Introduction

The tick species Rhipicephalus microplus (Canestrini) (Acari: Ixodidae) parasitizes cattle throughout the world. Due to its high prevalence and wide distribution, R. microplus is responsible for large losses related to animal mortality, in the levels of reduction in cattle breeding, reproductive disorders and transmission of disease-causing pathogens as babesia (Babesia bovis and Babesia bigemina) and anaplasma (Anaplasma marginale), responsible for the Bovine parasite sadness (Rodríguez-Vivas et al. 2014; Singh et al. 2014).

Currently, the control of R. microplus is predominantly done with the use of chemical acaricides, composed of active ingredients belonging to the classes of macrocyclic lactones, organophosphates, formamidines, synthetic pyrethroids, phenylpyrazoles and growth inhibitors (Reck et al. 2014). However, constant use against ticks, sometimes without adequate technical criteria, has resulted in the selection of resistant tick populations (Klafke et al. 2017). The development of new synthetic compounds is a long and expensive process, which reinforces the need for alternatives to control tick infestations (Chagas et al. 2012; Rashid et al. 2019).

Studies of sustainable alternatives have generated great interest from commercial, medical and scientific communities in various countries, as phytotherapy and biological control have the potential to reduce the use of chemotherapy agents currently used to control ectoparasite infestations (George et al. 2014; Politi et al. 2012). Achyrocline satureioides (Lam.) DC. is a species in the Asteraceae family, common in Brazil, occurring mainly from Minas Gerais to Rio Grande do Sul, growing spontaneously in pastures (De Souza et al. 2002; Retta et al. 2012). This species already presents activity against several insect species such as simuliids (borrachudo) reported by Dal Magro et al. (1998), Triatoma infestans (barber) (Rojas de Arias et al. 1995) and Myzus persicae (peach green aphid) (Saupe 2003) besides larvicidal activity against Aedes fluviatilis (Macêdo et al. 1997).

Another important strategy to control parasites involves biocontrol through entomopathogenic fungi. Combined with its high capacity to control pests, it also has the advantage of causing less environmental impact than chemical pesticides, being efficient and safe (Zimmermann 2007; Fernandes et al. 2008; Lacey et al. 2015). The mode of action of fungi on arthropods is the adhesion of conidia to the cuticle of terrestrial hosts (ticks, cockroaches, triatomines) and the formation of hyphae that penetrate the integument with the aid of secreted enzymes such as proteases, chitinases and lipases (Moraes et al. 2003; Santi et al. 2019). Toxins and organ destruction are what kills the ticks also with fungi.

Several studies have demonstrated the action of entomopathogenic fungi on the tick R. microplus in vitro particularly using Beauveria bassiana (Balsamo-Crivelli) Vuillemin [Hypocreales: Cordycipitaceae]. Strains of B. bassiana are among the most promising agents for use against ectoparasites of veterinary importance, such as the tick Amblyomma cajennense (Reis et al. 2008), Rhipicephalus sanguineus (Prette et al. 2005), and R. microplus (Sun et al. 2013; Fernandes et al. 2008, 2012).

The great potential of plant extracts (Minho et al. 2020) and entomopathogenic fungi (Santi et al. 2019) on ectoparasites has been demonstrated, but in most of these studies the extraction yields are low and the concentrations required to achieve this effect are high, which often makes its use on a large scale unfeasible. An alternative for this would be the joint use aiming at a synergistic effect; however, some natural pesticides can act negatively on the fungi, inhibiting their growth and causing genetic mutations, factors that can lead to reduced effectiveness (Islam and Omar 2012; Mohan et al. 2007; Ribeiro et al. 2012). To avoid such factors, testing is recommended to determine the minimum inhibitory concentration. Compatibility studies between pesticides and entomopathogenic fungi have been carried out almost entirely involving chemicals (Soares 2011), with a very small number of studies dealing with the compatibility of these microorganisms with natural pesticides (Luckmann 2013). Therefore, the objective of this study was to evaluate the efficacy of extracts from A. satureioides associated or not with the entomopathogenic fungus B. bassiana on different stages of the tick R. microplus.

