Developing green insecticides to manage olive fruit flies? Ingestion toxicity of four essential oils in protein baits on Bactrocera oleae

Industrial Crops & Products 143 (2020) 111884

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Developing green insecticides to manage olive fruit flies? Ingestion toxicity of four essential oils in protein baits on Bactrocera oleae

T

Roberto Rizzoa, Gabriella Lo Verdeb,*, Milko Sinacorib, Filippo Maggic, Loredana Cappellaccic, Riccardo Petrellic, Sauro Vittoric, Mohammad Reza Morshedlood, N’ Guessan Bra Yvette Fofiee, Giovanni Benellif a

CREA Research Centre for Plant Protection and Certification, S.S. 113 - km 245.500, 90011 Bagheria (PA), Italy Department of Agricultural, Food and Forest Sciences, University of Palermo, viale delle Scienze, Ed. 5, 90128 Palermo, Italy c School of Pharmacy, University of Camerino, via Sant’Agostino 1, 62032 Camerino, Italy d Department of Horticultural Science, Faculty of Agriculture, University of Maragheh, 55136-553 Maragheh, Iran e Department of Pharmacognosy, Université Félix Houphouët-Boigny, Abidjan, Cote d’Ivoire f Department of Agriculture, Food and Environment, University of Pisa, via del Borghetto 80, 56124 Pisa, Italy b

ARTICLE INFO

ABSTRACT

Keywords: attract and kill botanical insecticide Integrated Pest Management Pimpinella anisum Trachyspermum ammi Ocimum gratissimum Thymbra spicata

Effective and eco-friendly plant-borne insecticides for developing lure and kill control tools against tephritid flies are scarce. Herein, the activity of four essential oils (EOs) obtained from two Apiaceae, Pimpinella anisum L. and Trachyspermum ammi (L.) Sprague, and two Lamiaceae, Thymbra spicata L. and Ocimum gratissimum L., was evaluated against the olive fruit fly, Bactrocera oleae (Rossi), a key pest of olive groves. The EO chemical composition was determined by gas chromatography coupled with mass spectrometry (GC-MS) analyses. The four EOs incorporated in protein baits were tested for ingestion toxicity on B. oleae adults, mimicking lure and kill assays. Results showed concentration-dependent toxicity, with mortality rates ranging from 6.5% (P. anisum EO at 0.03% w/v concentration) to 100% (P. anisum EO at 0.5% w/v concentration, T. ammi EO at 1% w/v). The best efficacy was achieved by EOs from T. ammi and P. anisum, showing LC50 values of 633 ppm and 771 ppm, respectively, far encompassing currently published findings on the ingestion toxicity of EOs on tephritid adults. Thymol (58.3%), p-cymene (24.7%) and γ-terpinene (14.2%), and (E)-anethole (98.3%) were the major constituents of T. ammi and P. anisum EOs, respectively. Thymol (57.0%), p-cymene (12.4%) and γ-terpinene (6.9%), and carvacrol (41.4%) and p-cymene (41.2%) were the predominant components in O. gratissimum and Th. spicata EOs, respectively. Further field research on the efficacy of these EOs incorporated in food baits against the olive fruit fly is ongoing to boost their real-world application, contributing to develop alternative tools for the sustainable management of B. oleae.

1. Introduction The olive fruit fly, Bactrocera oleae (Rossi), is a monophagous and carpophagous tephritid species attacking fruits of different subspecies of Olea europaea L. (cultivated and wild). This fly is the key pest of olive groves worldwide and its harmfulness has long been documented in the Mediterranean area (Daane and Johnson, 2010). The B. oleae economic damage on fruits for table consumption results both from the fly oviposition stings on fruit surface and from the larval feeding activity, which causes a great depreciation of olives (Tzanakakis, 2006). About oil production and quality, the damage due to B. oleae consists mainly in premature fruit drop, yield decrease due to the larval trophic activity

⁎

(Neuenschwander and Michelakis, 1978), and oil quality deterioration (Gucci et al., 2012; Caleca et al., 2017). Different O. europaea cultivars show a variable susceptibility degree to the olive fly infestations and subsequent production losses (Rizzo et al., 2012; Malheiro et al., 2015a). Since the middle of the last century, the control of B. oleae has been mainly based on the use of chemical insecticides. In the last decades, olive fruit fly “attract and kill” tools based on the use of food attractants (protein baits) or semiochemicals coupled with an insecticide have been proposed as a novel control tool in tephritid Integrated Pest Management (IPM) (Petacchi et al., 2003; Wang et al., 2005; Canale et al., 2013). Notably, attract and kill formulations can be applied using

Corresponding author. E-mail address: [email protected] (G. Lo Verde).

https://doi.org/10.1016/j.indcrop.2019.111884 Received 15 April 2019; Received in revised form 16 October 2019; Accepted 18 October 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.

