Introduction

The southern cattle tick Rhipicephalus microplus is one of the most widely distributed ticks that constitutes a problem for the cattle industry in tropical and subtropical regions. This species causes the loss of approximately $30 billion annually worldwide (Estrada-Peña et al. 2006; Grisi et al. 2014). In massive infestations, this tick produces negative effects on its hosts by generating anaemia, tick worry, hide damage, injection of toxins, and the transmission of pathogens like Anaplasma marginale, Babesia bovis and Babesia bigemina (Miraballes and Riet-Correa 2018). Ticks are controlled with synthetic acaricides, such as organophosphates, carbamates, organochlorines, amidines, pyrethroids, fipronil, fluazuron, and macrocyclic lactones; however, this practice has resulted in the development of acaricidal resistance in several populations, environmental chemical contamination, effects on non-target species and risks to human health (Banumathi et al. 2017). An alternative for controlling tick infestations in cattle is the use of natural compounds. The main research interest has focused on components synthetized by the metabolism of plants that take part in the plant’s defense mechanisms against pests and pathogens. These metabolites—including steroids, alkaloids, terpenes, flavonoids, phenylpropanoids, amides and lignans—stand out as promising bioactive plant molecules against the emerging acaricidal resistance (Adenubi et al. 2018). Further studies suggest that extracts can be used alone or in combination with chemical compounds to enhance control methods (Singh et al. 2018; Khan et al. 2019). Moringa oleifera, commonly known as the horse radish tree, is a commercial crop and a widely distributed multipurpose tree (Khan et al. 2017). The glucosinolates and their breakdown products—such as isothiocyanates, thiocyanates, nitriles and thiocarbamates—are characteristic metabolites of the Moringa tree, which have an important role in the control of insect pests and nematodes. Several studies have documented the insecticidal activity of the Moringa species (Manzoor et al. 2015; Nwankwo et al. 2015; Dougoud et al. 2019).

Carica papaya belongs to the Caricaceae, a small family with four genera. This fruit is widely recognized as an important source of medicinal and insecticidal agents and is used in the treatment of various ailments due to its antimalarial, anti-inflammatory, hypoglycaemic and wound healing properties (Vij and Prashar 2015). Kovendan (2012) found larvicidal and pupicidal activity against the chikungunya vector Aedes aegypti, in a methanol extract of the leaf. During phytochemical screening, C. papaya leaves have been shown to contain many active components, such as papain, chymopapain, cystatin, alpha-tocopherol, ascorbic acid, flavonoids, cyanogenic glucosides and glucosinolates (Ekaiko et al. 2015).

Randia aculeata, commonly known as crucetillo, is a member of the Rubiaceae family and is used to counteract the effects of snake bites and other venomous animals, as well as for treatment of cancer, diabetes, inflammation and pain (Gallardo-Casas et al. 2012). Phytochemical analysis of R. aculeata has reported the presence of tannins (Torres-Fajardo et al. 2019) and alkaloids (Soto et al. 2001). On the other hand, Frame et al. (1998) reported the anti-Mycobacterium tuberculosis properties of R. aculeata. Possible anti-tick activity of Moringa oleifera root, C. papaya leaves, and R. aculeata seed and shell extracts have not been reported previously. The aim of this study was to evaluate the in vitro acaricidal activity of hexanic and hydroethanolic extracts of M. oleifera root, C. papaya leaves, and R. aculeata seed and shell on R. microplus larvae and engorged females.

Materials and methods

Biological material: plant material and extraction

Carica papaya leaves and R. aculeata fruits were acquired from local markets in the municipality of Veracruz and Paso de Ovejas, Veracruz, Mexico, in September 2018. Each fruit of R. aculeata was separated to obtain the seeds and shell. Plant materials were cleaned and air dried under shade in a well-ventilated place for 7 days at room temperature, then pulverized with a Hamilton-Beach mixer grinder. The M. oleifera root powder (from primary and secondary roots) was obtained from trees cultivated and processed as previously described by Alvarez-Roman et al. (2020). All extracts were prepared at 1:10 (w/v) ratio by adding the solvent n-hexane or hydroethanolic solution (EtOH–H2O, 80:20) to the powdered plant material. The extraction was carried out by maceration at room temperature. Each 24 h for 3 days, the contents were allowed to settle; the solvent was then collected, and a Whatman No. 1 filter paper was used to remove the solid material. The residue was re-extracted by adding the same volume of solvent. The extracts were reduced under vacuum using a Buchi Roto-Vapor at 26 °C. Finally, the concentrated extracts were lyophilized to remove traces of the solvent, collected in glass tubes and kept refrigerated at 4 °C in air-tight containers until required for biological assays.

