Introduction

Cassia volatile oil (Cinnamomum spp., Lauraceae) is toxic to several insects of agricultural interest (Park et al. 2000) and showed repellent activity against mosquitoes and fleas (Chang et al. 2006; Su et al. 2013), larvicide against various mosquitoes (Chang et al. 2006), and activity against eggs, larvae, and adults of Ctenocephalides felis felis (Dos Santos et al. 2020). Thyme volatile oil (Thymus vulgaris L., Lamiaceae) has an insecticidal action against several insects and mites of agricultural importance (Isman 2000; Kanat and Alma 2004) and of health importance (Park et al. 2000; Pavela 2007; Pavela et al. 2009). Oregano volatile oil (Origanum vulgare L., Lamiaceae) has insecticidal activity against insects that cause damage in agriculture and grain storage (Kim et al. 2010; Pavela 2012) and Diptera of health importance (Govindarajan et al. 2016; Xie et al. 2019).

Fleas of the Ctenocephalides (Siphonaptera, Pulicidae) genus are the most prevalent parasitic insects affecting dogs and cats worldwide. Its evolutionary cycle presents the stages of egg, larva, pupa, and adults and can last between 21 and 30 days under controlled conditions (Dryden and Rust 1994; Rust and Dryden 1997). The importance of these insects is due to the fact that they are hematophagous and, in large infestations, cause spoliation to the hosts and can cause anemia (Hinkle et al. 1991). In addition, they are responsible for the transmission of numerous pathogens of importance in veterinary medicine and public health. They can also cause allergic dermatitis causing intense itching, alopecia, and abrasions in the host (Rust 2017).

Flea control is carried out with the use of chemicals that eliminate adult forms present in animals. Recently, volatile oils of Schinus molle L., Anacardiaceae (Batista et al. 2016), Cinnamomum spp., and Ocimum gratissimum L., Lamiaceae (Dos Santos et al. 2020) have shown potential in the control of adults and immature stages of fleas, Ctenocephalides felis felis. However, there are still few studies that demonstrate the action of volatile oils against this parasite insect. Therefore, the aim of this study was to evaluate in vitro the insecticidal activity of the volatile oils of cassia, oregano, and thyme against the different stages of the flea C. felis felis and their residual effects.

Materials and Methods

Volatile Oils

Volatile oils of Cinnamomum cassia (L.) J. Presl (cassia), Origanum vulgare L. (oregano), and Thymus vulgaris L. (thyme) were purchased commercially (Ferquima Industry and Commerce Ltda., São Paulo, Brazil) and were kept in glass bottles protected from light and stored at − 20 °C, in order to guarantee their stability.

Dereplication of Chemical Constituents

The analysis was performed by gas chromatography (GC) equipped with a flame ionization detector (FID), and a split/split-less injector to separate and detect the constituents of the volatile oils of C. cassia, T. vulgaris and O. vulgare. The compounds were separated with a fused silica capillary column (5% phenyl 95% dimethylpolysiloxane), with 30 m × 0.25 mm (i.d.) × 0.25 μm (film thickness). Helium was used as the carrier gas at a flow rate of 1 ml/min. The column temperature was programmed as follows: 60 °C for 2 min followed by heating at 5 °C/min to 110 °C, followed by heating at 3 °C/min to 150 °C, and, finally, followed by heating at 15 °C/min until 290 °C and holding constant for 15 min. The injector temperature was 220 °C and the detector temperature was 290 °C. To separate and identify the substances, 1 μl of volatile oil samples diluted in dichloromethane (10 μl/ml), in the defined times, was injected in the gas chromatograph coupled to a mass spectrometer (GC-MS) QP-2010 Plus (Shimadzu, Japan). The flow of the helium gas carrier, the capillary column, and the temperature conditions for the GC-MS analysis were the same as described for the GC. The temperature of the injector was 220 °C and the interface temperature was 250 °C. Mass spectra were obtained with a quadrupole detector operating at 70 eV, with 40–400 m/z mass range and scanning rate equal to 0.5 scan/s. The identification of volatile compounds has been based on Linear Retention Indices (LRI) and mass spectra of the samples, compared with authentic standards injected under the same conditions, with the NIST database (2008) and the index Kovats, IK (Adams 2007). The IRL was calculated based on co-injection of alkanes series (Van Den Dool and Kratz 1963).