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specific devices as well as treating limited canopy areas of olive trees. The attract and kill method can be considered selective towards the target tephritid pest, with limited impact (varying according to the tested bait) on non-target species (Gregg et al., 2018). Traditionally, the products mixed to tephritid baits were organophosphates, such as dimethoate and fenthion (Roessler, 1989), whose side effects on beneficial insects are well known (Daane et al., 1990; Hoy and Dahlsten, 1984). It has been stressed that an overuse of pesticides can lead to severe problems on human health and the environment, and causes the development of cross- and multi-resistance in targeted insect populations (Sparks and Nauen, 2015). In this scenario, recent attract and kill efforts have been directed to replace synthetic pesticides with more environmental-friendly active ingredients of natural origin, such as spinosad (Vargas et al., 2002; Stark et al., 2004; Mangan et al., 2006; Piñero et al., 2009). Overall, a growing interest in environmentally-friendly control measures has been developed, including the use of mineral products like clays and copper (Saour and Makee, 2004; Caleca et al., 2010; Tsolakis et al., 2011; Thakur and Gupta, 2016) as well as natural products as biopesticides (Pavela and Benelli, 2016). An outstanding number of recent studies have highlighted that plant essential oils (EOs) have antifungal, antimicrobial, cytostatic and insecticidal activities (Bakkali et al., 2008; Miresmailli and Isman, 2014). Essential oils are natural mixtures of compounds characterized by low molecular weight, volatility and lipophilic nature, present in many plant families, among them Asteraceae, Apiaceae, Lamiaceae, Myrtaceae, Lauraceae and Rutaceae, showing a relevant insecticidal action (Maggi and Benelli, 2018; Pavela et al., 2019b). The use of natural products meets the main European policies promoting more sustainable crop production strategies (Directive 2009/128/EC) and follows the IOBC/WPRS “Guidelines for Integrated Production of Olives” (Malavolta and Perdikis, 2012). Moreover, the US FDA (Food and Drug Administration) and the EPA (Environmental Protection Agency) consider many of these natural products as GRAS (Generally Recognized as Safe), therefore selected ones can be of interest for real-world insecticide development (Miresmailli and Isman, 2014; Isman, 2019). While the potential of EOs as sources of green pesticides for realworld applications against several arthropods of agricultural (e.g., mainly aphids and moths) and public health importance (mainly mosquitoes and ticks) have been deeply explored (Stevenson et al., 2017; Benelli and Pavela 2018a, b), our knowledge about their efficacy against tephritid species remains patchy. Indeed, EOs insecticidal activity has been mostly investigated against the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) (e.g., Hamraoui and Regnault-Roger, 1997; Moretti et al., 1998; Sanna-Passino et al., 1999; Pavlidou et al., 2004; Chang et al., 2009; Papachristos et al., 2009; Siskos et al., 2009; Benelli et al., 2012, 2013; Faraone et al., 2012a,b; Ghabbari et al., 2018), while limited information is available on B. oleae (Canale et al., 2013) as well as on other Bactrocera flies (Chang et al., 2009; El-Minshawy et al., 2018), despite of their high economic importance. Furthermore, some plant extracts (e.g. Citrus aurantium) have been successfully tested for their insecticidal activity on olive fruit fly (Siskos et al., 2007, 2009). Repellent potential of some EOs against B. oleae has also been observed (Orphanidis and Kalmoukos, 1970). Notably, recent studies pointed out the useful potential of EOs as sources of compounds to be used in attract and kill control attempt (Canale et al., 2013; Barud et al., 2014; Malheiro et al., 2015b). On this basis, in the present study, we selected the EOs obtained from four plant species, i.e. Pimpinella anisum L., Trachyspermum ammi (L.) Sprague (Apiaceae), Thymbra spicata L. and Ocimum gratissimum L. (Lamiaceae), for which interesting results for a future use as botanical insecticides have been recently reported on various insect pests and vectors (Benelli et al., 2017, 2018; Pavela et al., 2018, 2019a,b). In detail, P. anisum (common name: aniseed), is an annual herbaceous plant characterized by medicinal and aromatic properties, which is