Materials and methods

Plant material

The plant material used was obtained through a scientific cooperation agreement signed between the School of Pharmaceutical Sciences of Araraquara (FCFAr-UNESP), Araraquara, SP, Brazil, and the Pluridisciplinary Center for Chemical, Biological and Agronomic Research (CPQBA- UNICAMP), Campinas, SP, Brazil. The species A. satureioides obtained from the CPQBA Medicinal Plants Collection, whose cultivation was under the responsibility of Prof. Dr. Ílio Montanari Jr., was collected in the city of Campinas-SP (22°48’S, 47°07’W), and have been registered and authorized for use by SISGEN under registration number AD73F75.

Production and fractionation of plant extracts

Plant material was dried in an oven (Fanem, Guarulhos, SP, Brazil) with air circulation at 40 °C and later ground in a knife mill (Fabbe, São Paulo, Brazil). Ethanol extracts (ASb) were obtained from aerial parts of A. satureioides by the method of maceration 1:10 in absolute ethanol (Merck, Darmstadt, Germany). After 7 days, the contents were filtered, rotoevaporated (Rotary Evaporator Fisatom, São Paulo, Brazil) and placed in an amber bottle. Fractionation was carried out by the liquid-liquid partition method with high, medium and low polarity solvents, generating the partitions ethyl acetate (ASac), hexane (ASh) and water (ASwater). For fractionation 2 g of ethanolic extract was solubilized in 200 mL of ethyl acetate (Merck), centrifuged and its supernatant placed in a separating funnel together with 100 mL of distilled water, remaining for 45 min with stirring every 15 min, removing and adding water 3×. Then the acetate fraction was rotoevaporated and resuspended in ethyl acetate, which was placed in a separating funnel together with 50 mL of hexane (Merck). Both fractions were rotoevaporated and stored in a beaker at a temperature of − 80 °C.

Fungus growth conditions

Beauveria bassiana strain 487 used in the trials was obtained from the Embrapa Cenargen Collection and registered in the ARSEF database (Agricultural Research Service Collection of Entomopathogenic Fungal Cultures, USA). Cultivation for maintenance and production of conidia was carried out in Petri dishes (10 × 2 cm) with Potato Dextrose Agar (PDA; Merck) for 7 days, kept in an oven (Fanem, Guarulhos, SP, Brazil) at 28 ºC.

Determination of minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC)

The MIC determination was performed according to the Clinical Laboratory and Standards Institute M38A2 (CLSI, 2008), with modifications, adapted in 96-well microplates and RPMI medium (Sigma Aldrich, Saint Louis, MO, USA) with 2% glucose buffered in MOPS (Sigma Aldrich) pH 7.2 and inoculum of 108 CFU/mL. Extracts and fractions were diluted in sterile water at a concentration of 40 mg/mL and 100 µL was added to the first well of the microplate containing 100 µL of RPMI medium (Sigma Aldrich). Serial dilution of the samples was then carried out, also including positive and negative controls. The inoculum was obtained by suspending the fungal cells in an aqueous solution of 0.1% Tween (Sigma Aldrich) for counting in a Neubauer chamber and the number of cells was adjusted to the cell concentration 108 CFU/mL. To test the acaricide effect of the fungal strains, they were added to microplates containing 100 mL of medium and 100 µL of fungal cell suspension. The microplate was kept for 7 days at 28 ºC in an oven with agitation at 120 rpm. Visual reading was performed for MIC determinations. After reading, a sample from each well was collected and inoculated in a Petri dish containing PDA medium, with the aid of a sterile toothpick. The Petri dish was kept in an oven at 28 °C for 5 days, and then a visual reading of growth inhibition was performed to determine the minimum fungicidal concentration (MFC). The MFC obtained supported the use of concentrations of crude extracts and fractions that were associated with the B. bassiana fungus without inhibiting it.

Preparation of fungal suspension for testing with Rhipicephalus microplus

To prepare the fungal suspension, the spores were scraped from the plates with the medium in which they were cultured and placed in 0.09% saline solution, after which, counting was performed in a Neubauer chamber to determine the volume needed to reach 106 spores/mL. In the suspension 10% of RPMI medium (Sigma Aldrich) was added so that the fungus had a source of nutrients to support its initial growth. The viability of the conidia was monitored by growing a sample of conidia inoculum placed in Petri dishes containing PDA medium (Merck), for 7 days, in an oven (Fanem) at 28 ºC.

