Cuticular Lipids as a First Barrier Defending Ixodid Ticks against Fungal Infection

The chemical composition of tick cuticles acts as a barrier to pathogens and may limit infection by entomopathogenic fungi. This study characterized the cuticular neutral lipids (NL) and hydrocarbons (HCs) of four ixodid ticks that are widely distributed in Brazil. HC extracts were analyzed by gas chromatography-mass spectrometry and used to challenge Beauveria bassiana IP361 and Metarhizium robertsii IP146; the effect of cuticular extracts in fungal growth were evaluated by disk diffusion and conidial viability assays. In addition, conidial germination on the tick cuticle was evaluated by scanning electron microscopy, and NL from ticks treated with fungi were assessed by thin layer chromatography. Six HCs were exclusively identified in Amblyomma sculptum. Additionally, cuticle extracts from Dermacentor nitens and A. sculptum inhibited the growth of M. robertsii IP146 and reduced conidial germination of B. bassiana IP361 to 70% and 49%, respectively; the same extracts also produced cytotoxic effects, with conidial death above 30% and 60%. Electron micrographs showed a delayed germination of conidia incubated for 48 h or 72 h on D. nitens and A. sculptum. The lipid profile of A. sculptum treated with fungi was not significantly altered; triacylglycerol was not detected in the cuticle extracts of any other tick species. Finally, A. sculptum and D. nitens cuticles have lipid components that may limit the development of M. robertsii.


Introduction
Ticks are distributed worldwide and can parasitize almost every class of vertebrates [1,2], and they are considered important vectors of a wide range of pathogens to animals and humans [3,4]. Ixodid tick species found in Brazil impose a high impact on public health and on the global economy: Amblyomma sculptum (formerly, Amblyomma cajennense sensu lato) (Acari, Ixodidae) transmits Rickettsia rickettsii (Rickettsiales: Rickettsiaceae) that causes the bacterial zoonosis known as Brazilian spotted fever [5,6], whereas Rhipicephalus microplus causes over three billion dollars in yearly economic losses in the cattle industry [7,8]. Rhipicephalus sanguineus s.l. and Dermacentor nitens are also vectors for many disease agents, becoming increasingly relevant to one health perspective [9][10][11].
The management of ticks and tick-borne diseases (TBDs) has been dependent on the use of chemical acaricides but the withdrawal of many pesticides due to health risks, pollution of the environment and development of resistance in tick populations has generated much interest in more sustainable, benign alternatives [9][10][11][12][13][14]. Particular attention has been given to entomopathogenic fungi, which are natural pathogens of insects, ticks and mites [12,13]. Entomopathogenic fungi have been used as mycoinsecticides against pests in the agriculture industry since 1888; in Brazil, their application exceeds three million hectares [10,15]. The efficiency of entomopathogenic fungi, such as Metarhizium anisopliae (Hypocreales: Clavicipitaceae) and Beauveria bassiana (Hypocreales: Cordycipitaceae), to control ticks has been tested under laboratory and field conditions with promising results [16][17][18][19][20]; however, the interaction between biological control agents and ticks still needs elucidation [21].
The variance in susceptibility of ixodid ticks to entomopathogenic fungi has been documented in the literature [22], but the underlying mechanism for this variance is poorly understood. Of the limited studies, differences in susceptibility are linked to tick gender, feeding, developmental stage and tick species [23,24]. Of particular interest is the different responses of ixodid ticks to the same entomopathogenic fungi strain such as that reported for B. bassiana Bb 986 (or ESALQ 986). Briefly, treated engorged females of R. microplus were very susceptible to B. bassiana Bb 986 (ca. 88% control efficacy at 10 8 conidia mL −1 ) with a low mean larval hatch (ca. 31%) [17], whereas A. sculptum engorged females had a low susceptibility to this fungal strain (ca. 42% control efficacy at 10 8 conidia mL −1 ) [25]. Treated engorged females of D. nitens (formerly, Anocentor nitens) also had a low susceptibility to Bb 986, with a high mean larval hatch (ca. 62% at 10 8 conidia mL −1 ) [26]. Conversely, engorged females of A. sculptum and R. microplus were equally susceptible to M. anisopliae s.l. Ma 959 (or ESALQ 959), with a high control efficacy (>96% at 10 8 conidia mL −1 ) [25]. Furthermore, the susceptibility to entomopathogenic fungi may also vary among different populations of the same tick species, as shown in R. microplus [27,28].
The arthropod cuticle is the first and possibly most important barrier to infection by entomopathogenic fungi [29,30] The tick cuticle is subdivided into the epicuticle and procuticle with the latter being composed of exocuticle (with several pore canals) and the endocuticle (secreted in highly organized overlaid lamellae) and is composed of an inner protein layer, an intermediate layer formed by lipids, and a wax-rich outer part produced by dermal glands [31]. These layers and their compounds act as a barrier to pathogens, and this may limit infections that influence tick survival [32][33][34][35]. On the other hand, entomopathogenic fungi such as Metarhizium spp. and Beauveria spp. have evolved specialized mechanisms for overcoming the arthropod's defense system by the enzymatic degradation of their integument [36] and invade their hosts by direct penetration through the cuticle and natural openings [19,37,38].
In the current study, we characterized and compared the neutral lipids and hydrocarbons from the cuticle of four ixodid tick species widely distributed and most frequently found in Brazil: (1) the common human-biting tick, A. sculptum; (2) the cattle tick, R. microplus; (3) a tropical lineage of the brown dog tick, R. sanguineus s.l.; and (4) the tropical horse tick, D. nitens. We also investigated the conidial germination of M. robertsii and B. bassiana s.l. on the cuticle of engorged females of the four ixodid tick species mentioned here, and evaluated the susceptibility of fungal isolates to the total lipid compounds extracted from the tick's cuticle.