cultivated in the Mediterranean area and Middle East and used as a typical flavouring in food industry. The fruits (schizocarps) from which EO is extracted can reach a 6% yield (Lubbe and Verpoorte, 2011). The aniseed EO has been reported as insecticide against the tephritids C. capitata, Bactrocera dorsalis (Hendel) and Bactrocera cucurbitae Coquilett (Chang et al., 2009). The powder and EO obtained from P. anisum showed to manage Tribolium castaneum (Herbst) beetles (Coleoptera: Tenebrionidae) on stored products (Nenaah and Ibrahim, 2011). Trachyspermum ammi (common name: ajwain) is an annual plant native to Egypt; it is also widely distributed and cultivated in arid and semiarid regions of Iraq, Iran, Afghanistan, Pakistan, and India (Ashraf, 2002; Bairwa et al., 2012; Vitali et al., 2016). The ajwain EO action was investigated towards the pinewood nematode Bursaphelenchus xylophilus (Steiner et Buhrer) (Park et al., 2007) and the Japanese termite, Reticulitermes speratus Kolbe (Seo et al., 2009). Thymbra spicata, commonly known as thyme, has a wide distribution in the coastal areas of East Mediterranean from Greece to Israel, extending to Iraq and Iran (Bräuchler, 2018). The economic importance of this plant species is due to the phenolic monoterpenes, especially carvacrol and thymol, which have been shown to be effective against several pathogens, including soil-borne plant pathogens, fungi infecting stored products, mycotoxic species and human pathogens (Yegen et al., 1992; Mueller-Riebau et al., 1995; Sampson et al., 2005; Barakata et al., 2013). Moreover, recent studies have shown insecticidal action of Th. spicata EO against adult turnip aphid, Lipaphis pseudobrassicae (Davis), the mite Tetranychus cinnabarinus Boisd., the fly Drosophila aurariaPeng, and the whitefly Bemisia tabaci Gen. (Konstantopoulou et al., 1992; Sampson et al., 2005; Sertkaya et al., 2010a, Sertkaya et al., 2010b). Ocimum gratissimum, known as African basil, is a perennial and aromatic species found in tropical and warm temperature regions of Africa, South Asia and South America (Gopi et al., 2006). Ocimum gratissimum EO has shown a repellent and insecticidal action, being very effective to control various insect species such as Aedes aegypti L., Musca domestica L., Culex quinquefasciatus Say, Spodoptera littoralis (Boisd.), Sitophilus oryzae (L.), T. castaneum, Oryzaephilus surinamensis (L.), Rhyzopertha dominica (F.), Callosobruchus chinensis (L.), and Phyllaphis fagi L. (Cavalcanti et al., 2004; Ogendo et al., 2008; Nguemtchouin et al., 2013; Yazdgerdian et al., 2015; Benelli et al., 2019). All these EOs can be obtained from cultivations in the respective countries so that they are promising as scalable ingredients of effective, eco-friendly and safe insecticidal formulations. Moreover, obtaining essential oils from commercial crop plants could provide suitable productions at low cost. On the above, this study aimed at evaluating for the first time the insecticidal activity of P. anisum, T. ammi, Th. Spicata, and O. gratissimum EOs on B. oleae. The EOs chemical composition was achieved by gas chromatography coupled with mass spectrometry (GC–MS), whereas their insecticidal activity on B. oleae adults was assessed through laboratory ingestion bioassays, where the four EOs were incorporated in protein baits. 2. Materials and methods 2.1. Plant material Trachyspermum ammi (schizocarps) was obtained from a cultivation conducted in the University of Maragheh (N 37°23′; E 46°16′, 1485 m a.s.l.), Iran, in August 2018. Pimpinella anisum (schizocarps) was obtained from a local market in Damasco, Siria, in August 2018. Ocimum gratissimum (leaves) was purchased in a local market sited in Abidjan, Ivory Coast, in August 2017. Thymbra spicata (leaves) was collected in Tayibe, South Lebanon (N 33°16′35′′; E 35°31′14′′, 800 m a.s.l.), in May 2018. For Th. spicata and O. gratissimum the botanical identification was performed by one of the authors (F. Maggi) and a voucher specimen was deposited in the Herbarium of the Floristic Research Centre of the Apennines, San Colombo, Barisciano, central Italy, and the National Floristic Center of the University of Félix Houphouët-Boigny, Ivory 2

