Acaricidal activity, mode of action, and persistent efficacy of selected essential oils on the poultry red mite (Dermanyssus gallinae)

Food and Chemical Toxicology 138 (2020) 111207

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Acaricidal activity, mode of action, and persistent efficacy of selected essential oils on the poultry red mite (Dermanyssus gallinae)

T

Mohaddeseh Abouhosseini Tabaria, Arash Rostamib, Aref Khodashenasb, Filippo Maggic,∗, Riccardo Petrellic, Cristiano Giordanid,e, Léon Azefack Tapondjouf, Fabrizio Papag, Yanting Zuog, Kevin Cianfaglioneh,i, Mohammad Reza Youssefij,∗∗ a

Faculty of Veterinary Medicine, Amol University of Special Modern Technologies, Amol, Iran Young Research Club and Elite, Babol Branch, Islamic Azad University, Babol, Iran c School of Pharmacy, University of Camerino, Camerino, Italy d Instituto de Física, Universidad de Antioquia, Calle 70 No. 52-21, Medellín, Colombia e Grupo Productos Naturales Marinos, Facultad de Ciencias Farmacéuticas y Alimentarias, Colombia f Laboratory of Environmental and Applied Chemistry, Faculty of Science, University of Dschang, Dschang, Cameroon g School of Science and Technology, University of Camerino, Camerino, Italy h EA 2219 Géoarchitecture, UFR Sciences & Techniques, Université de Bretagne Occidentale, Brest, France i School of Biosciences and Veterinary Medicine, University of Camerino, ì Camerino, Italy j Department of Veterinary Parasitology, Babol Branch, Islamic Azad University, Babol, Iran b

A R T I C LE I N FO

A B S T R A C T

Keywords: Dermanyssus gallinae Essential oils Acaricides Food safety

In this work, the essential oils (EOs) from Litchi chinensis, Clausena anisata, Heracleum sphondylium, Pimpinella anisum, Lippia alba, Crithmum maritimum and Syzygium aromaticum were tested for their contact toxicity against the poultry red mite, Dermanyssus gallinae, a deleterious ectoparasite of aviary systems. In addition, in order to give insights on their mode of action and effectiveness, the vapor phase and residual toxicity tests were also performed. Results showed that amongst all the tested EOs, that of S. aromaticum demonstrated the highest contact toxicity, with a LC50 value of 8.9 μg/mL, followed by C. maritimum and L. chinensis EOs, with LC50 values of 23.7 and 24.7 μg/mL, respectively. L. chinensis and C. anisata EOs showed higher vapor toxicity than the other EOs. L. chinensis and S. aromaticum EOs showed promising toxic effects up to 4 days post-application. Taken together, these results highlighted L. chinensis and S. aromaticum as two promising sources of biopesticides, able to cause severe contact, vapor and residual toxicity in the poultry red mites. Given the wide plant cultivation and uses in foodstuffs, cosmetics, flavour and fragrances, these EOs may be considered cheap and ready-to-use products as valid, eco-friendly alternatives to pesticides currently used in the aviary systems.

1. Introduction

rhusiopathiae, Escherichia coli, Streptomyces sp., Staphylococcus sp., Yersinia, Listeria, Pasteurella sp. and Rickettsia sp.), viruses (Newcastle disease virus, chickenpox virus, fowl typhoid, and fowl cholera), and parasites such as the genus Hepatozoon (Chirico et al., 2003; Moro et al., 2005, 2009; Zeman et al., 1982). From an economic point of view, this mite is responsible for losses of millions of dollars per year, due to its higher feed intake causing a lower egg quality and production, high mortality and expensiveness of the treatment costs (Kilpinen et al., 2005; Van Emous et al., 2006). As a chemical control, long term application of pesticides (e.g., organophosphates, organochlorines, carbamates, amitraz and pyrethroids) induced heritable resistance in mites, undesirable effects on non-target organisms, threatening both