Flea Origin

Fleas, Ctenocephalides felis felis (Bouché, 1935), in different stages (adult, eggs, larvae, and pupae) were used in this study and obtained from a laboratory colony maintained in cats without the introduction of external specimens and without exposure to insecticides since 1998. The flea colony has been approved by the Animal Use Ethics Committee from the Veterinary Institute, Universidade Federal Rural do Rio de Janeiro - protocol number 4313110419. This species is registered in the National System of Genetic Heritage Management and Associated Traditional Knowledge (SisGen) under number A710DC4.

Activity Against Adult Fleas

Adulticide activity evaluations were performed in glass test tubes (1 × 10 cm) containing ten adult fleas, five males and five females, not fed, aged 14 days per tube. Whatman 1 filter paper tapes (10 cm2, 80 g) were impregnated with volatile oil solutions prepared using acetone as solvent in different concentrations (n = 10) and after drying were introduced in each tube. The assay was performed six times for each concentration. Concentration ranges were 1.3 to 800.0 μg·cm−2 for cassia, 1.6 to 800.0 μg·cm−2 for thyme, and 3.1 to 800.0 μg·cm−2 for oregano. Tests were also performed with a placebo group (acetone) and a negative control group (no substance). The tubes were placed in a climatized chamber with a temperature of 27 ± 1 °C and a relative humidity of 75 ± 10% for a period of 48 h. Mortality assessments were performed at 24 and 48 h using motility as an assessment criterion.

Activity Against Immature Flea Stages

For activity evaluation against the immature stages of C. felis felis, each repetition was prepared as follows: 10 eggs less than 24 h old; 10 third instar larvae aged 5 to 7 days; and 10 pupae aged 15 days. The challenges were prepared in Petri dishes (60 × 15 mm), where a disc of Whatman filter paper no. 1 (80 g) 51 mm in diameter was impregnated with 0.451 ml of the test solution. The challenges were carried out in six repetitions per concentration. The concentration ranges used were 1.3 to 883.4 μg·cm−2 for cassia volatile oil, 3.4 to 883.4 μg·cm−2 for thyme volatile oil, and 1.7 to 883.4 μg·cm−2 for oregano volatile oil. Tests were also performed with a placebo group and a negative control group as described in item Activity of volatile oils against adult fleas. After the test, eggs, larvae, and pupae were incubated in a B.O.D. with temperature of 27 ± 1 °C and relative humidity of 75 ± 10%. Evaluation of ovicidal activity was performed according to the hatchability criterion in each repetition after 72 h of incubation. Eggs that did not hatch into larvae were considered dead. The evaluation of larvicidal activity was performed after 24 and 48 h of exposure, using the morphology criterion, in which larvae with blackish and opaque color and no movement were considered dead. For the evaluation of pupicidal activity, the number of adult fleas that emerged from the pupae was verified after 15 days of the test. Pupae that did not emerge as adult fleas were considered dead.

Inhibitory Activity of Flea Development

The evaluation of the inhibitory activity of the development of the biological cycle of C. felis felis was carried out using the same procedure as the ovicidal evaluation. After the test, the eggs were incubated with half a gram of diet used for the development of the larvae. The diet consisted of wheat flour, dehydrated bovine blood, and washed sand in a 1:1:5 ratio, as described by Cruz-Vieira et al. (2010) stored in a B.O.D. with temperature of 27 ± 1 °C and relative humidity of 75 ± 10%. For the evaluation of developmental inhibition, the number of adult fleas that emerged from the pupae was verified after 30 days of incubation.

Residual Efficacy

Evaluations of residual efficacy were performed using filter paper tapes (as described in activity against immature flea stages) impregnated at a concentration of 800 μg·cm−2 (concentration responsible for 100% of mortality) and challenged with ten adult fleas every 24 h for a period of 22 days. The criterion for assessing mortality was the same as that for assessing adult fleas.

Data Analysis

Mortality assessment for each concentration and for each test was determined by Abbott’s (1987) formula: Corrected mortality (%) = number of individuals killed in the treated group − number of individuals killed in the control group/(100 − number of individuals killed in the control group). The calculations of the lethal concentration (LC) 50 and LC90 for all evaluations performed were calculated through Probit analysis, using the IBM SPSS Statistics software, version 26.0 April 2019 computer program with a 95% confidence interval.