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Table 1 Chemical composition of the essential oils from Pimpinella anisum, Ocimum gratissimum, Trachyspermum ammi and Thymbra spicata. No

Componenta

RIb

RI Lit.c

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54

α-thujene α-pinene Camphene Sabinene β-pinene 1-octen-3-ol 3-octanone Myrcene 3-octanol α-phellandrene δ-3-carene α-terpinene p-cymene Limonene β-phellandrene 1,8-cineole (Z)-β-ocimene (E)-β-ocimene γ-terpinene cis-sabinene hydrate Terpinolene p-cymenene trans-sabinene hydrate Linalool 1,3,8-p-menthatriene trans-thujone Camphor Borneol Umbellulone terpinen-4-ol p-cymen-8-ol α-terpineol methyl chavicol coahuilensol, methyl ether thymol, methyl ether (Z)-anethole (E)-anethole thymol carvacrol α-cubebene α-copaene β-elemene (E)-caryophyllene α-trans-bergamotene α-humulene γ-himachalene germacrene D β-selinene α-selinene α-zingiberene 7-epi-α-selinene δ-cadinene caryophyllene oxide (E)-pseudoisoeugenyl 2-methylbutyrate

922 927 940 967 970 978 988 990 1000 1004 1009 1015 1022 1025 1026 1030 1038 1048 1056 1065 1086 1087 1097 1102 1109 1114 1140 1161 1171 1173 1184 1189 1196 1214 1235 1251 1285 1294 1304 1345 1369 1387 1410 1431 1444 1468 1472 1477 1486 1487 1507 1518 1572 1844

924 932 946 969 974 974 979 988 988 1003 1008 1014 1020 1024 1025 1026 1032 1044 1054 1065 1086 1089 1098 1095 1108 1112 1141 1165 1167 1174 1179 1186 1195 1219 1232 1249 1282 1289 1298 1345 1374 1389 1417 1432 1452 1481 1484 1489 1498 1493 1520 1522 1583 1841

Total identified (%) Chemical classes (%) Monoterpene hydrocarbons Oxygenated monoterpenes Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Phenylpropanoids Others

P. anisumd (%)

O. gratissimumd (%)

T. ammid (%)

Th. spicatad (%)

IDe

1.3 0.4 trf 0.1 0.1 tr tr 1.4

± 0.3 ± 0.1

0.2 ± 0.0 0.1 ± 0.0

± 0.0 ± 0.0

tr 0.7 ± 0.2

0.3 ± 0.1 0.9 ± 0.2 0.1 ± 0.0

± 0.3

0.3 ± 0.0

RI,MS Std Std Std Std Std RI,MS Std RI,MS Std Std Std Std Std RI,MS Std Std Std Std RI,MS Std RI,MS RI,MS Std RI,MS RI,MS Std Std RI,MS Std RI,MS Std RI,MS RI,MS RI,MS RI,MS Std Std Std RI,MS RI,MS Std Std RI,MS Std RI,MS RI,MS RI,MS RI,MS RI,MS RI,MS RI,MS Std RI,MS

0.1 ± 0.0 0.1 ± 0.0 1.1 ± 0.3 12.4 ± 1.0 0.6 ± 0.1 tr 0.2 tr 6.9 0.4 0.1 1.0 0.2 0.1 tr 0.1

0.8 ± 0.2 0.1 ± 0.0 98.3 ± 0.9

0.2 ± 0.0

tr

0.6 ± 0.1 99.9

0.2 99.7

a

0.1 0.1 1.2 0.2 0.1

± 0.0 ± ± ± ± ± ±

1.1 0.1 0.0 0.2 0.0 0.0

tr tr 0.2 ± 0.0 24.7 ± 2.9 tr 0.3 ± 0.1 tr 14.2 ± 2.1 tr tr tr tr

± 0.0 ± ± ± ± ±

0.0 0.0 0.3 0.0 0.0

0.1 ± 0.0 0.1 ± 0.0 0.7 ± 0.2 tr 0.1 ± 0.0 tr 1.4 ± 0.3 41.2 ± 3.9 0.7 ± 0.2 0.1 ± 0.0 5.5 ± 1.1 tr tr