Immersion test of females of Rhipicephalus microplus

Rhipicephalus microplus engorged females were collected from naturally infested cattle at Embrapa Pecuária Sudeste, São Carlos, SP, Brazil (21°57’42"S, 47°50’28"W). According to a resistance test carried out in 2019, the ticks in this herd were resistant to pyrethroids, organophosphates and amidines. The tests were conducted following the biocarrapaticidogram technique as described by Drummond et al. (1973), in which three homogeneous groups of 10 females per treatment/concentration were used.

The concentrations of the extract and fungus associations to be evaluated were determined using MFC values, which was found to be safe to start at 10 mg/mL. The treatments also had a negative control (water), solvent control (Tween 2%, Sigma Aldrich) and fungus control. The females of the test groups were immersed in the treatments for 5 min. Then they were gently dried and placed in Petri dishes that were placed in a biochemical oxygen demand (BOD) incubator (Eletrolab 2023) with an average temperature of 27 °C and > 80% RH for 18 days. The humidity kept high inside the incubator was obtained by the presence of 2 L of sterile water inside the BOD tray, monitored daily and replaced when necessary. After 18 days, the egg laying was evaluated, with the eggs being weighed and placed in adapted disposable syringes and kept in the incubator for another 18 days. Reproductive parameters were provided for calculation of the percentage of posture reduction (= oviposition index) and performed according to the equations (Drummond et al. 1973; Politi et al. 2012):

$$Posture\,reduction\,\left( \% \right) = \frac{{{M_{eggs}}}}{{{M_{females}}}} \times 100,\,and$$
$$Hatchability\,\,reduction\,\left( \% \right) = \frac{{Hatchabilit{y_{control}} - Hatchabilit{y_{treated}}}}{{Hatchabilit{y_{control}}}} \times 100,$$

where Meggs is the mass of the eggs laid (g), Mfemales is the mass of engorged females (g), Hatchabilitycontrol is the oviposition index of the control group, and Hatchabilitytreated is the oviposition index of the treated group.

The reproductive efficiency (%) and the product effectiveness (%) were calculated according to equations (Drummond et al. 1973; Politi et al. 2012):

$$Reproductive\,e\hspace{-4pt}f\hspace{-4pt}f\hspace{-4pt}iciency\,\left( \% \right) = \frac{{{M_{eggs}} + \% \,hatching \times 20,000}}{{{M_{females}}}},\,and$$
$$$$Product\,e\hspace{-4pt}f\hspace{-4pt}f\hspace{-4pt}iciency\,\left( \% \right) = \frac{{reproductive\,e\hspace{-4pt}f\hspace{-4pt}f\hspace{-4pt}icienc{y_{control}} - reproductive\,e\hspace{-4pt}f\hspace{-4pt}f\hspace{-4pt}icienc{y_{treated}}}}{{reproductive\,e\hspace{-4pt}f\hspace{-4pt}f\hspace{-4pt}icienc{y_{control}}}} \times 100.$$$$

The results are expressed as mean ± SD.

Sensitivity of larvae in contact test on impregnated paper

The larval packet test (LPT) of R. microplus larvae was performed as recommended by FAO (1971). In this methodology approximately 100 larvae from engorged females collected at Embrapa Pecuária Sudeste were placed on filter paper measuring approximately 10 ⋅ 8 cm, impregnated with different concentrations for samples, negative controls (water + Tween 2% and fungus) and positive control [cypermethrin (15%), chlorpyrifos (25%) and citronella (0.1%)]. Each of these papers impregnated with extracts and/or B. bassiana at a concentration of 106 spores/mL was folded to form a ‘sandwich’ and sealed with plastic pegs. The envelopes were conditioned in an incubator at 27 °C and 80% RH. Three replicates were performed for each concentration, as well as for the control groups. After 7 days the test was read differentiating live from dead larvae with the aid of a vacuum compressor.