Dermanyssus gallinae (De Geer, 1778) (Acari, Dermanyssidae), also known as the poultry red mite, is the most harmful ectoparasite of laying hens in many countries around the world (Sparagano et al., 2014; Tabari et al., 2017). To a lesser extent, it can also affect broilers, aviary birds, wild birds, and in some cases, poultry operators causing itching dermatitis (Chauve, 1998; Marangi et al., 2009). This obligatory blood-sucking ectoparasite causes blood loss leads to anaemia, irritation, stress, restlessness, blood-stained eggs, and, in the case of massive infestation, even death (Kirkwood, 1967). Moreover, this mite is likely a vector of many pathogenic agents such as bacteria (Erysopelothrix

∗

Corresponding author. Corresponding author. E-mail addresses: fi[email protected] (F. Maggi), youssefi[email protected] (M.R. Youssefi).

∗∗

https://doi.org/10.1016/j.fct.2020.111207 Received 26 November 2019; Received in revised form 7 February 2020; Accepted 14 February 2020 Available online 16 February 2020 0278-6915/ © 2020 Elsevier Ltd. All rights reserved.

Food and Chemical Toxicology 138 (2020) 111207

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2.2. Hydrodistillation

animals and human health and environmental issues (Flamini, 2003; Naqqash et al., 2016). On the above, there is an urgent need to substitute these chemicals through the adoption of safer alternative control strategies. Plant-derived essential oils (EOs) are liquid mixtures usually made up of small, lipophilic and bioactive compounds of terpenoid or phenylpropanoid nature, with many of them being used as natural acaricides devoid of detrimental effects on non-target organisms and environment (Benelli, 2015). Although botanical pesticide research has been mainly focused on mosquito and ticks (Pavela et al., 2016), studies on other arthropods of medical and veterinary importance are still in the preliminary phase. Several botanical pesticides are currently used in arthropod pest management. For instance, products based on the neem tree are quite common. Neem extracts are reported to have toxic effects against 200 species of arthropod pests (Choi et al., 2004). Plants can produce a broad range of secondary metabolites such as terpenoids, polyacetylenes, sugars, flavonoids and alkaloids which may act as antifeedants and repellents. In addition, they can also suppress the acetylcholinesterase activity leading to nervous system intoxication (Isman, 2006; Masoumi et al., 2016; Tabari et al., 2015; Vigan, 2010). Besides, they may act on other types of targets in the nervous system like nicotinic acetylcholine receptors (nAChR), octopamine receptors, tyramine receptors, sodium channels and γ-aminobutyric acid (GABA)gated chloride channels (Coats et al., 1991; Kostyukovsky et al., 2002; Nesterkina et al., 2018). On the above, we decided to document the potentially toxic effects of some plant EOs, notably those from Litchi chinensis Sonn., Clausena anisata (Willd.) Hook.f. ex Benth., Heracleum sphondylium L., Pimpinella anisum L., Lippia alba (Mill.) N.E.Br. ex Britton & P.Wilson, Crithmum maritimum L., and Syzygium aromaticum (L.) Merr. & L.M.Perry on D. gallinae. The selection of the above EOs was based on previous reports documenting their effectiveness against insect vectors and pests of public health and agricultural importance (Park and Shin, 2005; Benelli et al., 2018; Kamte et al., 2018; Pavela et al., 2017, 2018). Therefore, we evaluated the toxicity of these EOs through contact and fumigant assays on adult mites. Also, their duration of action was assessed through residual toxicity bioassays.