tr 0.1 ± 0.0 0.2 ± 0.0 0.1 ± 0.0

0.3 ± 0.1 tr tr

58.3 ± 3.4 0.6 ± 0.2

5.2 ± 1.0 41.4 ± 3.6

tr 0.3 ± 0.0 57.0 ± 3.6 1.2 ± 0.3 0.1 ± 0.0 0.6 ± 0.2 0.1 ± 0.0 3.2 ± 0.6 0.1 ± 0.0 0.4 ± 0.1

1.3 ± 0.3 tr

0.1 ± 0.0 4.7 ± 0.9 1.5 ± 0.3 0.4 ± 0.1 0.4 ± 0.1 0.4 ± 0.1

0.1 ± 0.0

98.9

99.9

99.7

26.0 61.0 11.5 0.4

40.7 59.2

51.0 47.1 1.3 0.1

0.1

0.1

Order of compounds is according to that from a HP-5MS (5% phenylmethylpolysiloxane, 30 m x0.25 mm, 0.1 μm) column. Temperature-programmed retention indices (RIs) using a mixture of C8-C30 alkanes. c Literature RIs taken from ADAMS and/or NIST17 libraries. d Relative peak area percentage as the mean of three independent measurements ± standard deviation. e Method of identification: Std, comparison with RI and MS of analytical standard (Sigma, Milan, Italy); RI, correspondence of the calculated values with those stored in ADAMS and NIST 17 libraries; MS, mass spectrum matching with ADAMS, NIST 17, FFNSC2 and WILEY 275 libraries. f traces, % < 0.1. b

3

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Coast, with the codes APP no. 59,011 and Adjanohoun and Aké-Assi no. 225, Akoupé, Fofie no. 05, respectively.

with pure emulsion alone. Five replicates for each concentration were performed. The number of dead insects was recorded daily, the last checking being carried out at 4 days. All tests were conducted inside a climatic room 21 ± 1 °C with a 16:8 (L:D) photoperiod.

2.2. Essential oil distillation The dried plant material, represented by schizocarps of P. anisum (200 g) and T. ammi (500 g), and leaves of O. gratissimum (400 g) and Th. spicata (930 g), was soaked overnight with 6 L of distilled water in a round flask (10 L of volume) and then subjected to hydrodistillation using a Clevenger-type apparatus until no more oil was condensed (approximately 3 h). Afterwards, the EO was separated from the aqueous layer and dehydrated using anhydrous sodium sulfate, then stored in 30 mL amber vials in darkness at 4 °C until use. The oil yields were determined on a dry weight basis (w/w); they were 1.6, 2.5, 2.2, and 2.0%, respectively.

2.6. Statistical analysis The experimental mortality was corrected with Abbott’s formula (Abbott, 1925) and transformed by arcsine√ before statistical analysis performed using ANOVA (two factors as fixed effects) followed by Tukey’s HSD test (p < 0.05). Ingestion LC50 and LC90 with associated 95% Confidence Interval (CI) and chi-squares, were estimated using probit analysis (Finney, 1978). MINITAB software was used for all statistical analyses (Minitab, Inc., State College, PA).

2.3. Gas chromatography-mass spectrometry analysis

3. Results and discussion

The four EOs were diluted 1:100 in n-hexane (Carlo Erba, Milan, Italy) then injected (2 μL) into a GC-MS system (Agilent 6890 N gas chromatograph equipped with a 5973 N single quadrupole mass spectrometer; Santa Clara, CA, USA) equipped with an auto-sampler 7863 (Agilent, Wilmingotn, DE) using the following analytical conditions; stationary phase: HP-5MS (5% phenylmethylpolysiloxane, 30 m, 0.25 mm i.d., 0.1 μm film thickness; Folsom, CA); mobile phase: He (99.99%) at 1 mL/min; oven temperature: 60 °C kept for 5 min, then increment up to 220 °C at 4 °C/min, finally to 280 °C at 11 °C/min; split ratio: 1:50; mass detector operating conditions: electron impact (EI) with acquisition in the range of 29–400 m/z. Identification and quantification methods were the same of those reported in our previous papers (Maggi et al., 2009; Zorzetto et al., 2015) using the software NIST Mass Spectral Search Program for the NIST/EPA/NIH EI and MSD ChemStation (Agilent, Version G1701DA D.01.00). For retention index determination, a mixture of n-alkanes (C8-C30, Supelco, Bellefonte, CA) was injected using the above reported conditions and used to calculate the arithmetic index (AI) by means of the Van den Dool and Kratz (1963). Mass spectra of the peaks were studied against the ADAMS, NIST 17, WILEY 275 and FFNSC2 libraries.

