Ticks are one of the most important ectoparasites in tropical and subtropical areas¹. In Mexico, 82 species of ticks have been recorded in both wild and domestic animals, Rhipicephalus microplus being the most predominant one in livestock. This tick generates economic losses related to reduced production levels, disease transmission, mortality, and high control costs^1,2. In recent years, the most widely used strategy for their control has been the application of chemicals known as ixodicides such as organophosphates, carbamates, formamides, synthetic pyrethroids, macrocyclic lactones, phenylpyrazolones, etc., which work adequately at recommended doses. However, the irrational use of these products has led to the selection of resistant tick populations, making control increasingly complex^3,4. For this reason, the availability of alternative methods has become a necessity for the producers, as well as for the consumers who demand pesticide-free products made with environmentally safe technology, such as plant extracts with active ingredients that exhibit insecticidal and pest control effects, especially in ecological and organic production systems^5,6,7.

In view of this situation, the control of ticks with extracts such plants as garlic and Mexican oregano has been widely studied in some species of livestock importance, using various methods of obtainment and application^8-11. On the other hand, commercial extracts from plants like cinnamon and marigold tend to have low toxicity to human health, slow development of resistance, and instability in the environment, and their active ingredients are rapidly metabolized by solar radiation and microclimatic humidity¹². For this reason, the objective of this research was to evaluate the ixodicidal effect of the extracts of two plants -cinnamon (Cinnamomum zeylanicum) and marigold (Tagetes erecta)- on the mortality of R. microplus ticks using the larval package technique (LPT) and the adult female immersion technique (AIT), and to determine the morphological damage caused at the microscopic level.

The present research utilized 7- to 15-d-old R. microplus tick larvae and adult ticks 21 to 23 d collected from bovines infested artificially. The susceptible reference strain from Moyahua, Zacatecas, Mexico, was used, having been provided by the ectoparasites and diptera laboratory of the National Center for Animal Health Testing Services (Centro Nacional de Referencia en Parasitología Animal y Tecnología Analítica, CENAPA) of the National Service for Agri-Food Health, Safety and Quality (Servicio Nacional de Sanidad, Inocuidad y Calidad Agroalimentaria, SENASICA).

Two plant extracts formulated by oil-in-water emulsion of C. zeylanicum 15 % (equivalent to 151.80 g a.i./L) and T. erecta 90 % (equivalent to 831.60 g a.i./L) in weight of active compound were assessed and used mainly to control botanical pests such as red spider mites (Tetranychus urticae, Oligonychus punicae) or whiteflies (Bemisia tabaci). In order to evaluate the ixodicidal effect of each of the extracts, solutions were prepared using the commercial concentration recommended as a reference (1.5 %).

In order to evaluate the ixodicidal effect of the extracts of C. zeylanicum and T. erecta 90 %, a Probit analysis was performed, using five different concentrations -6 %, 3 %, 1.5 %, 0.75 % and 0.375 %- and a control group of 0.375 %. The bioassay was performed under the larval immersion methodology (LIT)¹³. A solution was prepared in distilled water (according to the manufacturer's instructions) with the highest concentration of the extracts (6 %); serial double dilutions were then made with a dilution factor of 0.5, established by the technique up to the lowest concentration (0.375 %). In glass Petri dishes of 15 cm in diameter, a 12.5 cm Whatman No. 1 paper filter was placed, and 10 ml of each solution were added to the respective dish. Subsequently, ~ 400 larvae distributed on the paper were placed with a brush and covered with another paper filter simulating a larval immersion for 10 min. After the exposure time had elapsed, ~100 larvae were taken and transferred to 7.5 x 8.5 cm filter paper packets, which were secured with a BACO™ Bulldog-type clip. Each package containing the treated larvae was identified and kept in an incubator at a temperature of 28 ± 2 ºC and at a relative humidity of 80-90 % for 24 h. Three replicates were performed for each concentration. Finally, package readings were taken by counting the number of live and dead larvae, following the methodology described by FAO in 2004¹⁴.