Results

Dereplication of the volatile oil constituents was performed by GC analysis. This analysis demonstrated that the major constituents were for cassia, cinnamaldehyde (1, 91%); for thyme, thymol (2, 44.7%) and ocimene (3, 26.6%); and for oregano, carvacrol (4, 76.2%).

figure b

The complete dereplicated chemical composition of the volatile oils is shown in Table S1. Cassia volatile oil showed 100% insecticidal activity at 800 μg·cm−2 for adult forms at 24 and 48 h evaluations, 220.8 μg·cm−2 to inhibit the hatching of eggs, 883.4 μg·cm−2 and 110.4 μg·cm−2 for larvicidal activity after 24 h and 48 h of challenge; 110.4 μg·cm−2 for activity against pupae; and 16.5 μg·cm−2 for inhibition of development. Thyme volatile oil showed 100% adulticidal mortality at the same concentration of cassia volatile oil (800 μg·cm−2) after 24 h and at lower concentration (400.0 μg·cm−2) after 48 h. For immature forms, concentrations with 100% mortality were higher for eggs (331.2 μg·cm−2) and for larvae after 48 h (220.8 μg·cm−2). Pupal activity reached a maximum of 88.3% at the highest concentration tested (883.4 μg·cm−2) and development inhibition obtained 100% mortality at a concentration 10× greater than cassia volatile oil (165.8 μg·cm−2). Oregano volatile oil showed a 100% of mortality at a lower concentration (400.0 μg·cm−2) for the adult stage in both 24 and 48 h after exposure. However, the concentrations to reach 100% mortality for the egg, larva after 48 h, and pupa stages were higher (883.4 μg·cm−2). Development inhibition was also achieved in a higher concentration (441.7 μg·cm−2) (Table S2). LC50 and LC90 values for the egg, larva, pupa, adult, and development inhibition stages are shown in Table 1.

Table 1 Lethal concentration (LC50 and LC90) of Cinnamomum cassia, Thymus vulgaris, and Origanum vulgare volatile oils against different stages of flea
Full size table

Comparing the lethal concentration values obtained for the three volatile oils, it is possible to notice that the immature forms showed greater susceptibility with values of LC50 ranging from 2.3 to 6.2 μg·cm−2 for life cycle inhibition, from 3 to 94.8 μg·cm−2 for eggs, and from 10.3 to 57.7 μg·cm−2 for larvae. The same was observed for LC90 values, ranging from 11.4 to 97.4 μg·cm−2 for life cycle inhibition, from 18.2 to 400.0 μg·cm−2 for eggs, and from 25.8 to 641.7 μg·cm−2 for larvae. Cassia volatile oil presented the lowest values of LC50 and LC90 with relative potency (LC90) 2-fold higher for cycle inhibition, 8-fold higher for egg, and 3-fold higher for larva compared with thyme volatile oil, and 8.5-fold higher for cycle inhibition, 22-fold higher for egg, and 25-fold higher for larva compared with oregano volatile oil. Pupicidal activity showed less susceptibility with LC50 values ranging from 34.6 to 203.2 μg·cm−2 and LC90 ranging from 86.8 to 901.0 μg·cm−2. As well as observed for immature forms, cassia obtained the best results of LC50 and LC90 with a relative potency (LC90) 4-fold higher compared with oregano and 10-fold higher in relation to thyme volatile oil. Adulticidal activity showed LC50 and LC90 values ranging from 21.8 to 64.3 μg·cm−2 and from 87.2 to 483.0 μg·cm−2, respectively. Unlike the other stages, for adult forms, oregano obtained the best results for LC50 and LC90 with relative potency (LC90) 22-fold higher compared to thyme and 14-fold higher in relation to cassia volatile oil. The assessment of residual efficacy showed that thyme and oregano showed efficacy greater than 80% for up to 6 days; however, for thyme, the decline was faster (16 days) than that for oregano (22 days). For the cassia volatile oil, the efficacy was greater than 80% for 4 days; however, it decreased slowly (20 days) (Fig. 1).

