Bioactivity of Different Chemotypes of Oregano Essential Oil against the Blowfly Calliphora vomitoria Vector of Foodborne Pathogens

Simple Summary Calliphora vomitoria L. is a very common synanthropic blowfly. Since it is attracted by human food, it plays a main role in the transmission of foodborne diseases. Among aromatic plant essential oils (EOs), those of spices are the most suitable to protect food from insect pests. In the present work, we determined the bioactivity of three oregano EOs against C. vomitoria. The chemical analyses showed that the EOs belonged to three chemotypes, one with a prevalence of carvacrol and two with a prevalence of thymol. The bioassays showed that the bioactivity of the EOs significantly varies among chemotypes, with the thymol chemotype showing an overall higher efficacy compared to the carvacrol one. Abstract Blowflies play a substantial role as vectors of microorganisms, including human pathogens. The control of these insect pests is an important aspect of the prevention of foodborne diseases, which represent a significant public health threat worldwide. Among aromatic plants, spices essential oils (EOs) are the most suitable to protect food from insect pests. In this study, we determined the chemical composition of three oregano EOs and assessed their toxicity and deterrence to oviposition against the blowfly Calliphora vomitoria L. The chemical analyses showed that the EOs belonged to three chemotypes: one with a prevalence of carvacrol, the carvacrol chemotype (CC; carvacrol, 81.5%), and two with a prevalence of thymol, the thymol/p-cymene and thymol/γ-terpinene chemotypes (TCC and TTC; thymol, 43.8, and 36.7%, respectively). The bioassays showed that although all the three EOs chemotypes are able to exert a toxic activity against C. vomitoria adults (LD50 from 0.14 to 0.31 μL insect−1) and eggs (LC50 from 0.008 to 0.038 μL cm−2) as well as deter the oviposition (Oviposition Activity Index, OAI, from 0.40 ± 0.04 to 0.87 ± 0.02), the bioactivity of oregano EOs significantly varies among the chemotypes, with the thymol-rich EOs (TCC and TTC) overall demonstrating more effectiveness than the carvacrol-rich (CC) EO.


Introduction
Foodborne diseases are a significant public health threat and a major cause of morbidity worldwide [1]. Every year, both in developing and industrialized countries, a high percentage of people are subjected to illnesses caused by food bacterial contamination [2].

Essential Oils Extraction and Chemical Analysis
Inflorescences and leaves of the CC and TCC oregano (100 g) were dried at room temperature in the shadow, until constant weight, and then subjected to hydrodistillation. The hydrodistillation was performed according to the current European Pharmacopoeia [43] until there was no significant increase in the volume of oil (3 h). The oil was dried over anhydrous sodium sulfate (Na 2 SO 4 ) and stored under N 2 in a sealed vial until required. GC/EI-MS (Gas Chromatography/Electron Impact-Mass Spectrometry) analyses were performed with a Varian CP-3800 apparatus (Agilent Technologies Inc., Santa Clara, CA, USA) equipped with a DB-5 capillary column (30 m × 0.25 mm i.d., film thickness 0.25 µm) and a Varian Saturn 2000 ion-trap mass detector (Agilent Technologies Inc., Santa Clara, CA, USA). The operating conditions were as described in Giuliani et al. [44]. The oven temperature was programmed rising from 60 to 240 • C at 3 • C /min; injector temperature 220 • C; transfer-line temperature 240 • C; carrier gas He (1 mL/min). For each EO, the injection volume was 1 µL, after dilution in n-hexane HPLC grade at 5% v/v. Percentages of compounds were determined from their peak areas in their GC profiles. The identification of constituents was based on the comparison of their retention times with those of the authentic samples (when available), comparing their linear retention indices relative to the series of C9-C25 n-hydrocarbons. Computer matching was also used against a commercial [45] and a laboratory-developed mass spectra library, which was built up from pure substances and components of commercial essential oils of known composition and MS literature data [46].