3.1. Chemical analysis of the essential oils The chemical composition of the EOs from P. anisum, O. gratissimum, T. ammi and Th. spicata is depicted in Table 1, where a total of 54 volatile components are reported. The EO of P. anisum was almost entirely composed of phenylpropanoids (99.7%) among which (E)-anethole was by far the predominant one (98.3%). Methyl chavicol (0.8%), (E)pseudoisoeugenyl 2-methylbutyrate (0.6%) and (Z)-anethole (0.1%) were minor components. The only terpenes in this EO were γ-himachalene (0.2%) and α-zingiberene (< 0.1%). The P. anisum EO composition resulted quite similar to those reported for other accessions cultivated in the Mediterranean area (Iannarelli et al., 2017). Regarding the other three EOs, namely O. gratissimum, T. ammi and Th. spicata, they were mainly characterized by monoterpene hydrocarbons and oxygenated monoterpenes, with a noteworthy content of phenolic monoterpenes such as thymol and carvacrol. Notably, the O. gratissimum EO was dominated by oxygenated monoterpenes (61.0%), followed by lower contents of monoterpene hydrocarbons (26.0%) and sesquiterpene hydrocarbons (11.5%), with thymol (57.0%), p-cymene (12.4%), γ-terpinene (6.9%) and β-selinene (4.7%) as the most representative compounds. This composition was consistent with that reported in a recently published study and confirmed the membership of this oil to the thymol chemotype, which has been frequently accounted for O. gratissimum (Benelli et al., 2019). The T. ammi EO was characterized by oxygenated monoterpenes (59.2%) and monoterpene hydrocarbons (40.7%), with thymol (58.3%) as the predominant compound, followed by p-cymene (24.7%) and γterpinene (14.2%). These constituents are biogenetically related, being formed through the activity of thymol synthases CYP71D178, CYP71D179 and CYP71D182 (Morshedloo et al., 2017). The other compounds occurred in percentages in all cases below 1%. Based on these results, the chemical profile of T. ammi EO obtained from accessions growing in Iran and India appeared to be quite constant, with sporadically-occurring differences (Vitali et al., 2016; Bairwa et al., 2012). The EO of Th. spicata was characterized by comparable amounts of monoterpene hydrocarbons (51.0%) and oxygenated monoterpenes (47.1%), being p-cymene (41.2%) and carvacrol (41.4%) as the most representative compounds, respectively, whereas γ-terpinene and thymol gave minor contributions (5.5 and 5.2%, respectively). In this case, the carvacrol synthases CYP71D180 and CYP71D181 play a major role in the production of these monoterpenoids with respect to thymol synthases (Crocoll, 2011). Based on these results and previous reports (Hanci et al., 2003; Baydar et al., 2004; Kılıç (2006), it can be concluded that the carvacrol/p-cymene chemotype is a hallmark of Th. spicata.

2.4. Olive fruit fly rearing Insects used in this study were obtained from pupae of B. oleae collected in two Sicilian olive mills located in Sciacca (Agrigento) and Caccamo (Palermo). Pupae were preserved inside plastic boxes (30 × 30 x 15 cm) in dark climatic room at 8 °C. Groups of about 2000 pupae each were moved every week inside plastic cages (30 × 30 x 30 cm) that were then put in a climatic room at 21 ± 1 °C with a 16:8 (L:D) photoperiod, in order to obtain a progressive emergence of olive fly adults for laboratory assays. Flies were fed with an aqueous solution of 10% organic honey until the tests begin. 2.5. Ingestion toxicity bioassays Following the protocol described by Benelli et al. (2012) and Canale et al. (2013), bioassays were performed using groups of 10 adults (both sexes, 10–15 days old) randomly selected from the main rearing cages and placed inside transparent plastic boxes (450 mL), with a thin mesh on the top to allow air exchange. The olive fruit flies were fed with different concentrations of the tested EOs, mixed with 2 mL of an aqueous emulsion containing 2% of carboxy-methylcellulose sodium salt (Sigma-Aldrich®, medium viscosity), 12.5% of sucrose and 1% of the protein bait Nu-Bait® (Biogard). A small dish of sterile cotton wool was put inside a plastic cup, soaked with the emulsion containing the EOs, to avoid insects drowning, and placed inside each plastic box. The following concentrations (w/v) were tested for each EO: 0.03, 0.06, 0.125, 0.25, 0.5, and 1%, while in the control boxes olive flies were fed 4

