021a). Because of their simplicity of formulation and handling, NEs are considered one of the most promising nano-systems for pest control purposes. The insecticidal activity of EO-based NEs has been investigated on several insect species of agricultural and medical interest, such as aphids (Heydari et al. 2020; Pascual-Villalobos et al. 2017; Sciortino et al. 2021), mosquitoes (Jesser et al. 2020a; Mahran 2022; Pavela et al. 2021a), stored-product beetles (Giunti et al. 2019; Kavallieratos et al. 2022), and some Lepidoptera (Benelli et al. 2020; Jesser et al. 2020a; Louni et al. 2018). According to recent findings, a highly promising NE is that based on Carlina acaulis L. (Asteraceae) EO. Carlina acaulis EO is isolated from the roots of the plant and mainly includes carlina oxide, whose concentration usually overcomes 97% of the overall chemical composition and is the primary cause of the EO’s efficacy (Spinozzi et al. 2023a). In general, compounds belonging to the polyacetylenes’ family are well known as a phytoalexins, or plant defence molecules produced in reaction to living microorganisms, microbial products, and environmental stressors (Spinozzi et al. 2023b). Both C. acaulis EO and carlina oxide demonstrated a variety of biological activities, including a highly promising insecticidal potential (Benelli et al. 2019, 2022; Kavallieratos et al. 2020; Pavela et al. 2020; Rizzo et al. 2021), as well as antimicrobial and antiprotozoal activities (Herrmann et al. 2011; Stojanović-Radić et al. 2012; Spinozzi et al. 2023a). In addition, they have a limited impact on beneficial aquatic and terrestrial organisms (Benelli et al. 2022; Spinozzi et al. 2023a). Recent studies investigated C. acaulis EO and carlina oxide NE insecticidal activity and they both elicited high toxicity on 3rd -4th instar larvae of mosquitoes (Pavela et al 2021a), on 1st instar larvae of Lobesia botrana (Denis & Schiffermüller) (Lepidoptera: Tortricidae) (Benelli et al. 2020), on the invasive xylomicetophage ambrosia beetle Xylosandrus compactus (Eichhoff) (Coleoptera: Curculionidae, Scolytinae) (Gugliuzzo et al. 2023), and on the root knot nematode Meloidogyne incognita (Kofoid & White) (Tylenchida: Heteroderidae) (Ntalli et al. 2023). Although both NEs have been proven to be effective as green insecticides, their bioactivity has only been studied on a limited number of insect species. Hence, the objective of the present study was to assess the bioactivity of carlina oxide NE on the South American tomato pinworm, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae), a key pest of tomato crops both in greenhouses and in open field (Campolo et al. 2017). The endophytic habitat of T. absoluta larval stages shields them from the majority of chemical compounds (Biondi et al. 2018). This has led to an increased use of synthetic insecticides (Silva et al. 2011; Roditakis et al. 2015), that has also led to the development of resistance in T. absoluta (Roditakis et al. 2018).

For these reasons, recent research focused on eco-friendly tools to manage this moth pest, including botanical based NEs (Campolo et al. 2017; Ricupero et al. 2022; Salazar et al. 2022). In this framework, formulating effective and highly stable NEs can be a valuable strategy to enable the practical use of carlina oxide as an active component in the development of commercial green pesticides to manage the South American tomato pinworm. Here, through ultrasonic exposure, we develop two carlina oxide NEs (0.25% and 0.5% w/w carlina oxide) and characterised them using dynamic light scattering (DLS). Since carlina oxide is phytotoxic at high concentration for many plant species (Álvarez-Rodríguez et al. 2023), we assessed the potential phytotoxicity of carlina oxide NEs (0.25 and 0.50%) on tomato plants before starting our experiments. According to phytotoxicity trials, we investigated the insecticidal and repellent properties of the 0.25% (w/w) carlina oxide NE against T. absoluta eggs and larvae, as well as adults, respectively.