Calliphora vomitoria Rearing
Flies rearing was performed according to Bedini et al. [19], with minor changes. Larvae of C. vomitoria were purchased from a commercial supplier (Fish Company Arco Sport, Cascina (PI), Italy). Larvae were fed with minced beef liver and maintained under laboratory conditions (23 • C, 60-70% relative humidity, natural photoperiod) until pupation.
After the identification, adult flies were put in a 47.50 × 47.50 × 93.0 cm cage (BugDorm-4M4590DH Specimen Handling Cage, MegaView Science Co., Ltd., Taichung, Taiwan) in knitted mesh and polyester. Flies were fed with a solid diet (sugar and yeast 4:1) and water ad libitum. The sugar-yeast diet was previously shown to be successful in providing the protein amounts necessary to stimulate the oviposition in Calliphoridae flies [47,48]. The adult C. vomitoria population was maintained under laboratory conditions (23 • C, 60-70% relative humidity, natural photoperiod).
The toxicity evaluation of oregano EOs against C. vomitoria eggs was performed according to Bedini et al. [42], with minor changes. Eggs were obtained from adult females supplied with warm minced beef to stimulate the oviposition. Then, 4.5 × 4.5 cm squares of filter paper (surface 20.2 cm 2 ) were treated with 100 µL of 0.0 (control), 0.1, 0.2, 0.5, 0.75, and 1.0%. EtOH solution of oregano EO, corresponding to 0.0 (control), 0.005, 0.010, 0.025, 0.037, and 0.050 µL EO cm −2 . The ethanol was evaporated from the paper under airflow for 3-5 min before placing the eggs, and the squares of filter paper were wetted with 380 µL of water and placed in glass Petri dishes (10 cm of diameter). Finally, fifty newly laid eggs (0-12 h old) were collected and arranged, using a wet fine brush, on the surface of each of the paper squares. The eggs were incubated in Petri dishes kept in a climatic chamber (KW Srl, Siena (SI), Italy), in the dark, at 25 • C. Eggs hatching was recorded every 24 h for three days (24,48, and 72 h), counting the egg's chorions under a dissecting microscope (Nikon SMZ1500, Nikon Instruments Inc., Tokyo, Japan). At each check, the squares of filter paper were re-wetted with 380 µL of water. For each EO concentration, five replicates were performed. Eggs mortality values were corrected using Abbott's formula [49].

Oviposition Deterrence Bioassay
The EOs' protective effect on the meat was evaluated by oviposition deterrence assays performed according to Bedini et al. [19,42], with minor changes. One hundred and fifty unsexed adults of C. vomitoria, 10-14 days old, were placed into 47.50 × 47.50 × 93.0 cm cages (BugDorm-4M4590DH). Flies were fed with sugar and yeast (4:1) for the entire duration of the test. To stimulate the oviposition, the cages were provided with plastic cylinders (3.50 cm of diameter, 5.0 cm in height, surface area 9.61 cm 2 ) each containing 8 g of minced pork, added with water (20% w/w) to avoid desiccation. The meat surface was flattened and sprayed with 150 µL of oregano EOs solutions in EtOH, using a glass nebulizer. Tested concentrations of the three EOs were 0.0 (control), 0.5, 1.0, and 2.0%, corresponding to 0.0 (control), 0.08, 0.16, and 0.32 µL EO cm −2 . Four dishes, containing the four cylinders, each treated with one of the four EOs concentrations, were placed at each of the four inner corners of the cage, at about 5 cm from the edges. To prevent position biases, meat samples were placed in the same order in each corner of the cage. Cages were put under fluorescent lamps (14,000 lux) to provide even lighting and maintained at about 23 • C and 75% RH. A beaker (covered by a net) containing 500 mL of water was put in each cage to maintain the humidity. The whole experiment was replicated three times. Laid eggs were counted 24 h after the beginning of the assay (mean number of eggs laid per cage, 27,010 ± 8106), using the piece counter function of an analytical balance (KERN ABS-N, Kern & Sohn, Balingen, Germany).
The EOs' protective effect was expressed as Oviposition Activity Index (OAI), which was calculated using the following formula: where NC is the total number of eggs laid on the Control meat (treated with EtOH only) and NT is the total number of eggs laid on the treated meat [50].