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Table 2 Ingestion toxicity of the four essential oils formulated in protein baits on adults of the olive fruit fly, Bactrocera oleae. Essential oil and concentration (w/v)

0.03% (mortality% ± SD)

0.06% (mortality% ± SD)

Thymbra spicata Pimpinella anisum Trachyspermum ammi Ocimum gratissimum

10.20 ± 4.56 fg 6.52 ± 5.95 g 21.28 ± 16.13 fg 10.42 ± 5.71 fg

14.29 50.00 61.70 45.80

± ± ± ±

9.13 fg 19.75 cdef 19.33 bcde 30.70 defg

0.125% (mortality% ± SD) 16.33 71.70 70.21 70.80

± ± ± ±

8.54 efg 26.20 abcd 20.46 abcd 22.60 abcd

0.25% (mortality% ± SD) 48.98 93.48 95.74 85.40

± ± ± ±

7.22 cdef 5.95 ab 9.52 a 22.80 ab

0.5% (mortality% ± SD)

1% (mortality% ± SD)

75.51 ± 11.63 abcd 100 ± 0.00 a 97.87 ± 4.76 a 85.42 ± 13.98 abcd

87.76 ± 18.25 100 ± 0.00 a 100 ± 0.00 a 87.50 ± 13.58

ab

abc

Means ± SD within a column followed by the same letter do not differ significantly (ANOVA followed by Tukey’s HSD test, p < 0.05).

3.2. Insecticidal activity on olive fruit flies

through different mechanisms of action. The former can neutralize the P450 insect detoxification system (Afshar et al., 2017). (E)-Anethole was found to be effective towards the fruit flies C. capitata, B. dorsalis and B. cucurbitae (Chang et al., 2009). In addition, it did not affect the aquatic microcrustacean Daphnia magna Straus even after long-term exposure (Pavela, 2014). Thymol can inhibit acetylcholinesterase (AChE) (López et al., 2018) and also interacts with GABA and octopaminergic receptors (see Pavela and Benelli, 2016 and references therein). In the cell, thymol interacts with transmembrane proteins forming channels that increase membrane fluidity and permeability (Burt, 2004). Thymol is effective against many mosquito vectors and agricultural pests, with LC50 values in most cases below 100 ppm (Maggi and Benelli, 2018). To the best of our knowledge, no data are available on tephritid flies. However, earlier studies testing selected EOs through ingestion assays on adult medflies showed irreversible gut damage, with the best insecticidal action exerted by Thymus herbabarona Loisel. and Cinnamomum verum J.Presl EOs, which are rich in carvacrol and cinnamaldehyde, respectively (Moretti et al., 1998). Further research aimed at formulating the main components of the P. anisum and T. ammi EOs against B. oleae in highly stable nano- and micro-emulsions is ongoing (Pavela et al., 2019a). Moreover, despite the equal concentrations of thymol in O. gratissumum and T. ammi EOs, their effectiveness against the olive fruit fly was quite different, probably because of the action of other EOs components. Indeed, p-cymene and γ-terpinene resulted present in T. ammi with a double concentration compared with O. gratissimum EO. Furthermore, O. gratissimum and T. ammi EOs differ also for the number of EOs components (44 in O. gratissimum and 21 in T. ammi). However, further studies are needed to evaluate the action of the main EOs components and the possible occurrence of a synergistic or antagonist action. Safety concerns regarding these substances are quite minimal. Indeed, toxicity of (E)-anethole, γ-terpinene and thymol in rats after oral administration has been reported to be relatively low, especially when compared with that of synthetic insecticides, with LD50 values of 2090, 1680 and 980 ppm, respectively (Isman and Machial, 2006). Dermal toxicity of thymol in rats and rabbits exceeds 2,000 and 5,000 ppm, respectively (Park et al., 2017). Thymol is also used to protect honey bees against mites such as Varroa destructor (Anderson et Trueman) (Floris et al., 2004). Both (E)-anethole and thymol are also recognized as GRAS substances by the FEMA and United States FDA (Newberne et al., 1999; Guarda et al., 2011).