Plant laboratory rearing

Tomato plants used in all the experiments were cultivated at the CREA - Research Centre for Plant Protection and Certification (CREA-DC) of Palermo (Italy). San Marzano nano cultivar seeds (ITALSEMENTI S.N.C.®) were sown and grown in 1 L pots (10x10x15 cm) with peat, topsoil (Gramoflor®, GmbH & Co. KG) and expanded vermiculite (VIC, Italiana®) and were placed in fine mesh net cages (45x45x90 cm) to prevent other undesirable insect species. Water fertilization was done once every two weeks with Sangue di bue (COMPO Group) and no pesticides were used to manage pest infestations. The plants were placed in a chamber with controlled environmental conditions at 25 ± 2°C, 50 ± 10% relative humidity (RH) and a photoperiod (L:D) of 16:8. Plants of about 50 cm height and with at least five true leaves were used for testing and T. absoluta rearing.

Insect laboratory rearing

A laboratory stock culture, maintained at the CREA-DC of Palermo, supplied the insects utilised in this study. The original culture was set up in 2008, in the laboratory of the Department of Agriculture, Food and Environment (Di3A) of the University of Catania, with infested tomato plants from commercial farms in eastern Sicily. To reduce the genetic drift, once every 6 months, the colony is bred with new individuals collected in Sicilian tomato crops. The rearing is placed in a chamber with controlled conditions of temperature (25 ± 2°C) and relative humidity (50 ± 10%) with 16:8 L:D of photoperiod. Twice a week, 200 newly emerged adults of T. absoluta were launched in a screened cage with fine mesh (45x45x45 cm) with three plants. After three days, the adults were removed to avoid eggs of different age. Approximately after two weeks, when the 4th instar larvae became pupae, all the vegetal material was cut and put in another screened cage for the emergence of the new adults. 2nd instar larvae and newly emerged coetaneous adults were used for the experimental activities.

Extraction of carlina oxide

Carlina oxide was extracted from C. acaulis roots following the procedure reported by Rizzo et al. (2023) with a yield of 0.5% (w/w). The plant was purchased from Minardi & Figli S.r.l. (Bagnacavallo, Ravenna, Italy; https://www.minardierbe.it; batch no C-26102101, collected in 2021). The compound’s structure and purity (97.4%, Fig. 1) were confirmed by GC-MS and NMR analyses accordingly to protocols previously published and the data were linear to those reported in literature (Benelli et al. 2019). Once obtained, the compound was stored at -20°C protected from light before biological assays.

Preparation and characterization of tested carlina oxide nanoemulsions

Carlina oxide NEs were prepared through a high energy method by employing ultrasounds. Specifically, the corresponding amounts of carlina oxide to have a final concentration in the formulation of 0.25% and 0.5% (w/w) were mixed with ethyl oleate in a 3:1 weight ratio. Then, the oil phases were included dropwise in polysorbate 80 aqueous solutions with a final concentration of 0.16 and 0.33% (w/w) for the 0.25% and 0.5% NE, respectively, under high-speed stirring (Ultraturrax T25 basic, IKA® Werke GmbH and Co.KG, Staufen, Germany) for 5 min at 13500 rpm.

Finally, the two obtained emulsions were sonicated into a 2L-Ultrasound Extractor U2020 (170W, 230V, 50Hz) (Albrigi Luigi Srl Verona, Italy) for 40 min using the H + M (high power + homogenization) program to reduce the droplet size. The NEs’ particle size was determined through DLS using a Zetasizer nanoS (Malvern Instrument, Malvern, UK) following the procedure by Benelli et al. (2020). The stability of NEs, stored at 4°C in tight closed vials, was assessed by checking their mean droplet size (Z-average) and polydispersity index (PDI) at different time points: 0 day (T₀), 15 days (T₁), 30 days (T₂), and 90 days (T₃).

Phytotoxicity on tomato

To evaluate the potential phytotoxicity of the NEs, we applied two different doses of carlina oxide NE (0.25% and 0.5%) to tomato plants using a hand sprayer (V = 100 mL) from 20 cm until the solution ran off. We also included the respective control NE solutions (i.e., the NE without carlina oxide at the 0.25% and 0.5%) and a negative control (distilled water); each tested dose was repeated on 5 tomato plants. Treated plants were, then, placed in a greenhouse and checked 1, 3, 7, 14 days after the treatment. Following the methodology of Campolo et al. (2017), we noted the percentage of damaged leaves and the severity of the damage. The latter has been classified as: (i) 0, no damage; (ii) 1, leaf surface partially damaged with chlorosis but without necrosis; (iii) 2, leaves with evident necrosis; (iv) 3, dead leaves. The phytotoxicity index (P_i) was calculated as follows:

$${P}_{i}={\sum }_{j=0}^{n}\left(\frac{DL}{TL}x \frac{DC}{n-1}\right)$$

In which DL is the number of damaged leaves for each damage severity class j; TL is the total number of sprayed leaves; DC is the damage severity class; n is the number of damage severity classes.