The plant material cut into small pieces was put into glass flasks of 6–10 L capacity equipped with Clevenger-type modules and subjected to hydrodistillation for 3 h. The oil yield (w/w) was determined on a dry weight basis; it was 5.6, 0.3, 1.6, 2.9, 0.4 and 2.0% for S. aromaticum, L. chinensis, C. maritimum, P. anisum, H. sphondylium, and C. anisata essential oils, respectively. 2.3. GC-FID and GC-MS analyses An Agilent 4890D gas chromatograph coupled with an ionization flame detector (FID) was used. The separation was achieved on HP-5 capillary column (5% phenylmethylpolysiloxane, 25 m, 0.32 mm i.d.; 0.17 μm f.t.) (J & W Scientific, Folsom, CA). The oven temperature was taken 5 min at 60 °C, then raised to 220 °C at 4 °C/min up, finally up to 280 °C at 11 °C/min. The essential oils were diluted in hexane (1:100) and 1 μL of the solution was injected with a split ratio of 1:34. The temperature of the injector and detector was 280 °C. A mixture of nalkanes (C8-C30) (Supelco, Bellefonte, CA) was used under the above conditions to calculate the linear retention index (RI) of peaks. The quantitative values of peaks, as relative peak areas, were obtained by FID peak area normalization. GC-MS analysis was carried out by an Agilent 6890N gas chromatograph equipped with 5973N mass spectrometer (MS). A HP-5MS capillary column (5% phenylmethylpolysiloxane, 30 m, 0.25 mm i.d., 0.1 μm f.t.) (J & W Scientific) was used for separation. The oven temperature was set to 60 °C for 5 min, then raise to 220 °C at 4 °C/min, finally up to 280 °C at 11 °C/min, held for 15 min, and to 300 °C at 11 °C/min, held for 5 min. The carrier gas was helium at a flow rate of 1.0 mL/min. The temperatures of the injector and transfer line were 280 and 250 °C, respectively. The essential oils were diluted in hexane (1:100) and 2 μL of the solution injected into GC-MS with a split ratio of 1:50. The scan time was 75 min; the acquisition mass range was 29–400 m/z. The peak assignment was based on the correspondence of MS and RI of peaks with those stored in the commercial libraries ADAMS 2007 (Adams, 2007), NIST 17, FFNSC2 and WILEY 275 using the ChemStation software. In addition, whenever possible the co-injection of authentic standards (Sigma-Aldrich, Milan, Italy) available in the authors’ laboratory was used as an additional criteria.

2. Material and methods 2.1. Plant material

2.4. Mites

Fresh leaves of C. anisata were gathered in the village of Bafou (N 5° 32′ 30.0′′; E 10° 06′ 13.7″), Western Cameroon, in December 2016. A voucher specimen was archived in the Cameroon National Herbarium, Yaoundé, under the codex 44242/HNC. Schizocarps of P. anisum were obtained from a farm sited in Castignano (N 42°56′10″, E 13°35′00″, 496 m a.s.l.), central Italy, in September 2014. Schizocarps of H. sphondylium were obtained from plants growing in Montelago (N 43°06′30”; E 12°58′25″), central Italy, in July 2015. A voucher specimen was deposited in the Herbarium Universitatis Camerinensis (CAME), School of Bioscience and Veterinary Medicine, University of Camerino with the code CAME 25673. The flowering aerial parts of C. maritimum were collected in Le Conquet (N 48°20′49.31″, O 4°46′12.04″), Brittany, France in September 2016 and a voucher specimen was stored in the Herbarium of Géoarchitecture, Université de Bretagne Occidentale with the reference BRECK9. For L. alba, we directly bought the relative EO from Centro de Investigación de Excelencia – CENIVAM, Bucaramanga, Santander, Colombia. Cloves buds S. aromaticum were purchased from the herbal company Minardi & Figli (https://www.minardierbe.it) in July 2013. The fruits of L. chinensis (origin: Madagascar) were purchased from a market in Rome, Italy, in July 2013.

Red mite (D. gallinae) colonies used in this study were collected from a laying hen farm near Quchan, Khorasan Razavi province, Iran, in June 2019. The collected specimens were transferred to a sealable glass container and held at 25 ± 1 °C and 55 ± 5% relative humidity (R.H.). Mites were stored for 24 h before the experiment phase. 2.5. Contact toxicity The toxicity caused by direct contact was assayed based on the protocol designed by Tabari et al. (2015). We prepared different dilutions of EOs in ethanol (50 μL) corresponding to concentration of 5, 10, 20, 50, 100 and 200 μg/mL. Whatman No. 1 filter papers were impregnated with corresponding dilutions of EOs, and after 3 min drying in a fume cupboard were added to Petri dishes (4.8 cm diameter × 1.4 cm). Then, alive mites of all motile stages (n = 30) were transferred into each Petri dish, and all were sealed with parafilm™. Controls were treated with only 50 μL of ethanol. All assays were carried out in the same aforementioned conditions of temperature and humidity. Three replicates were performed at the same time for all the tested groups of mites. Observations were made by stereomicroscope, 24 h after treatment and the number of dead mites was recorded. Permethrin was used as a positive control. 2