The four EOs investigated in this study showed noteworthy insecticidal activity against the olive fruit fly when incorporated in protein baits (Table 2). A significant effect of the tested EOs (F5, 96 = 74.24, p < 0.001), their concentration (F3, 96 = 23.78, p < 0.001) and the interaction tested EO*concentration (F15, 96 = 2.15, p = 0.013) were observed. The best efficacy was achieved testing the EOs from T. ammi and P. anisum, reaching 50% of mortality at 0.06%, and 100% at 1% and 0.5% concentrations, respectively. The efficacy of both EOs was confirmed by the LC50 values, which were 633 ppm and 771 ppm, respectively, as well as by LC90 values, which were 2,131 ppm and 1,981 ppm, respectively (Table 3). Notably, the bioactivity of these two EOs against the olive fruit fly was substantially higher if compared with earlier results testing Hyptis suaveolens (L.) Poiteau, Rosmarinus officinalis L. and Lavandula angustifolia Mill. EOs incorporated in protein baits, which achieved LC50 values of 4,922, 5,107 and 6,271 ppm, respectively (Canale et al., 2013). Moreover, the three EOs mentioned above, as well as the Thuja occidentalis L. EO had comparable toxicity on adult medflies in ingestion toxicity assays, where the T. occidentalis oil resulted the most effective, with an LC50 of 5,371 ppm (Benelli et al., 2012). Tea tree, Melaleuca alternifolia (Maiden & Betche) Cheel EO also showed an ingestion LC50 value of 0.269% (w/w) on adult medflies (Benelli et al., 2013). However, toxicity reported in the latter researches were significantly lower if compared with results obtained testing T. ammi and P. anisum on olive flies in the present study. Interestingly, herein the oils from O. gratissimum and Th. spicata were endowed with significantly lower potency. It could be assumed that in the first case the lower insecticidal activity can be due to the presence of sesquiterpenes (almost 11%), which are known to possess a minor effect on some insects (Benelli et al., 2019b). For Th. spicata, the lower efficacy can be ascribable to the higher content of carvacrol than thymol; indeed, carvacrol has been reported to be less active than thymol on some insects (Park et al., 2017). Furthermore, the lower efficacy of this EO may be related to possible antagonistic effects occurring between active constituents of the EO (Pavela, 2015). Earlier research showed that T. ammi and P. anisum are two of the most promising EOs to be employed in insecticidal formulations (Pavela et al., 2019b). These two EOs showed to be effective against several vectors and pests of economic importance (Benelli et al., 2017, 2018; Chang et al., 2009; Seo et al., 2009; Tabari et al., 2017). The major compounds of the two EOs, namely (E)-anethole and thymol, act

Table 3 Lethal concentrations of the four essential oils formulated in protein baits on adults of the olive fruit fly. Essential oil

LC50 (95%CI) (ppm)

LC90 (95%CI) (ppm)

Intercept ± SE

Slope ± SE

Goodness of fit χ2 (d.f.)

Thymbra spicata Pimpinella anisum Trachyspermum ammi Ocimum gratissimum

2,509 (2,010-3,155) 771 (629-919) 633 (489-782) 925 (670-1,208)

12,519 (8,690-21,469) 1,981 (1,593-2,708) 2,131 (1,654-3,066) 6,365 (4,407-10,915)

−6.240 −9.022 −6.816 −4.538

1.836 3.125 2.433 1.530

6.872 (4) p = 0.143 n.s. 4.067 (4) p = 0.397 n.s. 4.943 (4) p = 0.293 n.s. 15.482 (4) p = 0.004

LC = lethal concentration killing 50% (LC50) or 90% (LC90) of the exposed population; 95% CI = 95% confidence interval; n.s. = not significant. 5

± ± ± ±

0.707 1.167 0.920 0.592

± ± ± ±

0.208 0.390 0.308 0.184

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R. Rizzo, et al.

4. Conclusions

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