On the other hand, the immersion test of R. microplus adult females (AIT) was performed using the methodology described above¹⁵. Only the commercial concentration recommended for C. zeylanicum and T. erecta according to the norm NOM-006-ZOO-1993 was used to evaluate their toxicity on female weight and inhibition of oviposition and hatching. Ticks were placed in beakers with 30 ml of 1.5 % solution of both extracts and kept in immersion for one minute while being subjected to circular movements. Subsequently, they were removed in order to eliminate the excess product and placed in Petri dishes to be incubated at 28 ± 2 ºC and 80-90 % relative humidity. On d 14, the eggs were separated and weighed to determine the percentage of oviposition inhibition between the treated and control groups. Finally, 1 g vials of duly identified eggs were placed and incubated for 26 d until hatching. The shells and eggs within 3x3 quadrants were counted in order to determine the percentage of hatching.

At the end of the immersion test, a morphological analysis was performed using R. microplus tick larvae to determine potential morphological damage to the cuticular structure after exposure to C. zeylanicum and T. erecta extracts. Ten (10) larvae per treatment were selected from 6 %, 3 %, 1.5 % and 0.75 % concentrations and analyzed by stereo microscopy (Leica microscope) at the helminthology laboratory of the National Center for Disciplinary Research on Animal Health and Safety (Centro de Nacional de Investigación Disciplinaria en Salud Animal e Inocuidad, CENID-SAI, INIFAP).

Larval mortality parameters were obtained (% mortality = number of dead larvae x 100/number of total larvae)¹⁶. Data were subjected to analysis of variance and to the Kruskal-Wallis test in order to determine significant differences between treatments and controls. Additionally, the data generated in the bioassays from the Probit analyses were submitted to the Polo Plus program (LeOra Software, Petaluma, CA), with the purpose of calculating the lethal concentration 50 (LC₅₀) with a 95% confidence interval.

Probit analysis of the ixodicidal effect of the plant extracts applied using the larval immersion technique made it possible to determine a gradual mortality from the highest concentration (6 %) to the lowest (0.375 %), as shown in Table 1. The extract of C. zeylanicum produced a significant mortality (P<0.05) at concentrations of 6 % and 3 %, obtaining results of 100 and 97.8 %, respectively. On the other hand, the mortality resulting from the 1.5 % concentration was 64.2 %, which was not statistically significant, similarly to the lowest concentrations that exhibited 0 % mortality. In contrast, the extract of T. erecta showed no biological activity in any of the concentrations applied through the Probit methodology, yielding mortality percentages of 0 %, indicative of a null toxic effect on R. microplus ticks larvae.

Table 2 shows the values obtained when analyzing the mortalities of each of the concentrations used in the Probit analysis. With the results of each concentration, an analysis was carried out using the Polo PC program to calculate the LC₅₀ for each of the replicates. The total number of R. microplus tick larvae used in the first repetition was 1,413; it was 1,556 in the second repetition, and 1,529 in the third repetition. The LC₅₀ ranges obtained in each repetition were a minimum of 1.37 and a maximum of 2.41, with a 95% confidence interval.

The results for the inhibition of oviposition and hatching indicate that there was no statistically significant difference for any of the extracts evaluated with respect to the control group (Table 3). The percentage of oviposition inhibition was 3.88 % and 0 % for C. zeylanicum and T. erecta, respectively. The percentage of hatching inhibition was 0 % for both extracts.

Morphological analysis by stereo microscopy showed remarkable effects on R. microplus larvae after treatment with C. zeylanicum extract. It was observed that, as the concentration of the extract increased, the structural damage became more noticeable. In panel A of Figure 1, the larva exhibits evident morphological alterations in the gnathosome (mouthparts), characterized by a reduction in the size of the pedipalps and hypostome, a diminished amount of cuticle and a change in the coloration of the legs. In addition, the shield present in the gnathosome shows an orange coloration that differs from panel D or the control group, suggesting inadequate development and an affected gnathosome. Panel B and C show that the idiosome (body) is whiter and more transparent, indicating an adverse effect on the intestinal caecum that is not adequately appreciated, possibly due to the toxicity of the extracts at the concentrations used. No morphological damage in panel D or the control group.

A= 6 % concentration (the black arrows show structural damage). B= 3 % concentration. C= 1.5 % concentration; D= control larva (water).