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2.6. Vapor phase toxicity

3.2. Contact toxicity

To find out whether the mode of action of the EOs on red mite was by contact toxicity or vapor toxicity, the protocol described by Tabari et al. (2015) was used. In brief, 20 mites were put in a 2 mL container and two ends of the container were closed by using a mesh barrier which limited the direct contact of mites with the tested EOs. These containers were placed in 15 mL vessels containing EO treated filter papers with concentrations equal to their contact LC50 values in 50 μL of ethanol calculated based on 15 mL volume of the vessel container. Mite containing vessels were either closed with a layer of parafilm™ (Closed) or left uncovered (Open). Mortality rates were determined after 24 h by pushing mites with a pin. If no reaction was recognized under a loop, mites were regarded as dead.

Toxicity data of the seven EOs tested on adult D. gallinae by direct contact are shown in Table 2. Based on the LC50 values, the S. aromaticum EO was the most toxic, showing a LC50 value of 8.9 μg/mL, followed by C. maritimum and L. chinensis EOs, with LC50 values of 23.7 and 24.7 μg/mL, respectively. Permethrin as positive control resulted in a LC50 value of 35.1 μg/mL, which was significantly higher than that of S. aromaticum and L. chinensis, but not C. maritimum. Treatment with EOs of P. anisum, C. anisata and L. alba resulted in a LC50 value of 47.5, 59.0, and 77.7 μg/mL, respectively. Finally, the EO of H. sphondylium had a weak effect on D. gallinae, resulting in 50% lethality at concentrations higher than 200 μg/mL. When comparing the LC90 values of the tested EOs, S. aromaticum was the most potent acaricidal agent against D. gallinae, with a value of 19.7 μg/mL. P. anisum and L. chinensis were the next two efficacious acaricidal EOs though their LC90 values were significantly higher (78.0 and 121.9 μg/mL, respectively). Permethrin LC90 value was 210.2 μg/mL.

2.7. Residual toxicity The assay was performed according to a previously described method (Masoumi et al., 2016). In brief, aluminum tray surfaces were sprayed with each tested compound at its contact LC50 values. After different time periods, i.e. 1, 2, 4, and 6 days post spraying, mites were exposed to treated surfaces, and 24 h mortality rates were recorded.

3.3. Vapor toxicity All the tested EOs displayed significant toxic effects in the closed vapor phase in comparison with the control vessels (P < 0.05, Fig. 1). There was also a significant reduction of toxicity in EOs tested when using the open methods (P < 0.05). The L. chinensis and C. anisata EOs showed higher fumigant toxicity in comparison with the other EOs with moratlity rates of 78 ± 3.66 and 68 ± 2.8%, respectively. There was no significant difference between the vapor phase toxicity of EOs of P. anisum, L. alba, C. maritimum and S. aromaticum (P > 0.05) (see Fig. 2).

2.8. Statistical analyses Mite mortality data were subjected to Probit analysis to determine LC50 and LD90 values. Tukey's HSD was used to test the significant difference between EOs in vapor toxicity assays. Values of P < 0.05 were considered significant. All the statistical analyses were carried out using SPSS software, version 18 (Chicago, IL).

3.4. Residual toxicity EOs were tested for the persistence of their toxic effects on D. gallinae at 1, 2, 4, and 6 days. Considering the high LC50 value in the contact toxicity assay, the H. sphondylium EO was not further tested for residual toxicity. As can be seen in Table 2, all tested EOs showed significant toxic effects at 1 and 2-days post spraying in comparison with the control (P < 0.05). EOs of L. chinensis, C. anisata, and S. aromaticum demonstrated a prolonged toxicity in comparison with the other EOs at day 2 post spraying. Notably, the toxic effects of L. chinensis and S. aromaticum EOs lasted for up to 4 days post spraying (P < 0.05). At a 6-day time point, none of the EOs was able to keep its toxicity on the poultry red mite.