Data Analyses
The median lethal dose (LD 50 ) of the EOs against C. vomitoria adults and median lethal concentration (LC 50 ) of the EOs against C. vomitoria eggs were calculated by Log-probit regression [51]. Log-probit regression curves were compared by relative median potency (RMP) after having checked for their parallelism [52]. Significant differences among the LD/LC values of the EO chemotypes were determined by estimating the confidence intervals of RMP. The differences were considered statistically significant when values in the 95% confidence interval of relative median potency analyses were = 1.0. To confirm the Probit results (and in the meantime to provide a more synthetic output of the results), bioassays data were also processed by one-way between-groups univariate analysis of covariance (ANCOVA), with the EOs as a fixed factor and the dose/concentration as a covariate to control its effects in the model. The mean response for each factor (EOs), adjusted for the dose/concentration, was reported as estimated marginal (EM) means, and significant differences among them were determined by post hoc comparisons using Bonferroni corrections for multiple comparisons [20,21,53,54]. The EOs protective effect data were processed by the Kruskal-Wallis test with the OAI as a factor. Means were separated by Dunn-Bonferroni pairwise comparisons [55]. Statistics were performed by SPSS 22.0 software (IBM SPSS Statistics, Armonk, North Castle, NY, USA).

Toxicity Bioassays
The three oregano chemotypes EOs showed a clear toxic action by direct contact when administered on the thorax of adult flies (Figure 1).

Toxicity Bioassays
The three oregano chemotypes EOs showed a clear toxic action by direct contact when administered on the thorax of adult flies (Figure 1). ANCOVA indicated statistically significant differences among the EOs chemotypes (F 2,26 = 7.742, p = 0.002). Estimated marginal (EM) means showed that the most effective chemotype was the TTC, while the TCC was the less effective (Table 2). Table 2. Adjusted estimated marginal (EM) means of the mortality of Calliphora vomitoria adults exposed to the essential oils extracted from different oregano chemotypes. In particular, the post hoc tests indicated a significant difference between TTC and TCC (Bonferroni pairwise comparison, p = 0.002), but no differences between TCC and CC nor between TTC and CC were evidenced (Bonferroni pairwise comparison, p = 0.269 and 0.150, respectively) ( Table 2). Consistently, LD 50 of the three EOs chemotypes, calculated by Probit analysis, were 0.141, 0.240, and 0.312 µL insect −1 for TTC, CC, and TCC, respectively (Table 3, Figure 2).   In detail, the estimated marginal (EM) means (Table 4) showed that the most effective chemotype was the TTC, while the CC was the least effective one.  In detail, the estimated marginal (EM) means (Table 4) showed that the most effective chemotype was the TTC, while the CC was the least effective one. The EM means post hoc tests indicated that the differences among the ovicidal activity of the three EO chemotypes are significant (Bonferroni pairwise comparison, p < 0.001). LC 50 of the three EOs chemotypes were 0.038, 0.008, and 0.013 µL cm −2 for CC, TCC, and TTC chemotypes, respectively (Table 5, Figure 4).   The RMP analysis showed that the CC chemotype EO was significantly less effective in killing C. vomitoria eggs than the other two oregano chemotypes EOs (TTC vs. CC RMP = 0.333 (0.093-0.702); TCC vs. CC RMP = 0.225 (0.049-0.521).

Oviposition Deterrence Bioassay
Oviposition deterrence assays indicated that all the EOs are able to strongly affect the oviposition behavior of C. vomitoria females. Mean OAI values ranged from 0.41 to 0.88 ( Figure 5), depending on the concentration and the EO chemotype.  . Dose-effect relationship of the mortality (mortality %) of Calliphora vomitoria eggs exposed by contact to carvacrol (CC), thymol/p-cymene (TCC), and thymol/γ-terpinene (TTC) oregano chemotypes essential oils as predicted by the Probit model.