Preparation of stock and test concentrations

Initially, a stock solution of Triton X-100 2% (Sigma–Aldrich) was prepared in absolute ethanol (ETH-TX 2%). The stock solution was then water diluted up to 1% ethanol in 0.02% Triton X-100 solution (diluent), and extracts were dissolved up to 100, 50, 25, 12.5, 6.25, 3.12 and 1.56 mg/mL.

Tick preparation

Engorged females were collected from naturally infested cattle that had not received acaricidal treatment for 20–30 days, on a farm of the municipality of Saltabarranca, Veracruz, Mexico. After collection, ticks were identified by morphological characteristics following Walker et al. (2007). The ticks were then immersed in a 2% sodium hypochlorite solution (for use in subsequent molecular analyses), dried and selected according to integrity, motility and degree of engorgement. The larvae used in the larval packet test (LPT) came from engorged females. These ticks were attached dorsally in Petri dishes and placed at 27 ± 1 °C and 80% RH for egg laying. After 18 days of oviposition, the egg masses were removed, placed in plastic tubes and kept at the same temperature and humidity conditions previously described until hatching of the larvae. Bioassays were conducted in sextuplicate for each concentration of the extracts, in both the larval and engorged female tests.

Larval packet test (LPT)

Bioassays were performed according to FAO (2004). The packets were made of filter paper and impregnated with 1 mL of each plant extract concentration. Packets were also impregnated with 1 mL of diluent, 1% ethanol and 0.02% Triton X-100 (negative control), and amitraz at a concentration of 0.0002% (positive control). Approximately 100 larvae were placed in each packet (six per concentration), which was immediately sealed with paper clips and put in a BOD chamber at 27 ± 1 °C and > 80% RH, where they remained for 24 h. After this interval, the packets were opened, and the live and dead larvae were counted in each replicate to calculate mortality. Larval mortality was corrected using Abbott's formula (1925).

Adult immersion test (AIT)

The adult immersion test (AIT) was performed as described in the literature (Drummond et al. 1973; FAO 2004; Sharma et al. 2012). Each replicate (six per concentration) consisted of a group of 10 females, with homogeneous weight, immersed for 5 min in 10 mL extract solution at each of the concentrations evaluated. The controls were amitraz at a concentration of 0.0002% (positive control) and diluent, 1% ethanol and 0.02% Triton X-100 (negative control). After immersion, the engorged females were dried on a paper towel and mounted dorsally in Petri dishes with two-sided tape. The plates were kept in the BOD chamber at 27 ± 1 °C and > 80% RH. Ticks were examined with a stereoscope, and mortality counts were recorded daily. Ticks were confirmed dead based on signs of haemorrhagic skin lesions, cuticular darkness and lack of Malpighian tube movement. After 14 days, the eggs were weighed and transferred to tubes, which were identified and sealed. Tubes were then placed in the incubator under the same conditions for larval hatching and were read visually after 16 days of incubation. Reading was performed by a single technician who had no knowledge about the treatment, to avoid biased estimation according to the procedure described by Drummond et al. (1973) and Figueiredo et al. (2018).

The egg production index (EPI), the reduction in oviposition (RO), reproduction efficiency index (REI) and the efficiency of the extract (EP) were calculated according to the following formulas:

EPI (%) = (weight of eggs/weight of engorged female) × 100 (Bennett 1974),

RO (%) = [(EPI control group − EPI experimental group)/EPI control group] × 100 (Roulston et al. 1968),

REI = (egg mass weight × % egg hatching/engorged females weight) × 20,000 (Drummond et al. 1973), and.

EP (%) = [(REI control − REI treated)/REI control] × 100 (Drummond et al. 1973).

Statistical analysis

Data were analyzed by one-way ANOVA, followed by Tukey test to separate means, using STATISTICA v.10 software (α = 0.05). The lethal concentrations (LC50 and LC90) were calculated by probit analysis (Finney 1962) using Stata Graphics v.18. For all analyses of variance and probit, the assumptions of normality and homogeneity of variance were checked and no data transformation was required.

Results

Larval packet test (LPT)

All extracts showed an acaricidal effect against the larvae of R. microplus. Especially, the hydroethanolic extract of R. aculeata seed at 1.56–100 mg/mL had the highest acaricidal effect (93–100%), followed by R. aculeata shell (91.2%), M. oleifera root (77.7%) and C. papaya leaves (75.5%) at 100 mg/mL (Table 1). For the hexanic extracts at 100 mg/mL, the R. aculeata seed also showed the highest acaricidal effect (82.2%), followed by R. aculeata shell (81.2%), M. oleifera root (78.2%) and C. papaya leaves (61.7%). Mortality was significantly different after larvae ticks were exposed to extracts in a dose dependent manner, compared with the control groups exposed to diluent (Table 1). Ascending concentrations of tested extracts were evaluated to obtain lethal doses that were then subjected to probit analysis. The LC50 and LC90 for hydroethanolic and hexanic extracts are shown in Table 2.