Figure 1 Morphological damage identified in R. microplus larvae treated with different concentrations with C. zeylanicum extract 

In the present study, C. zeylanicum extract was observed to exhibit ixodicidal activity on R. microplus larvae. In particular, it was determined that the use of this extract at a 6 % concentration produced 100 % larval mortality. These findings are in agreement with previous studies where the efficacy of a chemotype derived from C. verum against R. microplus ticks was evaluated by LPT and AIT, observing that the use of essential oils with benzyl benzoate showed efficacy against R. microplus in its larval stage¹⁷. Likewise, previous findings¹⁸, were similar to those obtained in the present investigation, where they evaluated the compounds (E)-cinnamaldehyde and α-bisabolol derived from cinnamon essential oils and obtained a 100 % larval mortality at concentrations of 2.5, 5 and 10 mg/ml, concluding that the essential oils and their compounds have high acaricidal activity; however, they report low toxicity in adult ticks filled with R. microplus.

On the other hand, it has been reported that the essential oils of C. zeylanicum act on adult ticks filled with R. microplus in pure form, reaching mortalities of 100 %, 97 %, and 62 % at concentrations of 10, 5, and 1 mg/ml, respectively¹⁹. However, in the present study, when evaluating the commercial concentration recommended of 1.5 %, no effect was observed on the inhibition of oviposition and hatching of adult ticks. It is important to note that evaluations above the recommended concentration were not performed, since the norm NOM-006-ZOO-1993 states that, if this concentration does not have a 98 % mortality rate in adult ticks, the efficacy is considered negative.

However, it should be noted that the use of the commercial concentration of 1.5 % is for the control of phytophagous insect pests. In this work, a strategy of concentrations was standardized, considering that these arthropods are hematophagous and are at different stages of development. The 6 % concentration of C. zeylanicum, which is lethal to R. microplus tick larvae, proves that a higher amount is required than for the target species. In addition, no effectiveness was observed on adult ticks, which could indicate that this product does not have the potential to biologically affect the ectoparasite in the adult stage. However, previous studies assessed the combination of three essential oils, demonstrating that increasing the concentration of C. zeylanicum resulted in greater effectiveness in controlling adult stage ticks. These findings suggest that the use of higher concentrations of C. zeylanicum may be an effective strategy to combat R. microplus ticks at all life stages. However, further research is needed to determine the optimal concentrations and to evaluate their potential side effects, environmental effects, and economic feasibility²⁰.

Further studies using different concentrations on adult ticks are needed, as, in this case, only the commercial concentration recommended was used. These additional studies will make it possible to evaluate the efficacy of the product at different doses and to determine whether there are more effective concentrations for the control of adult ticks. Thus, it will be possible to obtain a better understanding of the response of ticks to different concentrations and to establish more precise guidelines for their control.

Furthermore, in the present study, no distillation or chromatography was performed to separate the compounds from the evaluated extract. However, it has been reported that the main biocomponent of C. zeylanicum is eugenol, which could be involved in its toxicity against R. microplus tick larvae²¹. In addition, the plant C. zeylanicum is known to possess certain properties, notably its insecticidal and acaricidal activity^6,18,20, which suggests that it is an alternative method for the biological control of ticks. In contrast, assessment of the T. erecta extract showed no toxicity on R. microplus larvae or adults. In this regard, no acaricidal activity of T. erecta extract against ticks has been reported. To date, there are only studies where the ixodicidal effect of T. minuta essential oil against R. microplus tick infestation was evaluated, and statistically significant results were obtained in the productive parameters (number and weight of the ticks, oviposition, and viability of larvae), showing an efficacy of 99.9 % at a concentration of 20 %²². Therefore, future research is needed to isolate, identify and characterize the compounds of T. erecta extract in order to determine whether it has ixodicidal properties against R. microplus ticks.

The efficacy shown by the plant extract of C. zeylanicum for the control of R. microplus tick larvae highlights the potential use of this natural product as a biological control method and as an economical and sustainable alternative for tick control. However, additional studies are needed to evaluate different methods of plant extraction; in vivo tests and toxicity studies must yet be performed in order to elucidate its effect on adult R. microplus ticks and the possible risk of using it on domestic animals.