3. Results 3.1. Chemical profiles of EOs The chemical compositions of the seven EOs tested in this work are presented in Table 1. The S. aromaticum EO chemical profile was dominated by the phenylpropanoid eugenol (81.0%) with a minor contribution by its acetate ester (eugenol acetate, 15.5%). The sesquiterpene (E)-caryophyllene was another component occurring at a noteworthy level (2.6%) in this EO. The L. chinensis EO chemical profile was made up of sesquiterpene hydrocarbons (91.4%), with α-zingiberene (38.7%), ar-curcumene (16.9%) and β-selinene (16.2%) as the main constituents. Other noteworthy components were β-sesquiphellandrene (5.9%) and (E)-caryophyllene (3.6%). The L. alba EO was characterized by monoterpenes (75.7%), with carvone (35.2%) and limonene (32.0%) as the most abundant constituents. Germacrene D (14.8%) was the most abundant compound in the sesquiterpene fraction. The major groups dominating the C. maritimum EO composition were the phenylpropanoids (60.4%) and monoterpenes (39.5%), with dill apiole (55.7%), γ-terpinene (14.0%) and carvacrol methyl ether (11.8%) as the main components. The EO of H. sphondylium was almost all made up of aliphatic compounds (94.8%), with esters (89.9%) as the major fraction. Octyl acetate (61.0%) was the most abundant compound followed by octyl butyrate (9.4%) and octyl hexanoate (8.3%). The P. anisum and C. anisata EOs were characterized by phenylpropanoids (98.8 and 84.0%, respectively), with (E)-anethole (94.8 and 64.6%, respectively) as the predominant compound. Methyl chavicol (2.6%) and (E)-methyl isoeugenol (16.1%) were other noteworthy compounds in these EOs, respectively.

4. Discussion Arthropod pests are increasingly developing resistance to synthetic pesticides due to the extensive application of these products during the last decades (Naqqash et al., 2016; Tabari et al., 2017). Some of these pesticides can contaminate food commodities, soil, water, and even air by their residues which may impact human health and other life forms (Aktar et al., 2009). Growing demands for residue-free food products has led market trend toward alternative pesticides, such as nature-derived pesticides (Pavela and Benelli, 2016; Pavela et al., 2016; Semmler et al., 2011). Plant-derived EOs have distinct advantages over synthetic chemicals. For instance, their residues are easily biodegradable in foods and the environment. Besides, thanks to their diverse action sites, resistance to these substances is not easy to occur (de Oliveira Monteiro et al., 2010; Pavela and Benelli, 2016; Pavela et al., 2016). Despite these advantages, there are some challenging facts, including EOs stability and persistence in the field and determination of their exact chemical composition (Turek and Stintzing, 2013). Plant-EOs were found to exert repellent, antifeedant, ovicidal, and toxic effects using both fumigation and filter paper assays. In contact toxicity testing 0.07 mg/cm2 of clove (S. aromaticum) bud and leaf EOs, 100% mortality has been reported; however, reduction of concentration 3

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Table 1 Chemical composition of the selected essential oils assayed against the poultry red mite Dermanyssus gallinae.a Componentb

RI exp.c

RI lit.d

Peak area percentage (%)e S. aromaticum

isopropyl-2-methyl butyrate α-pinene sabinene n-octanal p-cymene limonene γ-terpinene n-octanol methyl chavicol (3Z)-3-octenyl acetate octyl acetate thymol, methyl ether carvone piperitone (E)-anethole piperitenone eugenol β-bourbonene β-elemene 7-epi-sesquithujene octyl butyrate (E)-caryophyllene α-himachalene amorpha-4,11-diene γ-himachalene germacrene D β-selinene ar-curcumene α-zingiberene (E)-methyl isoeugenol β-bisabolene myristicin β-sesquiphellandrene eugenol acetate octyl hexanoate agarospirol dillapiole β-acorenol α-bisabolol xanthorrhizol (E)-pseudoisoeugenyl 2methylbutyrate Oil yield (%, w/w) Total identified (%) Grouped compounds (%) Monoterpene hydrocarbons Oxygenated monoterpenes Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Phenylpropanoids Others