Table 1 Mortality (%) of Rhipicephalus microplus larvae exposed to different concentrations of hydroethanolic (Ha) and hexanic (He) extracts of Randia aculeata seed and shell, Moringa oleifera root and Carica papaya leaves, or to 1 mL of diluent with 1% ethanol and 0.02% Triton X-100 (negative control)
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Table 2 LC50 and LC90 values (with 95% confidence intervals in parentheses) [mg mL−1] for Rhipicephalus microplus larvae exposed to hydroethanolic (Ha) and hexanic (He) extracts of Randia aculeata seed and shell, Moringa oleifera root and Carica papaya leaves
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Adult immersion test (AIT)

Mortality caused by the hydroethanolic extracts (Table 3) and hexanic extracts (Table 4) of R. aculeata, M. oleifera and C. papaya varied from 55–85.5% to 0–75%, respectively, when tested at 1.56–100 mg/mL. Hatching of the eggs was blocked only by (the higher concentrations of) hydroethanolic extracts of R. aculeata seed. Other extracts were only partially able to block the hatching; however, newly hatched larvae did not survive and died within a few hours. All concentrations of hydroethanolic extract tested caused significant mortality of adult engorged ticks and reduction in the mass of eggs laid, when compared to the control. Consequently, there was a significant reduction in the reproduction efficiency index. Adult mortality caused by the hydroethanolic extract of the R. aculeata seed varied from 17.5 to 85.5%—and mortality by R. aculeata shell extracts varied from 15 to 75%—when tested at concentrations ranging from 1.56 to 100 mg/mL, respectively. Moringa oleifera root extracts at 50 and 100 mg/mL had lower efficacy, and caused 55–66% mortality.

Table 3 Effect of hydroethanolic extracts of Randia aculeata seed and shell, Moringa oleifera root and Carica papaya leaves on mortality (%), egg mass (g), egg production index (EPI, %), reduction in oviposition (RO, %), reproduction efficiency index (REI), larval hatching (%) and efficacy of the extracted product (EP, %) in engorged females of Rhipicephalus microplus
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Table 4 Effect of hexanic extracts of Randia aculeata seed and shell, Moringa oleifera root and Carica papaya leaves on mortality (%), egg mass (g), egg production index (EPI, %), reduction in oviposition (RO, %), reproduction efficiency index (REI), larval hatching (%) and efficacy of the extracted product (EP, %) in engorged females of Rhipicephalus microplus
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Hexanic extracts of R. aculeata seed at 12.5–100 mg/mL caused 47.5–75% mortality and significantly inhibited reduction in oviposition (47.3–65.1%), when compared to the control ticks (Table 4). Hexanic extracts of C. papaya leaves did not cause mortality at any concentration. Adult ticks treated with higher concentrations of the various hexanic extracts—all except C. papaya extracts—laid egg masses that were significantly lower in weight than the egg masses of control ticks, in spite of the low adult mortality in the treated ticks. The LC50 and LC90 for hydroethanolic and hexanic extracts are shown in Table 5.

Table 5 LC50 and LC90 values (with 95% confidence intervals in parentheses) [mg mL−1] for Rhipicephalus microplus adults exposed to hydroethanolic (Ha) and hexane (He) extracts of Randia aculeata seed and shell, Moringa oleifera root and Carica papaya leaves
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Discussion

Plants have an important role in traditional medicine, as it has been shown that they provide many metabolites that can intervene in the biological processes and the life cycle of ticks, and they are considered an important part of ethnoveterinary medicine (Zaman et al. 2012; Chagas et al. 2014). The potential use of plant extracts for the control of arthropods of veterinary importance has been reviewed by Ghosh et al. (2015) and Rosado-Aguilar (2017), and a few plants were identified as being promising against ticks. In the present study, the anti-tick potential of three more plants was identified, to contribute to the development of plant-based acaricides for the control of tick species.

Omoregie et al. (2018) found that an alcoholic extract of M. oleifera root showed the presence of bioactive compounds: alkaloids, tannins, flavonoids, steroids, triterpenoid glycosides, saponins and anthraquinones. These findings are similar to those reported by Sholapur and Patil (2013). In addition, the insecticidal activity of M. oleifera extract on termites (Nasutitermes cornige) has been reported by Paiva et al. (2010) and Muhammad (2012). Coelho et al. (2009) and Agra-Neto et al. (2014) documented that aqueous extracts from M. oleifera seeds delayed the larval development of A. aegypti, and a water-soluble seed lectin was able to kill A. aegypti larvae by promoting morphological alterations in the digestive tract, causing an imbalance in digestive enzyme activities. Oliveira et al. (2011) reported great insecticidal activity of the seed coagulant M. oleifera lectin (cMoL) on flour moth (Anagasta kuehniella). The insecticidal activity of this plant may be due to the presence of phenolic compounds such as β-amyrin, β-sitosterol, kaempferol and quercetin (Pontual et al. 2012), which have negative effects on insects, as they decrease fertility and shorten their life span (Dawkar et al. 2013).