P_i ranges from 0 (no damage) to 1 (dead leaves).

Contact toxicity on eggs

Based on the results obtained in phytotoxicity trials, we assessed the ovicidal effect of carlina oxide NE (0.25%) by directly spraying the NE on the eggs and measuring the percentage of egg mortality. In a cage (45x30x45 cm) with a fine mesh net, healthy tomato sprouts were exposed to 100 unsexed newly emerged mated adults of T. absoluta for 24 h. After 48 h, the sprouts with coetaneous laid eggs were sprayed as above described and were let dry for 60 min in laboratory conditions. After drying, with a fine brush a total number of 70 treated eggs per treatment were moved in new healthy tomato sprouts placed in a two-cup experimental arena as described by Biondi et al. (2012) to wait for the hatching. Every two days after the treatment the sprouts were checked to verify egg hatching, the overall egg hatching after 11 days was noted. At the same time, we tested a negative control (i.e., distilled water) and a control NE (i.e., the NE without carlina oxide).

Topical toxicity on larvae

The larvicidal effect of carlina oxide NE (0.25%) was evaluated in topical toxicity trials. The larval survival (%) and the adult emergence (%) were recorded. In addition, the negative control and the control NE were tested. For each treatment, 50 T. absoluta 2nd instar larvae from the insect rearing were collected and isolated in absorbent paper sheet (to prevent larval drowning caused by excess of solution after the treatment). Subsequently, 15 mL of NE were sprayed on larvae as above described (see “Phytotoxicity on tomato” paragraph). After 15 min, the treated larvae were moved with a fine brush and placed in healthy tomato sprouts arranged in the two-cup experimental arena as described above. The experimental arenas were checked at 24, 48, and 72 h after the release of the larvae for the evaluation of larval survival and after 12 days for adult emergency.

Ingestion toxicity on larvae

Ingestion toxicity tests were performed to assess the larvicidal effect of NE carlina oxide. The tested NE and the parameters noted were the same as above mentioned. Healthy tomato sprouts were treated as described above (see “Phytotoxicity on tomato” paragraph) and dried in laboratory conditions for 60 min. The treated sprouts were fixed in the two-cup experimental arena and 50 T. absoluta larvae were released on each sprout. As for the previous experiment of the larval toxicity by topical contact, the experimental arenas were checked at 24, 48, and 72 h after the release of the larvae for the evaluation of larval survival and were checked after 12 days for adult emergency.

Translaminar toxicity on larvae

The potential translaminar effect of the NE on T. absoluta larvae within the tomato leaf was assessed. The same experimental setting was used as in the previous paragraph for this test. However, the larvae were released on healthy sprouts 24 h before the treatment to give them time to get inside the leaves. The experimental arenas were checked at 24, 48, and 72 h after the treatment of the tomato sprouts for the evaluation of larval survival.

Ovideterrence trials

The repellent activity of NE carlina oxide on T. absoluta adults was evaluated in the two-choice assays. Three different theses were compared: (i) distilled water vs carlina oxide NE, (ii) distilled water vs control NE and (iii) carlina oxide NE vs control NE. For this trial, 24 h before the experiment, T. absoluta newly emerged adults were collected and placed inside a screened cage for the mating. Tomato sprouts, with two true leaves, were sprayed (see “Phytotoxicity on tomato” paragraph) and let dry for 30 min in laboratory conditions. After drying, the sprouts were placed in a plastic box (45x30x45 cm) aerated with a fine mesh net, and a total number of 10 adults were released inside to lay eggs. After 72 h, adults were removed and the total number of laid eggs on each tomato sprout was noted. Five replicates (cages) for each two-choice assay were performed for a total of 50 tested T. absoluta adults.

