Toxicity and oviposition deterrence of essential oils of Clinopodium nubigenum and Lavandula angustifolia against the myiasis-inducing blowfly Lucilia sericata

Cutaneous myiasis is a severe worldwide medical and veterinary issue. In this trial the essential oil (EO) of the Andean medicinal plant species Clinopodium nubigenum (Kunth) Kuntze was evaluated for its bioactivity against the myiasis-inducing blowfly Lucilia sericata (Meigen) (Diptera Calliphoridae) and compared with that of the well-known medicinal plant species Lavandula angustifolia Mill. The EOs were analysed and tested in laboratory for their oviposition deterrence and toxicity against L. sericata adults. The physiology of EO toxicity was evaluated by enzymatic inhibition tests. The antibacterial and antifungal properties of the EOs were tested as well. At 0.8 μL cm-2, both EOs completely deterred L. sericata oviposition up to 3 hours. After 24 h, the oviposition deterrence was still 82.7% for L. angustifolia and the 89.5% for C. nubigenum. The two EOs were also toxic to eggs and adults of L. sericata. By contact/fumigation, the EOs, the LC50 values against the eggs were 0.07 and 0.48 μL cm-2 while, by topical application on the adults, LD50 values were 0.278 and 0.393 μL per individual for C. nubigenum and L. angustifolia EOs, respectively. Inhibition of acetylcholine esterase of L. sericata by EOs (IC50 = 67.450 and 79.495 mg L-1 for C. nubigenum and L. angustifolia, respectively) suggested that the neural sites are targets of the EO toxicity. Finally, the observed antibacterial and antifungal properties of C. nubigenum and L. angustifolia EOs suggest that they could also help prevent secondary infections.


Introduction
Myiasis, the parasitic infestation of live mammals by fly larvae (maggots), is an extension of the carrion-feeding habits of blowflies [1]. Gravid females of myiasis-inducing flies such as botfly (Oestridae) and blowfly (Calliphoridae) are attracted and stimulated to lay their eggs on open wounds or even natural body openings of living mammals' body by a variety of cues, a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 respectively; oven temperature programmed from 60˚C to 240˚C at 3˚C/min; carrier gas helium at 1 mL/min; injection of 0.2 μL (10% hexane solution); split ratio 1:30. Constituents identification was based on comparison of retention times with those of authentic samples, by comparing their LRIs with the series of n-hydrocarbons and using computer matching against commercial (NIST 2014 andAdams 2007) and home-made library mass spectra (built up from pure substances and components of known oils and mass spectra literature data) [39; 40].

Oviposition deterrence bioassay
One hundred and fifty 10-to 14-day-old unsexed adults of L. sericata were put in a 75 cm × 75 cm × 115 cm cage (model BugDorm-2400 model of Insect Rearing Tent) with net on the sides and mesh ends at the front. Oviposition was stimulated by polyethylene embedding moulds filled with lamb meat (5 g) mixed with 1 mL of water to prevent desiccation. The meat was gently flattened down and treated with a glass nebulizer with 100 μL of 0, 2, 5, and 10% ethanol solution of the EO, equal to 0.4, 1, and 2 μL cm -2 . Four groups each of them composed of four meat-moulds treated with the four different EO concentrations (0, 2, 5, and 10%) were put at each corner of the cage about 10 cm from the edge. A beaker containing 500 mL of water, covered by a net, was put in the cage to increase humidity. The eggs laid onto the meat were counted, 3 and 24 h after the start of the test, under a dissection microscope. Large aggregates of eggs were counted by an analytical balance equipped with a piece-counter feature. The test was performed in three replicates. The cages were placed under a bank of fluorescent lamps to provide even lighting and kept at about 23˚C and 75% RH.
Oviposition deterrence was calculated using the following formula: Where, OD% = percent oviposition deterrence, NC = total number of eggs on the control meat and NT = total number of eggs on the treated meat [41; 42].