884 926 966 1005 1022 1025 1056 1071 1196 1200 1218 1235 1245 1254 1287 1337 1360 1377 1385 1387 1392 1410 1442 1457 1470 1472 1478 1479 1492 1499 1505 1519 1520 1530 1585 1620 1627 1638 1672 1754 1833

880 932 969 998 1020 1024 1054 1071 1195 1200 1219 1232 1239 1249 1282 1340 1356 1387 1389 1391 1394 1417 1449 1451 1481 1484 1477 1479 1493 1491 1505 1517 1521 1521 1575 1632 1622 1637 1685 1753 1841

IDf

L. chinensis

C. maritimum

P. anisum

H. sphondylium 1.4 ± 0.3 0.2 ± 0.0

2.3 ± 0.2 4.7 ± 0.6 Tr 3.5 ± 0.4 tr 14.0 ± 1.8

2.4 0.1 0.2 0.1 1.3

± ± ± ± ±

0.5 0.0 0.0 0.0 0.3

L. alba

C. anisata

Trg Tr

0.2 ± 0.0 0.6 ± 0.1

Tr 32.0 ± 3.0 Tr

2.9 ± 0.6 0.4 ± 0.1 2.4 ± 0.5

2.6 ± 0.5

2.0 ± 0.4 3.0 ± 0.6 61.0 ± 3.1

11.8 ± 2.0 35.2 ± 2.8 1.1 ± 0.2 94.8 ± 2.1

64.6 ± 2.9

Tr Tr

2.1 ± 0.4 Tr 1.8 ± 0.4 1.0 ± 0.2

0.1 ± 0.0 0.1 ± 0.0

9.4 ± 1.5 0.2 ± 0.0

0.3 ± 0.1

0.8 ± 0.2

0.8 ± 0.2 0.2 ± 0.0

0.2 ± 0.0

14.8 ± 2.2

2.2 ± 0.5

Tr

Tr 0.1 ± 0.0

Tr 0.1 ± 0.0 16.1 ± 2.6 0.3 ± 0.1

81.0 ± 3.4

1.3 ± 0.3 2.6 ± 0.5

3.6 1.0 1.3 1.2

± ± ± ±

0.7 0.2 0.3 0.3

Tr

16.2 ± 1.1 16.9 ± 0.9 38.7 ± 2.4

Tr

1.1 ± 0.2

Tr 0.6 ± 0.2 0.2 ± 0.0

4.4 ± 0.9 5.9 ± 0.8

Tr

15.5 ± 1.9 8.3 ± 1.4 1.4 ± 0.3 55.7 ± 3.8 2.0 ± 0.4 1.3 ± 0.2 1.0 ± 0.2 1.3 ± 0.3

5.6 99.8

2.9 0.2 96.6 0.1

0.3 99.2

91.4 7.5 0.3

1.6 100.0

2.9 99.9

0.4 97.5

NDh 99.2

2.0 99.6

26.5 13.0 Tr

Tr

0.7 0.3 0.9

32.9 42.8 21.9 1.3

60.4 Tr

98.9

9.6 0.3 5.2 0.2 84.0 0.2

1.2

0.6 94.9

0.3

RI,MS RI,MS,Co-I RI,MS,Co-I RI,MS,Co-I RI,MS,Co-I RI,MS,Co-I RI,MS,Co-I RI,MS RI,MS RI,MS RI,MS,Co-I RI,MS RI,MS,Co-I RI,MS RI,MS,Co-I RI,MS RI,MS RI,MS RI,MS,Co-I RI,MS RI,MS,Co-I RI,MS,Co-I RI,MS RI,MS RI,MS RI,MS RI,MS RI,MS RI,MS RI,MS RI,MS RI,MS,Co-I RI,MS RI,MS RI,MS RI,MS RI,MS RI,MS RI,MS RI,MS RI,MS