Fig. 1
figure 1

Pulicidal residual efficacy of volatile oils of Origanum vulgare (a), Cinnamomum cassia (b), and Thymus vulgaris (c) at 800 μg·cm−2

Full size image

Discussion

The major constituent of cassia volatile oil was cinnamaldehyde, as previously described by Su et al. (2013). Thyme volatile oil presented an oxygenated aromatic terpene, thymol (2), and a non-oxygenated terpene, ocimene (3), as the major constituents, according to the results of Thompson et al. (2003). Despite the fact that the oregano volatile oil may present as major constituents the isomers thymol (2) and carvacrol (4), the sample analyzed in this study represented a chemotype with carvacrol (4), as described by Milos et al. (2000). Mortalities observed in negative control and placebo groups in egg, pupae, and inhibition of development evaluation can be explained by the fact that during the biological cycle of C. felis felis, the total emergence of adults can vary between 70 and 100%. This loss in the cycle is considered normal, as it occurs due to the non-hatching of some eggs, larval mortality, and death of the flea inside the pupary (Rust and Dryden 1997; Rust 2005). Comparing the LC50 values between the different stages, it appears that the pupal and adult stages were the least susceptible. In the cases of oregano (LC50 = 98.4 μg·cm−2) and thyme (LC50 = 203.2 μg·cm−2), pupa was the least susceptible and, in the case of cassia, the adult stage was less susceptible (LC50 = 74.7 μg·cm−2). Our results corroborate with those described by Blagburn and Dryden (2009), in which adults and pupae were less susceptible to ectoparasiticide.

All evaluated volatile oils presented great activity against immature stages (egg, larva, and pupa) and in the life cycle inhibition mainly for cassia that presented the best results compared to the remaining volatile oils evaluated. The greater susceptibility of immature forms (egg and larva) to the volatile oil of cassia (cinnamaldehyde, 1; 91%) has already been evidenced by dos Santos et al. (2020); however, our results of ovicidal (LC50 = 3.0 μg·cm−2), larvicide (LC50 = 17.2 μg·cm−2), and adulticide (LC50 = 74.7 μg·cm−2) activity were higher than those demonstrated by dos Santos et al. (2020) which obtained lower values of LC50 for egg (LC50 = 1.8 μg·cm−2), larva (LC50 = 0.4 μg·cm−2), and adult (LC50 = 41.9 μg·cm−2) stages. The difference observed between our results and those reported by dos Santos et al. (2020) may be related to the difference between the species of the genus Cinnamomum, which despite having cinnamaldehyde (1) as the major constituent, present differences in their chemical composition. In addition, factors such as parasite generation, environmental conditions, time of exposure, and evaluation techniques can influence the susceptibility of fleas to an ectoparasiticide (Blagburn and Dryden 2009). Other volatile oils with greater activity have been reported in the literature mainly against larvae such as Laurus nobilis L., Lauraceae (LC50 = 0.5 μg·cm−2) and Ocimum gratissimum L., Lamiaceae (LC50 = 1.2 μg·cm−2), inter alia (Dos Santos et al. 2020). Volatile oils containing eugenol as major constituent, such as Syzygium aromaticum (L.) Merr. & L. M. Perry, Myrtaceae, and O. gratissimum, also showed excellent results in inhibiting the biological cycle, LC50 = 0.3 μg·cm−2 (Lambert et al. 2020), and in ovicidal activity, LC50 = 1.8 μg·cm−2, respectively (Dos Santos et al. 2020). Adulticide activity evaluation after 24 h showed that the best results were obtained with oregano (LC50 = 33.5 μg·cm−2) compared to thyme (LC50 = 64.5 μg·cm−2) and cassia (LC50 = 74.7 μg·cm−2) volatile oils.

The assessment of the residual efficacy of volatile oils should be highlighted, since there is a lack of knowledge about how these compounds remain active in the environment after use. Ellse and Wall (2014) state in their work that the use of these volatile compounds may be limited to flea control due to minimal or no residual concentrations in the host, being an important factor in preventing flea reinfestations in the environment. Our results showed that the pulicidal activity of the evaluated volatile oils remained above 80% for up to 4 days in a concentration of 800 μg·cm−2.

Conclusion

The volatile oils of cassia (cinnamaldehyde, 1), thyme (thymol, 2), and oregano (carvacrol, 4) showed insecticidal activity against the immature and adult stages of the flea C. felis felis, demonstrating their insecticidal potential. The residual efficacy of these products was also demonstrated for the first time, an effect highly pursued by the pharmaceutical industry for the development of phyto-pesticides for veterinary medicine.