Toxicity bioassays
To evaluate the toxicity of the EOs against eggs of L. sericata, fifty freshly laid eggs (0-12 h old) were placed in a Petri dish (5 cm Ø) the lower surface of which was covered with a black filter paper (Hahnemüehle black filter paper, grade 551) treated with 0.5%, 1.0%, 2.0%, 5.0%, 10.0%, and 20.0% EtOH solutions of the EOs equal to 0.04, 0.08, 0.16, 0.40, 0.80, and 1.60 μL cm -2 . As control, 50 eggs were placed on filter paper treated with 100 μL of EtOH only. In all of the treatments, before placing the eggs, the ethanol was evaporated by exposing the treated paper to an airflow for 3-5 min and, then, the paper was wetted with 0.4 mL of water. The tests were performed at room temperature (about 23˚C). Egg-hatching was recorded after 48 h. Four replicates per concentration were performed.
To evaluate toxicity against adults of L. sericata, twenty unsexed flies were treated by topical applications of the two EOs. 2 μL ethanol EO solution was applied onto the thorax of each fly with a hand micro-applicator (Burkard Scientific Ltd, Uxbridge, UK) [43]. The EOs were tested at the doses of 0.1, 0.2, 0.4, and 1 μL of EO per insect. Three replicates per dose (60 treated flies) were run. The control flies were treated with 2 μL of ethanol. The treated insects were kept in small Plexiglas cages, 20 cm diameter, 30 cm long (10 insects per cage), with water and sugar ad libitum under laboratory conditions (23˚C, 75% RH). The mortality of the flies was checked daily for 4 days and values were corrected using the Abbott's formula [44].

Acetylcholine esterase inhibition assay
The Acetylcholine esterase (AChE) of L. sericata was extracted as described by Seo et al. [45] with few modifications. An aliquot (300 mg) of adult insects were homogenized in 4 mL of buffer (10 mM Tris-HCl, pH 8.0) containing 0.5% (v/v) Triton X-100 and 20 mM NaCl. The homogenate was centrifuged at 17,000 g at 4˚C for 15 min and the supernatant containing AChE was filtered through glass wool to remove any excess lipid. The total protein content was quantified by the Protein Assay Kit II (Bio-Rad) and the extracted AChE was used in the AChE assays.
Inhibition of AChE was determined by the colorimetric method of Ellman et al. [46] with few modifications using acetylthiocholine (ACh) as substrate. The protein content of the AChE extract was diluted to 0.1 mg mL -1 and the reaction mixture consisted of 500 μL of diluted AChE extract (which contained 0.05 mg protein mL -1 ) and 50 μL of EOs for each concentration (2,5,25,50,100,125, and 250 mg L -1 dissolved in 5% (v/v) acetone). Controls were prepared by adding acetone at the same concentrations and without EOs. The tube was set in incubator at 25˚C for 5 min before adding 100 μL of 0.01 M 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB; dissolved in phosphate buffer pH 7.0) and 2.4 mL of phosphate buffer (pH 8.0). The mixture was gently stirred and incubated for another 10 min at 25˚C before adding 40 μL of 75 mM ACh (dissolved in 0.1 M phosphate buffer pH 8.0), then the mixture was incubated for 20 min at 25˚C. The activity of AChE was measured by spectrophotometry using an Ultrospec 2100 Pro spectrophotometer (GE Healthcare Ltd, England) at 25˚C by the increase of absorbance at 412 nm. Inhibition percentage of AChE activity was calculated as follows: where SAT is the specific activity of the enzyme in the treatment group and SAC is the specific activity of the enzyme in the control group. Residual percentage of AChE activity was calculated as (SAT/SAC) x 100. Three replicates were measured for each EOs concentration.

Antimicrobial activity assay
The EOs were individually tested against Escherichia coli ATCC 10536, Staphylococcus aureus (ATCC BAA-1026), Bacillus subtilis (ATCC 11774), Salmonella enterica subsp. enterica serovar Abaetetuba (ATCC 35640), and Candida albicans (ATCC 10231). All the strains were purchased from the American Type of Culture Collection (ATCC, Manassan, USA) and maintained in the Laboratories of the Universidad Técnica del Norte, Ecuador. E. coli, S. aureus, and B. subtilis strains were grown on nutrient agar; C. albicans strain was grown on malt agar; S. enterica was grown on trypticase soy agar.
The antibacterial activity of the EOs was determined by the agar disc diffusion method as follows: active microbial suspensions were made from 24-h-old agar plates using sterile saline solution up to a concentration of approximately 1-2 x 10 7 CFU mL -1 . The microbial suspension was spread on the surface of Mueller Hinton agar (MHA, Oxoid) plates using a sterile cotton swab in order to have uniform microbial growth. Under aseptic conditions, filter paper discs (diameter 6 mm, Whatman paper No.1, Oxoid) were put on the agar plates (one disc per Petri dish, in order to avoid any possible additional activity), then 10 μL of each EO dilutions (corresponding to 10, 5, 2.5, 1.25, and 0.63 μL EOs per disc) were put on the discs. The control discs contained 10 μL of methanol. The inoculated plates were then incubated at 37˚C for 24 h to allow microbial growth. Microbial inhibition zones were measured using a digital calliper and given in millimetres (mm). Six repetitions of each treatment were made.
The minimum inhibitory (MIC) and lethal (MLC) concentrations were determined by the broth dilution method in test tubes as follows: 5 mL of 10 7 UFC mL -1 microbial broth was incubated in tubes containing 50 μL of decreasing concentrations of the oil (10, 5, 2.5, 1.25 and, 0.63 μL EOs per tube). The MIC was estimated as the lowest EOs concentration that inhibited any visible microbial growth [47]. To determine the MLC, 0.1 ml of the cell suspensions from the tubes showing no growth were sub-cultured on nutrient agar plates for bacteria and on malt agar plates for yeast to find out if such inhibition was reversible or permanent. The MLC was calculated as the highest EO dilution (lowest concentration) at which no growth occurred on the plates. Three repetitions of each treatment were made.