Data analysis

Contingency analysis was used to evaluate differences in hatchability rates among treatments (p = 0.05). Data from topical, ingestion, translaminar toxicity, and adult emergence experiments were not normally distributed (Shapiro-Wilk test, p > 0.01) and homoscedastic (Levene test, p > 0.01). As a result, the Kruskal-Wallis test was used to assess treatment differences (p = 0.05). Because ovi-deterrence data were normally distributed (Shapiro-Wilk test, p > 0.01) and homoscedastic (Levene's test, p > 0. 01), two-choice assay findings were subjected to paired t-tests (p = 0.05) (Tomè et al. 2013). The phytotoxicity index (P_i) was analysed with univariate analysis of variance, with concentrations and time after the treatment as fixed factors and P_i as dependent variable. The Steel-Dwass test and Tukey test were utilized as a post-hoc analysis when suitable (p < 0.05). For statistical analysis, JMP Pro 17 and SPSS® V. 20 (IBM) were employed, whereas GraphPad PRISM (10.0.3) was used for graphing.

Characterization of tested carlina oxide nanoemulsions

The nanometric size range of the carlina oxide NE oil droplets was assessed by DLS measurements. Both 0.25% and 0.5% (w/w) formulations showed a monomodal size distribution with Z-average size of 113.87 ± 1.32 nm and 127.27 ± 0.47 nm, respectively (Fig. 2). The quality of the two systems with respect to the size distribution was checked on the bases of the PDI. The latter showed values of 0.15 ± 0.01 and 0.16 ± 0.01 for 0.25 and 0.5% (w/w) NEs, respectively, which confirmed that the systems had a low grade of polydispersity.

The physical stability of carlina oxide NEs was evaluated for a storage period of 3 months, after which the sizes of the dispersed phases showed a slight variance. For instance, the average particle size of 0.25% NE obtained after 3 months (133.77 ± 6.05 nm) of storage is increased by only 20 nm compared to the T₀. Similarly, 0.5% NE particle size slightly increased from 127.27 ± 0.47 (T₀) to 145.63 ± 5.92 (T₃). Moreover, the PDI values obtained for the NEs at T₃ were really similar to those at T₀, confirming that this type of formulations remains stable for at least a period of 3 months. The Z-average size and PDI values of oil droplets in the two NEs obtained from the measurements at T₀, T₁, T₂, and T₃ are shown in Fig. 3.

Phytotoxicity of carlina oxide nanoemulsions

The toxic effect of the different concentrations of carlina oxide NE on plants was expressed as the phytotoxicity index (P_i) and it is shown in Fig. 4. No phytotoxicity was recorded in the distilled water treatment (P_i=0) and a slight effect was observed for control NE 0.25% (P_i=0.006) and control NE 0.5% (P_i=0.022). Carlina oxide NE at the concentration of 0.25% had slight toxic effects on tomato plants (P_i=0.170), compared to the higher concentration of 0.5%, where heavier toxic effects were recorded (P_i=0.443). Only the concentration at 0.5% of carlina oxide NE showed significant damage in the leaves with evident necrosis and dead leaves. The data analysis highlights that the phytotoxicity index significantly depends on the treatment (F_4,25=386.75; p < 0.001) and on the days after the treatment (F_3,20=5.961; p < 0.001). In all the treatments, the new vegetation grown in the 14 days after the beginning of the trial, did not show any phytotoxic effect.

Contact toxicity on eggs

Eggs sprayed with carlina oxide NE 0.25% (w/w) showed lower hatching rates after 11 days (χ² = 133.042, p < 0.0001). Most eggs failed to hatch when exposed to carlina oxide NE (unhatched: 85.7±2.0%), whilst a high percentage of eggs hatched when treated with control NE (hatched eggs: 99.0±0.5%) and distilled water (hatched eggs: 81.0±5.5%) (Fig. 5).

Topical toxicity on larvae

Topical toxicity results indicated that carlina oxide NE 0.25% (w/w) was scarcely effective till 72 h post-treatment if compared to positive and negative controls (24 h: χ² = 2.333, d.f.=2, p = 0.311; 72 h: χ² = 2.533, d.f.=2, p = 0.282) (Fig. 6a, b). No significant reduction in adult emergence was found after 12 days (χ² = 0.267, d.f.=2, p = 0.875) (Fig. 6c).

Ingestion toxicity on larvae

Carlina oxide NE 0.25% (w/w) showed a toxic effect after 24 h (χ² = 27.671, d.f.=2, p < 0.0001) in ingestion toxicity trials (Fig. 7a). Larval mortality was observed only in treated specimens. Adult emergence differed significantly from positive and negative control after12 days (χ² = 24.728, d.f.=2, p < 0.0001) (Fig. 7b).