a

For sake of clarity, only components occurring at percentages above 1% were included. Compounds are listed in order of their elution from a HP-5 column. c Linear retention index on HP-5 column, experimentally determined using homologous series of C8-C30 alkanes. d Linear retention index taken from Adams, 2007, or NIST 17 and FFNSC2. e Relative percentage values are means of three determinations ± SD. f Identification methods: Co-I, based on co-injection with authentic compound (Sigma-Aldrich); RI, based on comparison of calculated RI with those reported in ADAMS, FFNSC 2 and NIST 17; MS, based on comparison with WILEY, ADAMS, FFNSC2 and NIST 17 MS databases. g Tr, % below 0.1%. h ND, not declared by manufacturer. b

to 0.02 mg/cm2 caused a significant decrease in the lethal activity (Kim et al., 2004). In the present study, clove EO showed significant toxicity against the poultry red mite with LC50 value of 8.9 μg/cm3 appearing as the most potent EO tested in contact toxicity assay. On the other hand, in the vapor phase toxicity assay, the EO from litchi (L. chinensis), was the most potent acaricidal agent, resulting in nearly 80% mortality. In this case, the mortality rate of clove EO was less than 50%. Thus, it can

be concluded that the toxicity of clove EO is due to direct contact of mites with the EO, and this was limited in the vapor phase assay. On the other hand, the high mortality rate of mites in litchi treated vessels can clarify the presence of toxic compounds from litchi EO which can exert their toxicity without direct contact with mites. The effectiveness of S. aromaticum EO against D. gallinae can be attributed to its main component eugenol (Lee et al., 2019). This 4

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Table 2 Contact toxicity of the seven essential oils on the poultry red mite, Dermanyssus gallinae, based on 24 h exposure time. EO

LC50a

CI95

LC90a

CI95

Chi

p-value

Df

L. chinensis C. anisata H. sphondylium P. anisum L. alba C. maritimum S. aromaticum Positive control Permethrin

24.7 ± 1.6 59.0 ± 3.7 ˃200 47.5 ± 5.2 77.7 ± 7.7 23.7 ± 3.8 8.9 ± 0.4

21.7–28.2 52.1–66.9

78.0 ± 8.0 170.0 ± 17.7

65.0–97.4 141.9–213.4

2.228 (ns) 4.259 (ns)

0.693 0.372

4 4

34.8–51.3 64.7–95.6 18.2–33.7 7.9–9.9

121.9 ± 21.1 468.9 ± 19.3 290.5 ± 65.8 19.7 ± 1.7

85.9–139.1 358.9–623.8 258.1–515.3 16.9–24.2

7.174 1.572 0.922 4.001

(ns) (ns) (ns) (ns)

0.126 0.813 0.631 0.405

4 4 3 4

35.1 ± 3.1

29.5–41.7

210.2 ± 15.8

157.8–304.3

6.178 (ns)

0.186

4

a Lethal concentrations (LC50 and LC90) values in μg/mL and CI95–95% confidence intervals; essential oils activity is considered significantly different when the 95% CI fail to overlap. Chi-square value, not significant (ns) at P > 0.05 level.