Statistics and data analysis
The median lethal dose (LD 50 ) of the EOs against L. sericata adults was calculated by Logprobit regression. Significant differences between the LD 50 values of the two EOs were determined by estimating the confidence intervals of relative median potency (RMP). The differences were considered statistically significant when values in the 95% confidence interval of relative median potency analyses were 6 ¼ 1.0. The percentages of oviposition deterrence, ovicidal activity, and residual AChE activity were arcsine transformed prior to statistical analysis and processed using GLM with one factor (EO) and dose as covariate. P < 0.05 was used for the significance of differences between means. The IC 50 values of AChE activity (the inhibitory concentration needed to inhibit 50% of the enzyme activity, negative Hill slope) were calculated by nonlinear regression to a four-parameters logistic equation (variable Hill slope). The differences in the sizes of the inhibitory zones formed by the EOs against different microbial strains were tested by the Kruskal-Wallis test and the means were separated by Dunn-Bonferroni pairwise comparisons. Data were processed by SPSS 22.0 software (IBM SPSS Statistics, Armonk, North Castle, New York, USA) and by GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA). The individual data points behind means and variance measurements for the quantitative analyses presented in the tables are available in S1 Appendix.

Chemical composition of the EOs
The GC-MS analysis of the EO of C. nubigenum identified 33 constituents accounting for 99.6% of the whole oil. In the EO of L. angustifolia 27 constituents were identified, accounting for 99.2% of the whole oil ( Table 1). The principal chemical constituent of the EO of C. nubigenum was carvacrol (32.9%), followed by pulegone (25.4%), whereas linalool (35.2%) and linalyl acetate (33.4%) were the main compounds in the EO of L. angustifolia. Other important volatiles were p-cymene (9.1%) and iso-menthone (6.4%) for the EO of C. nubigenum, and αpinene and borneol (3.6 and 3.5%, respectively) for the EO of L. angustifolia (Table 1).
Monoterpenes, in both their oxygenated and hydrocarbon forms (74 and 19.7%, respectively), represented the main chemical class for the EO of C. nubigenum. Monoterpenes were also the most abundant chemical class of compounds in the EO of L. angustifolia, as they accounted for up to 94.6% of the total composition, mostly in their oxygenated form (82.4%) ( Table 1).

Ovicidal activity
The EOs of C. nubigenum and L. angustifolia were found to have a definitely toxic effect on the eggs of L. sericata. The ovicidal activity was dependent on the EOs (F 1,45 = 38.354; P < 0.001) and on the dose (F 1,45 = 74.261; P < 0.001). The most effective EO was the C. nubigenum one with an LC 50 value of 0.07 μL cm -2 while the LC 50 value of the EO of L. angustifolia was 0.48 μL cm -2 ( Table 3).
The RMP analysis showed that such differences in toxicity were significant (L. angustifolia vs C. nubigenum RMP = 6.899 (2.635-36.081)). More specifically, the C. nubigenum EO did reduce the egg-hatching up to 97.3% with a dose of 1.6 μL cm -2 , with no significant differences among the concentrations from 0.16 to 1.6 μL cm -2 , while the maximum reduction in the hatching of the eggs treated with L. angustifolia EO was 84.3% with no significant differences among concentrations, from 0.4 to 1.6 μL cm -2 (Fig 2).

Adulticidal activity
The two EOs showed to have a clear adulticidal activity, by topical application, against the fly L. sericata even at low doses. More specifically, the LD 50 values of the EOs were 0.28 to 0.39 μL per individual for C. nubigenum and L. angustifolia, respectively (Table 4). Relative toxicity, calculated by RMP analysis, showed that the EO of C. nubigenum was significantly more effective than the L. angustifolia one (L. angustifolia vs C. nubigenum RMP = 1.417 (1.125-1.836)).