Oviposition Deterrence Bioassay
Oviposition deterrence assays indicated that all the EOs are able to strongly affect the oviposition behavior of C. vomitoria females. Mean OAI values ranged from 0.41 to 0.88 ( Figure 5), depending on the concentration and the EO chemotype. No significant difference among the EOs chemotypes was observed at 0.08 and 0.16 μL cm −2 . On the contrary, at the highest concentration tested (0.32 μL cm −2 ), the Kruskal-Wallis test showed a significant difference among the EO chemotypes (χ2 = 6.033; df = 2; p = 0.049). The Dunn-Bonferroni pairwise comparisons of the OAI values indicated that the protective effect of the TTC was significantly higher than the one of TCC and CC oregano EOs (TTC vs. TCC, p = 0.019; TTC vs. CC, p = 0.045).

Discussion
Insects vectors of microorganisms are responsible for the loss and spoilage of a huge quantity of food and the spread of foodborne disease [1]. Spices EOs are widely used as a food ingredient for their aroma and preservative properties [56], and their toxic and repellent activity against food insect pests is reported in the literature [19,20,22,57], but very little is known about their use against synanthropic flies. Here, for the first time, we tested three oregano EOs chemotypes as insecticides and repellents against the blowfly C. vomitoria, which is a synanthropic fly vector of foodborne diseases.
The chemical analyses of the three oregano EO chemotypes tested in this work showed that phenols are the main components of the EOs. According to the classification by Napoli and Ruberto [58], two of the tested EOs (TCC and TTC EOs) belong to the thymol chemotype, while the CC EO belongs to the carvacrol chemotype. Thymol and carvacrol are reported as the major components of oregano EOs [59][60][61]. Vokou et al. [62] analyzed the chemical composition of the EOs extracted from oregano samples collected in twenty-three localities in Greece. Their results showed that despite the presence of EOs composed for over 90% by thymol or carvacrol, all the intermediate combinations between the two phenols can be found. On the contrary, thirty-six samples of O. vulgare collected from more than twenty localities in Turkey showed a prevalence of carvacrol in their compositions (carvacrol from 23.4 to 78.7%) [63,64]. The relationship between the increase of the content of one phenol to the decrease of the other one indicates that there is a biosynthetic correlation between thymol and carvacrol [65].
Regarding the lesser components, Kokkini et al. [66] suggested that differences in the No significant difference among the EOs chemotypes was observed at 0.08 and 0.16 µL cm −2 . On the contrary, at the highest concentration tested (0.32 µL cm −2 ), the Kruskal-Wallis test showed a significant difference among the EO chemotypes (χ 2 = 6.033; df = 2; p = 0.049). The Dunn-Bonferroni pairwise comparisons of the OAI values indicated that the protective effect of the TTC was significantly higher than the one of TCC and CC oregano EOs (TTC vs. TCC, p = 0.019; TTC vs. CC, p = 0.045).