Translaminar toxicity on larvae

In translaminar toxicity trials, after 24 h, larval mortality was recorded on 0.25% (w/w) NE-treated sprouts (χ² = 26.238, d.f.=2, p < 0.0001) (Fig. 8). Control NE mortality rates increased after 72 h, but it still significantly differed from the NE-treated sprouts (χ² = 25.530, d.f.=2, p < 0.0001) (Fig. 7b).

Ovideterrence effect

In two-choice experiments, carlina oxide NE 0.25% (w/w) do not cause considerable egg-laying deterrence in mature females of T. absoluta (Fig. 9). There was a significant difference in the number of eggs laid between the NE-treated sprout and the negative control (distilled water) (F_1,9=19.997, p = 0.002). By contrast, there were no differences in the number of eggs laid on sprouts treated with carlina oxide NE over the NE control (F_1,9=0.613, p = 0.456).

Nanoformulations can improve the stability and efficiency of botanical insecticides by addressing difficulties such as EO volatility and poor water stability, as well as gradually and effectively spreading the insecticide over the target plant, potentially reducing the impact on non-target species (Pavoni et al. 2020). NEs can be prepared with different techniques; the high energy methods are the most widespread. Earlier, carlina oxide NEs were developed using the high-pressure homogenization methodology (Benelli et al. 2020; Pavela et al. 2021a; Kavallieratos et al. 2022; Ntalli et al. 2023). In this study, we relied to ultrasounds to assist in the preparation of two formulations. Ultrasounds are a high energy method commonly used for creating nanoformulations (Bamisaye et al. 2023). Although different methodologies have been used for the preparation of carlina oxide NEs, the nanometric size of dispersed phase was achieved in both cases. For instance, the Z-average size of the oil droplets in this study was remarkably similar to size measured for the NE prepared using a high-pressure homogenization method (Pavela et al. 2021a). Both had a size of around 127 nm and 142 nm, respectively, when the concentration of oil and surfactant was the same. Herein we also focused whether the NEs prepared with ultrasounds had long-lasting stability. The slight variance of Z-average and PDI values obtained from DLS analysis at different time points confirmed that the ultrasounds are suitable for the preparation of relatively stable carlina oxide NEs.

EOs and their major compounds have a significant impact on plant biological activities by directly interacting with the lipid layers of biological membranes (Campolo et al. 2017, Synowiec et al. 2017). As herbicides, EOs applied to the leaves can cause noticeable damage in just a matter of hours (Álvarez-Rodriguez et al. 2023). Our study found that the phytotoxic effect of carlina oxide NEs increased with the dosage and became more pronounced over time. Additionally, the P_i index showed a significant increase 24 h after treatment. At the highest concentration (0.5% w/w), NE-treated plants showed damaged and dead leaves, and evident necrosis on the leaf surface. Instead, at the lowest concentration (0.25%), the leaf damage was considerably less. For this reason, carlina oxide NE (0.25%) was chose for further investigations.