AChE inhibition
The AChE inhibitory activity of the two EOs is shown in Fig 3. The ANOVA showed significant differences between the inhibitory activity of the two EOs (F 1, 28 = 60.140; P < 0.001), with a significant effect of the dose (F 6, 28 = 315.589; P < 0.001) and the interaction oil x dosage (F 6, 28 = 4.512; P = 0.003). Generally speaking, the EO of C. nubigenum was found to be a stronger in vitro inhibitor of L. sericata AChE (IC 50

Antimicrobial activity
Both the EOs showed to have significant antibacterial activity, the intensity of which varied depending on the microbial strain (F 4, 139 = 11.745; P < 0.001) and the EO concentration (F 1, 139 = 239.925; P < 0.001) but not on the EO (F 1, 139 = 0.320; P = 0.857) and with no significant interaction between the EO and the microbial strain (F 4, 139 = 1.591; P = 0.180).  The microbial growth inhibiting effect of the EOs as measured by the agar disc diffusion method is presented in Table 5. At the highest dose (10 μL disc -1 ), the zone of inhibition of the EO of C. nubigenum ranged from 14.7 ± 0.7 to 45.0 ± 0.0 mm for S. abaetetuba and C. albicans, respectively. Similarly, the microbial growth inhibiting effect of the EO of L. angustifolia ranged from 14.7 ± 0.3 and 45.3 ± 1.7 mm for S. abaetetuba and E. coli, respectively. A post-hoc pairwise comparison showed that S. abaetetuba was the most resistant strain (Bonferroni, P � 0.001). Consistently, the MIC and MLC values showed that the most generally susceptible microbial pathogen was the C. albicans with MIC and MLC values of C. nubigenum EO of 0.63 and 2.5 μL mL -1 , respectively and MIC and MLC values of L. angustifolia EO of 1.25 and 5 μL mL -1 , respectively ( Table 6). The most resistant microbial strain was S. abaetetuba with values of 10 μL mL -1 or above for both the EOs (Table 6).