aromaticum, C. maritimum, P. anisum and C. anisata, respectively, were active against D. gallinae. As reported in literature (Afshar et al., 2017), although the mechanism of action of phenylpropanoids is not completely understood, it is assumed that they act as inhibitors of cytochrome P450 detoxicative enzymes. S. aromaticum and C. maritimum revealed to be rich of eugenol and dillapiole whose acaricidal properties were highlighted above. Regarding (E)-anethole-containing EOs, namely P. anisum and C. anisata, the higher was the content of the active compound (94.8 vs 64.6%, respectively), the higher was the toxicity of the EO on the poultry red mite (LC50 of 47.5 and 59 μg/mL, respectively, Table 2). Notably, (E)-anethole was previously found quite active against the larvae of Rhipicephalus microplus Canestrini and Dermacentor nitens Neumann (Senra et al., 2013). Determining the mode of action of any chemicals using in this field is necessary for red mite control since it will give us valuable knowledge on the proper formulation and delivery means. Concerning the life cycle and feeding behavior of D. gallinae, it prefers feeding during dark hours for short period and most of the time it hides in poultry house cracks and clefts. This is a favorable property for an acaricide to exert its efficacy by vapor toxicity without direct contact with mites (George et al., 2009). The fumigant activity against D. gallinae has also been reported for EOs of Artemisia sieberi Besser, Thymus vulgaris L., Leptospermum scoparium J.R.Forst. & G.Forst., and Mentha pulegium L. (George et al., 2009; Tabari et al., 2017). These findings revealed that the mode of action for some EOs was mostly through the vapor form, although the final effect is the result of their multitarget action.

phenylpropanoid has demonstrated potent acaricidal effects on mites, where the position of the double bond in the side chain of the molecule plays a pivotal role for the bioactivity (Pasay et al., 2010; Sparagano et al., 2013). Eugenol is also effective to treat bee products against varroosis (Girisgin et al., 2014). Regarding the mode of action, it has been assumed that its functional group may interfere with mitochondrial respiration of the target mite (Tewary et al., 2006). The most active EOs after S. aromaticum were those from C. maritimum and L. chinensis. The former was characterized by the phenylpropanoid dill apiole which was previously shown to display contact toxicity similar to that of eugenol on the two-spotted spider mite Tetranychus urticae C.L. Koch. (Araújo et al., 2012). In addition, dill apiole was also capable of affecting the food preference of mites. γTerpinene, another C. maritimum EO main component, is a monoterpene hydrocarbon occurring at noteworthy percentages in several plant EOs that showed effectiveness as acaricidal agents (Cetin et al., 2010; Lima et al., 2018; Villarreal et al., 2017). The L. chinensis EO was characterized by sesquiterpenes such as α-zingiberene and ar-curcumene. These compounds were reported as two main factors affecting plant resistance against mites and other arthropod pests (Antonious and Kochhar, 2003; Lima et al., 2016; Maluf et al., 2001). This EO may be interesting as a new source of acaricidal agents since it is obtained from a part (peel) which is a by-product obtained during the litchi processing. Our study put in evidence that some EOs containing phenylpropanoids such as eugenol, dill apiole and (E)-anethole, namely S.

Fig. 1. Fumigant toxicity of essential oils in the vapor form in closed and open containers. Data are presented as Mean ± SEM. Different superscript letters show a significant difference. ANOVA, post hoc Tukey P < 0.05. 5

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Fig. 2. Mortality rates of Dermanyssus gallinae after application of the essential oils at different time points post spraying. Data are displayed as Mean ± Standard error; Capital letters indicate significant difference between different treatments, Small letters indicate significant difference among a treatment (P ≤ 0.05, ANOVA, post hoc Tukey).

References

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5. Conclusions This work highlighted that several plant EOs of economic importance (clove bud) or obtained as a by-product during fruit processing (litchi), are promising miticides to be used for maintaining poultry health and productivity without impacting the environment. Further research should be carried out in order to develop stable formulations of these natural products for real-world applications.

Author contribution statement Conceptualization: M.R.Y., F.M. Methodology: M.A.T., A.R., A.K., M.R.Y., F.P., Y.Z. Investigation: M.A.T., A.R., A.K., R.P., C.G., L.A.T., F.P., Y.Z. Resources: M.R.Y., F.M. Data curation: M.A.T., A.R., A.K., L.A.T., K.C. Writing – Review & Editing: M.A.T., A.R., A.K., L.A.T., K.C., L.A.T., R.P. Visualization: M.R.Y., F.M. Supervision: M.R.Y., F.M.

Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements The authors wish to thank the University of Camerino (Fondo di Ateneo per la Ricerca, FAR) for financial support. 6

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