Larvae immersion test

In the larval immersion test (LIT), 300 larvae were added to 1 mL of the various solution concentrations to be tested that contained plant extracts and/or B. bassiana fungus. In this solution, 0.02% Triton was added to ensure immersion of the larvae. These larvae were immersed for 10 min. After this period, the larvae were placed in filter paper envelopes, sealed and conditioned in an Eletrolab EL2023 BOD incubator at 27 °C and 80% RH. After 48 h the test was read differentiating live from dead larvae with the aid of a vacuum compressor (Sabatini et al. 2001). Three replicates were performed for each concentration, as well as for the control groups: negative controls (water + 0.02% Triton and fungus) and positive control [cypermethrin (15%), chlorpyrifos (25%) and citronella (0.1%)].

Statistical analysis

Statistical analyses were performed using ANOVA followed by comparison of means using Tukey’s honestly significant difference test (α = 0.05) using ASSISTAT free software (https://assistat.software.informer.com/). The Probit procedure (SAS Institute, Cary, NC, USA) was used to calculate the lethal concentrations of 50% (LC50) to reflect the treatment concentration that resulted in 50% parasite mortality and their respective 95% confidence intervals (95% CI).

Results

Immersion test of females of Rhipicephalus microplus

The immersion test of engorged females evaluated the effectiveness of extracts and associations on the female, egg laying and egg hatching. An increase in A. satureioides efficacy was observed when associated with entomopathogenic fungus (Table 1). This was mainly observed in the highest percentages. The negative control composed by 2% Tween was significantly different from most of the treatments, presenting 0% efficacy. The hexanic partition (ASh) at 10 and 5 mg/mL + fungus reached 40.6 and 43.5% efficacy, respectively, causing reduction of larval hatching and reproductive efficiency. For ASb, ASac and ASwater, the efficacy percentages were slightly but significantly higher when associated with the fungus B. bassiana strain 487. Reduction of posture was not so significant, reaching a maximum of 28.1% for ASac at 10 mg/mL + fungus. When the larval hatching parameter was evaluated, the extract/fungus association was also not highly effective, highlighting again ASh at 10 and 5 mg/mL + fungus.

Table 1 Mean (± SD) parameters (%) obtained in the immersion test of engorged Rhipicephalus microplus females in various extracts of Achyrocline satureioides associated or not with the fungus Beauveria bassiana (106 cells/mL)
Full size table

Sensitivity of larvae in contact test on impregnated paper

In the larval packet test, the extracts of A. satureioides at all concentrations, when associated with the fungus B. bassiana strain 487, showed strong effects (Table 2), with the highest mortalities being found in the association ASb at 20 mg/mL + fungus (100%) and ASh at 20 mg/mL + fungus (71.5%). All concentrations of ASb associated with fungus were as lethal as the positive control [cypermethrin (15%), chlorpyrifos (25%), citronella (0.1%)]. No statistical difference was found among ASac and ASh at 2.5–10 mg/mL + fungus; larval mortality of these combinations was the same as that of the fungus-only control (41.7%). These results indicate increased activity against tick larvae of the fungus in association with A. satureioides, especially of ASb + fungus. The LC50 calculated (with 95% CI in parentheses) were ASb + fungus 0.30 (0.17–0.53) mg/mL, ASac + fungus 5.80 (3.53–9.52) mg/mL, and ASh + fungus 6.23 (4.19–9.27) mg/mL.

Table 2 Larval packet test: mean (± SD) mortality (%) of Rhipicephalus microplus larvae in contact with papers impregnated with Achyrocline satureioides extracts of different polarities associated or not with the fungus Beauveria bassiana
Full size table

Larvae immersion test

In the larval immersion test, the ASh partition showed the highest mortality from 20 to 5 mg/mL + fungus (Table 3). In association with the fungus B. bassiana, all extracts at the higher concentration caused 100% mortality of the larvae, equal to the positive control and significantly higher than the fungus control. The latter caused 64.5% mortality, demonstrating again the potential of the association. The LC50 calculated in this test (with 95% CI) were ASb + fungus 2.86 (2.21–3.70) mg/mL, ASac + fungus 3.61 (3.13–4.16) mg/mL, and ASh + fungus 2.65 (2.51–2.79) mg/mL.