Discussion
Health safety and environmental concerns about synthetic pesticides have led to restrict their use and look for safe alternatives to control insects and other pests. EOs, classed as botanical pesticides, have shown to have remarkable potential as effective eco-friendly biocides and successful insect pest repellents. To date, however, very few studies have been performed on their use against myiasis-inducing species of blowflies. In this study, we tested the EOs extracted from C. nubigenum and L. angustifolia. While L. angustifolia is a well-known aromatic plant, the EO of which has been investigated in many trials in the past, only a few reports have been published about the composition of the EO of C. nubigenum [48; 49]. Monoterpenes, particularly the oxygenated ones, seem to be the main components in all the reported essential oil compositions of C. nubigenum specimens. Ruiz et al. [48] hydrodistilled samples of C. nubigenum from Ecuador and the EO was mainly rich in oxygenated monoterpenes, the most abundant of which was carvacryl acetate (38.1%). This was followed by carvacrol, accounting for 29.0%. Gilardoni et al. [49] also described the composition of the EO extracted from both dried and fresh plants from Ecuador and the specimens had a pulegone chemotype (37.11 and 72.79% in the dried and fresh samples, respectively). In the dried sample, it was followed by menthone with a relative abundance of 11.57%, whilst, only traces of it could be found in the fresh sample, where the second most abundant compound was linalool (7.81%).
Although different in their chemical composition, the EO of C. nubigenum and L. angustifolia EOs, showed to have a clear toxic activity against L. sericata. In this respect, the results showed that C. nubigenum and L. angustifolia are toxic by contact and/or fumigation against the eggs and adults of the fly and can inhibit its oviposition as well. To the best of our knowledge, this is the first report about the toxic and oviposition deterrent activity of C. nubigenum against insects. On the contrary, the effectiveness of the EO of L. angustifolia has been observed in several trials against Diptera [50; 51; 52], Lepidoptera [53] and Coleoptera species [54; 55]. In our experiment, we found the EO of C. nubigenum to be more toxic than that of L. angustifolia. This could be due to the different chemical composition of the two EOs. Because of such interspecific chemical variability, Bedini et al. [6] found a very different level of toxicity when comparing two EOs extracted from Artemisia annua and A. dracunculus against the blowfly Calliphora vomitoria.
In this trial, the EO of C. nubigenum was found to be more effective than that of L. angustifolia also in terms of oviposition deterrence. Even though both the EOs did almost completely deter oviposition for up to 3 h after the treatment, the EO of C. nubigenum was the most persistent one maintaining a good oviposition deterrence activity even at the lower dose after 24 hours after the treatment. In keeping with our results, a strong oviposition deterrence was also previously observed for the EOs of A. annua and A. dracunculus against the blowfly Calliphora vomitoria [6] and for Lucilia cuprina, in media treated with Melaleuca alternifolia EO [56]. In comparison, the effectiveness of the EO of C. nubigenum (OD% = 72.61 at 2% EO) seems to be in line with those of A. annua and A. dracunculus EOs (OD% = 69.31 and 96.77, respectively, at 2.5% EO). The different effectiveness of the two EOs indicates that the EOS oviposition deterrent effect may depend not only on the different chemical composition of the EOs but also on the target species. Moreover, the complexity of the insects' sensory system makes it difficult to understand how the chemical information encoded in the deterrent molecules is perceived by the insect (through olfactory, gustatory or other receptors on the ovipositor or perhaps tarsi) and triggers its behavioural response.
To the best of our knowledge, no study concerning the AChE inhibitory activity of the EOs from C. nubigenum and L. angustifolia has been reported so far. Our results (IC 50 = 67.450 and 79.495 mg L -1 for L. sericata and L. angustifolia, respectively) are in agreement with the AChE inhibitory activity of Salvia lavandulaefolia Vahl., Eucalyptus camaldulensis Dehnhardt and Ocimum canum Sims, with (IC 50 of 50.0, 18.0 and 36.0 μg/mL, respectively) [57] while, the AChE inhibitory effect of C. nubigenum and L. angustifolia turned out to be stronger than the one observed for the A. annua and A. dracunculus EOs (IC 50 of 202.6 and 472.4 μg/mL, respectively) [6]. This finding suggests that one of the effects of the EOs extracted from C. nubigenum and L. angustifolia against L. sericata is the inhibition of AChE activity. Therefore, the enzymatic test based on the inhibition of AChE could be a useful quick tool for further researches into the effectiveness of each single compound of both the EOs against L. sericata.
Flystrike flies usually lay their eggs near wounds or on moist, attractive areas of the sheep. Such preference for wounds and the ability of the larvae to abrade and penetrate in the tissues makes these flies also a source and carrier of microbial infections. EOs can prevent the spread of pathogens through their well-known antimicrobial activity. As expected, we found that C. nubigenum and L. angustifolia showed to have clear toxic, bacteriostatic and mycostatic activities against several pathogens. In particular, we found that the Gram-negative S. abaetetuba was the overall more resistant pathogen. To the best of our knowledge, there are no previous reports on the susceptibility of S. abaetetuba to essential oils of aromatic plant. However, Sahu [58], found that S. abaetetuba was susceptible to ethanolic extracts of four Ocimum species and a clear antimicrobial effect of EOs against Salmonella species were observed [59; 60]. On the contrary, we observed a high susceptibility of the pathogenic fungus C. albicans to essential oils. This is consistent with previous trials that found anti-candida properties in plants used in the Brazilian traditional medicine [61] and in EOs extracted from other aromatic plants such as Myrtus communis [62], Mentha piperita [63], Origanum spp. EOs [64; 65], Artemisia annua and A. dracunculus EOs [6].
Extensive evidence suggests that the observed antimicrobial effect of the EOs is due to their interaction with the cytoplasmic membrane of microorganisms. The hydrophobicity of the EOs [66] enables their chemical components to accumulate in cell membranes, interfering with their structures and increasing their permeability [67; 68; 69; 70]. The leakage of intracellular constituents and the impairment of the microbial enzymatic system can then lead the cell to death [71; 72; 73]. However, antimicrobial activity of the EOs, as well as their toxic and oviposition deterrent activity against insects cannot be attributed to one particular or specific mechanism [74]. In fact, such toxic effects (and most likely their toxicity to insects) can be due to a large number of different chemical components of the EOs, the synergistic or antagonistic effects of which do not always let the actual biological effect correlate with the type and quantity of the main components of the EOs [75; 76].

Conclusions
The prevention of parasitic infections is a priority in animal husbandry. Using the EOs of C. nubigenum and L. angustifolia as protectant against the myiasis-inducing blowfly L. sericata may broaden the very narrow spectrum of eco-and animal welfare-friendly alternative options to synthetic pesticides and surgical procedures to control flystrike.
Besides, the exploitation of indigenous aromatic plants such as C. nubigenum EO may be a valuable additional resource for the economy of rural Andean communities. However, further studies are needed to test the actual applications of our laboratory results and to establish the right doses and the best methods to formulate and deliver such EOs, in the attempt to extend their effectiveness and minimize the number of treatments.
Supporting information S1 Appendix. Excel spreadsheet containing raw data from the study. Each sheet contains the individual data obtained in a particular trial, as noted. (XLSX)