Discussion
Insects vectors of microorganisms are responsible for the loss and spoilage of a huge quantity of food and the spread of foodborne disease [1]. Spices EOs are widely used as a food ingredient for their aroma and preservative properties [56], and their toxic and repellent activity against food insect pests is reported in the literature [19,20,22,57], but very little is known about their use against synanthropic flies. Here, for the first time, we tested three oregano EOs chemotypes as insecticides and repellents against the blowfly C. vomitoria, which is a synanthropic fly vector of foodborne diseases.
The chemical analyses of the three oregano EO chemotypes tested in this work showed that phenols are the main components of the EOs. According to the classification by Napoli and Ruberto [58], two of the tested EOs (TCC and TTC EOs) belong to the thymol chemotype, while the CC EO belongs to the carvacrol chemotype. Thymol and carvacrol are reported as the major components of oregano EOs [59][60][61]. Vokou et al. [62] analyzed the chemical composition of the EOs extracted from oregano samples collected in twentythree localities in Greece. Their results showed that despite the presence of EOs composed for over 90% by thymol or carvacrol, all the intermediate combinations between the two phenols can be found. On the contrary, thirty-six samples of O. vulgare collected from more than twenty localities in Turkey showed a prevalence of carvacrol in their compositions (carvacrol from 23.4 to 78.7%) [63,64]. The relationship between the increase of the content of one phenol to the decrease of the other one indicates that there is a biosynthetic correlation between thymol and carvacrol [65].
Regarding the lesser components, Kokkini et al. [66] suggested that differences in the contents could be related to the harvesting season. In fact, oregano samples collected in autumn in three distinct geographic areas of Greece had a lower amount of γ-terpinene and a higher amount of p-cymene compared to the summer ones.
Regarding our results, the bioassays showed higher toxicity by contact against C. vomitoria adults of the TTC, which contains thymol and γ-terpinene as main components (36.70 and 19.70%, respectively). A traditional application of oregano against flies is known in Albania, where dried grounded oregano leaves are traditionally used to keep flies away from houses and facilities where foods are processed [67]. Actually, Xie et al. [41] observed the toxicity of O. vulgare EO against the housefly Musca domestica L. (Diptera: Muscidae). In agreement with our observations, Karpouthsis et al. [68] showed that the O. vulgare subsp. hirtum and Coridothymus capitatus (L.) EOs containing higher percentages of carvacrol (74.56 and 81.46%) were less effective than the thymol-rich EO of Satureja thymbra L. (Lamiaceae) as insecticide agents against Drosophila melanogaster (Diptera: Drosophilidae) larvae.
In previous studies focused on the application of EOs extracted from aromatic plants, which are generally used as spices, Bedini et al. [20] observed LD 50 by contact against C. vomitoria as 0.44, 1.10, and 1.97 µL insect −1 respectively for garlic (Allium sativum L.), rosemary (Rosmarinus officinalis L.), and sage (Salvia officinalis L.) EOs, with significantly higher toxicity exerted by the garlic one. Similarly, tarragon (Artemisia dracunculus L.) EOs showed clear toxicity against C. vomitoria with an LD 50 of 0.49 µL EO insect −1 [19]. The three oregano EOs tested in this research showed higher toxicity than the garlic, rosemary, and sage EOs, with the TTC (0.14 µL insect −1 ) that was about four times more toxic than the A. sativum EO (0.44 µL insect −1 ) but very close to that of the tarragon. In line with the toxicity toward adults, ovicidal tests showed that the two oregano thymol-type EOs (TCC and TTC) were significantly more effective than the carvacrol one.
The higher effectiveness of the thymol-type EOs was also confirmed by the oviposition deterrence tests. All EOs were able to strongly affect the oviposition behavior of C. vomitoria females, but after 24 h, the protection effect of 0.32 µL cm −2 of the TTC EO was significantly higher than the other two EOs. The complete protection of the meat from C. vomitoria, up to 24 h, oviposition was also obtained by A. sativum EO, but at a much higher concentration (1.25 µL EO cm −2 ) [20], while a stronger protective effect was observed for the spice A. dracunculus EO that was able to completely deter C. vomitoria oviposition from 0.05 µL EO cm −2 [19].
One of the main mechanisms at the bases of the toxicity of EOs against blowflies is the inhibition of acetylcholinesterase [19,42], which is an important enzyme in neuronal and neuromuscular communication, whose only difference between its insect and mammalian counterparts is a single residue, making AChE an insect-selective target for newly developed insecticides [40]. For this reason, EOs may represent an effective specific ingredient in the formulation of pesticides and repellents that is effective against the target insect pest and safe for humans and suitable to be used for food protection as well. Although their strong smell may interfere with the sensory quality of food, EOs extracted from spices, thanks to their established acceptance as ingredients in food, may overcome such withdraw.

Conclusions
Our results indicate that oregano EOs may represent an effective tool for the control of blowflies. Our data showed that the EOs bioactivity against C. vomitoria varies depending on the chemical composition of the EO chemotypes. Thus, an extensive EOs chemotyping, coupled with the specific bioassays, may lead to defining standards of EO chemical compositions that are able to ensure the constant and reliable activity needed for the formulation of insecticides or repellents based on spice EOs to be used for the protection of food.