Phytochemical analysis of methanolic extracts of C. papaya stems have shown the presence of alkaloids, tannins, flavonoids, carbohydrates and triterpenes (Rashed et al. 2013). Literature is scarce about the insecticidal activity of C. papaya leaf extract; however, Zobayer and Hasan (2013) reported great insecticidal activity against Aphis gossypii. Phytochemical analysis of methanol, ethanol and water extracts and fractions of C. papaya leaf revealed the presence of alkaloids, flavonoids, saponins, steroids and tannin (Yusha’u et al. 2009; Rashed et al. 2013). Larvicidal activity of C. papaya against the mosquitoes Culex quinquefasciatus and Anopheles stephensi was reported by Rawani et al. (2009). Methanol extract of C. papaya seeds caused 82.2% larval mortality and 93.3% adult mortality on R. microplus (Shyma et al. 2014). These higher acaricidal activity of the C. papaya seed extracts—compared to our study with hydroethanolic and hexane leaf extracts—may be due to a synergistic effect of the active components, or perhaps to more sensitive ticks. Other possible differences are related to the organospecific production profile of the plant or the solvent used, the phase of the plant at the time of collection, or the variety of C. papaya tested (Pandey et al. 2014).

Phytochemical analysis of R. aculeata revealed the presence of tannins (Torres-Fajardo et al. 2019), among other chemical constituents. Tannins have been shown to produce anthelminthic activities, and it is possible that tannin can bind to free proteins in the gastrointestinal tract of the host animal or to glycoproteins on the cuticle of the parasite, which may cause death (Niezen et al. 2002; Athanasiadou et al. 2001). Antoun et al. (1993) tested R. aculeata against Plasmodium falciparum without effect. To our knowledge, this is the first report of anti-tick activity of hydroalcoholic and hexane extracts of the seed and shell of R. aculeata. The acaricidal effect of hexane extracts in our study are not similar to previous studies, in which the non-polar fraction had higher anti-tick activity than the polar fraction. The acaricidal efficacy of this assay may be attributed to the organic solvents working better as the cuticle of the tick is mainly formed externally by waxes and internally by proteins (Cherry 1969); hence, the more non-polar a chemical compound, the greater its ability to penetrate the cuticle (Chagas et al. 2002).

The current study revealed that the mean mortality of larvae and adult ticks increased with increasing dosage (concentration) and exposure time, after in vitro treatment. This result is in line with the findings of Fouche et al. (2017), in which the mortality effect of extracts was found to be dose and exposure time dependent. Our study also showed that hydroalcoholic extracts of R. aculeata seed and shell, C. papaya leaves and M. oleifera root induced significant acaricidal effects against R. microplus, compared with the negative control. Plants have a diversity of defense mechanisms to decrease insect attacks, both constitutive and inducible, while insects have evolved strategies to overcome these plant defenses. The mode of action and target site for insecticidal activities of plants have been studied often (Akhtar et al. 2009; Moreau et al. 2012; Ribeiro et al. 2012; Cardoso et al. 2020). Most of the work has been carried out by studying the effects of extracts and essential oils, their lethal doses and time to achieve lethal effects, but modes of action at a molecular level are generally unknown. Secondary plant metabolites, such as terpenoids and alkaloids, are reported as candidates for effective alternative insecticidal compounds. These metabolites can be linked to structural proteins, enzymes, receptors, ion-channels, nucleic acids and other cellular components, damaging the arthropod’s structure (Lopez et al. 2010). Phytochemicals can serve as a model for the development of chemically synthesized derivatives with enhanced activity (Kostyukovsky et al. 2002; O’Callaghan et al. 2019). Nevertheless, details of the specificity of the metabolites with insecticidal activity are scarcely known. In reality, it is likely that the mechanism will behave like a web, in which pathways and molecules will interact.

We can conclude that extracts of R. aculeata seed and shell, C. papaya leaf and M. oleifera root have in vitro acaricidal activity. In addition, seed and shell extracts of R. aculeata were shown to have a high acaricidal effect at various life stages of R. microplus, thus justifying the testing of these plants against other arthropods. Further in vivo studies including these plants should be undertaken to evaluate the effect on host animals. Furthermore, the identification of active ingredients presents in R. aculeata seed and shell that caused adult tick mortality, decreased egg production and the inhibition of egg hatching will be fruitful. These studies will further help to confirm factors that increase the acaricidal activity. The acaricidal properties of the tested extracts could make them a valuable component in the development of a sustainable strategy for arthropod pest management in agriculture and the cattle industry.