Table 3 Larval immension test: mean (± SD) mortality (%) of Rhipicephalus microplus larvae immersed in extracts of Achyrocline satureioides of different polarities associated or not with the fungus Beauveria bassiana
Full size table

Discussion

This is the first study involving the biological effect of A. satureioides extracts in association with B. bassiana strain 487 on R. microplus ticks. The compatibility of the extract and partitions with the fungus was evaluated by the minimum inhibitory concentration assay. The tick life cycle consists of several stages, in which the behavior and morphological constitution present remarkable differences, which may alter the efficacy of the compounds evaluated for tick control. The results presented here demonstrated that the extracts, from the concentration of 20 mg/mL (106 spores/mL) down, would be viable for the association with B. bassiana, causing no damage to the growth of the fungus and showing a positive interaction.

In the immersion test of engorged females, the highest concentrations of ASh + fungus presented the best results, decreasing larval hatching and reproductive efficiency. In the moment of oviposition, females not only produce an egg mass, they also secrete a ‘protective wax’ by Gene’s organ that in nature serves to protect eggs from solar radiation and attack by fungi and bacteria (Vasconcellos et al. 2018; Matos et al. 2019) – possibly this wax did not allow the growth of B. bassiana.

Treatment for tick control is usually based on eliminating the animal parasites; however, one of the major problems is the high prevalence of larvae in pastures, representing high chances of reinfestation, perpetuating the parasitic cycle. To evaluate the efficacy of extracts and partitions against larvae, two methodologies were used, the larval packet test (FAO 1971) and the larval immersion test (Sabatini et al. 2001). In the larval packet test, ASb + fungus demonstrated high potential against R. microplus larvae, whereas ASac and ASh from 10 mg/mL down, had their effect mainly due to the fungus presence – mortality was quite similar (40.8–51.0%) to the fungus control (41.7%). The study by Barci et al. (2009) with the fungal strain of B. bassiana IBCB66 demonstrated that the fungus at a concentration of 107 CFU/mL presented 38.1% of larval mortality on R. microplus, whereas 100% mortality was achieved only at 109 CFU/mL. This comparison demonstrates that the fungal strain used in our study had almost twice the mortality at a lower concentration, as we used 106 CFU/mL, reinforcing the hypothesis that the fungus associated with the extract significantly increases the mortality rate.

In the larval immersion test, ASac + fungus until 10 mg/mL and ASh + fungus until 5 mg/mL presented the best biological effect instead of ASb. All the other results seem to be an effect of the fungus alone. The difference among tests may be due to better contact of the treatment with the parasite in the larval immersion test, as it does not allow fast volatilization of the compounds, as in the larval packet test. Moreover, it seems that compounds extracted by the hexanic solvent may present better effect on R. microplus in immersion tests, although in the adult immersion test the ASb at highest concentration also showed a small effect. Studies with associations among extract/fungus to control the cattle tick are rare, but a study using an association of Melia azedarach (Meliaceae) with B. bassiana in an emulsion showed an apparent synergistic effect in the control of infestations of R. microplus in cattle (Sousa et al. 2011). In the present study, the efficacy of the association on larvae can be explained by the adherence of extracts to the cuticle of the larvae. At higher concentrations, the solution tends to be thicker, thus facilitating adherence to the larvae, offering a higher possibility of the fungus spores to penetrate, whereas when placed in more fluid solutions, the spores end up not adhering properly.

In the in vitro tests in the present study, it is noted that lower concentrations of the association of A. satureioides extracts + fungus cause higher percentages of mortality than A. satureioides extracts alone, even at higher concentrations. This was true mainly for R. microplus larvae. One explanation for this curious finding may be a hormesis effect (Southam et al. 1943). Hormesis – i.e., a stronger effect by a lower dose – has been found in various groups of organisms, ranging from bacteria and fungi to higher plants and animals (Calabrese 2005). This effect becomes interesting, as it allows a decrease in the concentration used.

The results showed that the association between the ASb and ASh + fungus presents potential use against R. microplus larvae, suggesting a sustainable alternative for the cattle tick control. These results open new perspectives for studies that will allow the development of acaricide formulations with those compounds for future evaluation in vivo.