From an entomological point of view, we observed an acute effect of carlina oxide NE on larvae at 24 and 72 h, as well as a long-lasting effect on egg hatching and adult emergence. Our results suggests that carlina oxide NE (0.25% w/w) is effective towards both eggs and larvae of T. absoluta. After 11 days, less than 15% of eggs hatched after topical treatments with carlina oxide NE. If compared with other nano-systems, carlina oxide NE seems to be more effective. Indeed, less than 50% of eggs reached the adult stage when sprayed with sweet orange-EO nanoparticles (NPs) at the highest dose (Campolo et al. 2017). Tuta absoluta larvae survival rates in topical, ingestion and translaminar toxicity trials were also assessed. Topical experiments revealed no significant impact of carlina oxide NE on T. absoluta larval mortality, probably due to a low ability of NE carlina oxide to permeate the insect body (Tortorici et al. 2022). However, the potential ability of T. absoluta larvae to detoxify the NE should be also considered (Ghanim & Abdel Ghani 2014). By contrast, the results of ingestion and translaminar experiments were encouraging. In both experiments, T. absoluta larval mortality was significantly higher carlina oxide NE-treated sprouts than the ones sprayed with control NE and distilled water. Translaminar toxicity results indicate that using NEs as a nano-system is highly recommended. Indeed, the toxic effect on T. absoluta larvae in translaminar tests utilizing citrus peel EO-NP was considerably less effective than the respective EOs (Campolo et al. 2017). The toxic effect could be due to carline oxide’s ability to damage insect cells by forming carbocations that react with -SH groups and amino groups of proteins and other molecules (Kavallieratos et al. 2022). In addition, the phototoxicity of carlina oxide induced by UV exposure may enhance this potential (Kavallieratos et al. 2022). Furthermore, Spinozzi et al. (2023a) suggested different hypothesis for carlina oxide insecticidal mechanism of action, such as inhibition of acetylcholinesterase, modulation of gamma-aminobutyric acid receptors A (GABA-A), and inhibition of octopamine and P450 cytochromes receptors. Unfortunately, the mechanism of action of this carlina oxide remains still unknown. Lastly, carlina oxide NE has a weak ovideterrence effect; indeed, when compared its control (control NE) no significant differences were found. A possible explanation is the presence of ethyl oleate as a stabilizer component of both NE and its control which acts as an attractant or a repellent depending on the dose (Hafez et al. 2014; Chen & Dai 2015). The strong repellent effect of the NE is probably due to the potential bioactive properties of this polyacetylene. Indeed, active ingredients, such as EOs, can elicit a repellent effect towards several arthropod species (Benelli et al. 2021). Further studies are still required to assess the repellent and/or attractive activity of carlina oxide NE using electroantennography (EAG) and olfactory bioassays, which might be a promising element to better understand the compound's potential. An oviposition repellence caused by NEs has also been observed when tomato sprouts were treated with LC₅₀ dose of Allium sativum EO-based NE (GEO-NE) (Ricupero et al. 2022).

Overall, our results support the possible use of carlina oxide NE as an effective ovicide and larvicide against T. absoluta, in the framework of the severe insecticide resistance quickly developed by this important tomato pest worldwide (Guedes et al. 2019; Desneux et al. 2022). One may argue that the phytotoxicity detected on tomato plants may represent a problem for practical implementation. To face this challenge, we are focusing on other nano-objects to allow highly stable carlina oxide formulation, such as zein nanocapsules and polymeric micelles (Athanassiou et al. 2018; Sanchez-Gomez et al. 2022). Further research is still required to understand its method of action and bioactivity on non-target species (Jesser et al. 2020b; Giunti et al. 2022; Yerguerdan et al. 2022), with a focus on tomato biological control agents and pollinators.

Acknowledgements

The authors are grateful to Antonello Tucci (CREA-Research Centre for Plant Protection and Certification -_Palermo) and Milko Sinacori (Department of Agricultural, Food and Forest Sciences (SAAF), University of Palermo) for their help during plant and insect rearing. The study was partially funded by Agritech National Research Center and received funding from the European Union Next-GenerationEU (PIANO NAZIONALE DI RIPRESA E RESILIENZA (PNRR) – MISSIONE 4 COMPONENTE 2, INVESTIMENTO 1.4 – D.D. 1032 17/06/2022, CN00000022; Spoke 2 Task 2.2.4 Biopesticides and biostimulants) (R. Rizzo). This manuscript reflects only the authors’ views and opinions, neither the European Union nor the European Commission can be considered responsible for them.

Author Contributions

ST, FM, GB, and RR conceived and designed the study. ST, VZ, DRP, MF, ES, FM, GB, and RR conducted the experiments and/or analysed data. ST, VZ, DRP, MF, ES, and RR wrote the original draft. All authors revised the manuscript and approved its final version.

Declaration of competing interest

The authors report no declarations of interest.

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Toxicity and repellent activity of a carlina oxide nanoemulsion toward the South American tomato pinworm, Tuta absoluta

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Abstract

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Key Message

Introduction

Materials and methods

Plant laboratory rearing

Insect laboratory rearing

Extraction of carlina oxide

Preparation and characterization of tested carlina oxide nanoemulsions

Phytotoxicity on tomato

Contact toxicity on eggs

Topical toxicity on larvae

Ingestion toxicity on larvae

Translaminar toxicity on larvae

Ovideterrence trials

Data analysis

Results

Characterization of tested carlina oxide nanoemulsions

Phytotoxicity of carlina oxide nanoemulsions

Contact toxicity on eggs

Topical toxicity on larvae

Ingestion toxicity on larvae

Translaminar toxicity on larvae

Ovideterrence effect

Discussion

Declarations

References

Additional Declarations

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