Fungal Isolates, Cultivation and Preparation of Fungal Suspensions
Two fungal isolates were investigated in the current study: M. robertsii IP 146 and B. bassiana s.l. IP 361. IP code refers to fungi deposited in the Fungal Research Collection of the Laboratory of Invertebrate Pathology, at the Institute of Tropical Pathology and Public Health of the Federal University of Goiás. These fungal isolates originated from Goiás State, Center-West Brazil; IP 146 was isolated from a soil sample by using a selective medium for entomopathogenic fungi [39] and IP 361 was isolated from a live A. sculptum engorged female collected from a naturally infested horse [40]. Both IP 146 and IP 361 are very effective against the tick R. microplus [20].
The isolates were cultured in Petri plates (90 × 10 mm; Cralplast ® , Cral Prod. for Lab. Ltda., Cotia, SP, Brazil) on potato dextrose agar medium (Difco Laboratories, Sparks, MD, USA) supplemented with 1 gL −1 yeast extract (Bacto™ Yeast Extract, Sparks, MD, USA) in a 12 h-light/12 h-dark cycle for 15 days at 27 ± 1 • C and with a relative humidity (RH) > 80%. Fresh conidia were harvested from the plates with a spatula and suspended in 10 mL of Tween 80 ® 0.01% (v/v) (Sigma-Aldrich ® , São Paulo, SP, Brazil). The conidial suspensions were vortexed for one minute, filtered through sterile cheese cloth (Cremer S.A. © , Blumenau, SC, Brazil), and the conidia were quantified by using a hemocytometer at 400× magnification in a Leica DM750 light microscope (Leica Microsystems, Wetzlar, Germany); the conidial concentrations were then adjusted to 1 × 10 5 conidia mL −1 . The viability of conidia for each suspension was assessed by the percent germination, following the methods of Braga [41] and Bernardo [19]. All tests were conducted using suspensions of conidia with a viability higher than 95%.

Collection of Ticks
Engorged females of R. sanguineus s.l. and R. microplus were obtained from colonies maintained on artificially infested rabbits (Oryctolagus cuniculus), and dairy cattle, respectively. Engorged females of A. sculptum and D. nitens were collected from naturally infested horses held in the Center for Zoonosis Control in Goiânia. The hosts were not treated with acaricides for at least 30 days before tick collections.

Cuticular Hydrocarbons Identification
Thirty engorged females of each tick species were individually vortexed in 0.5 mL in n-hexane for liquid chromatography (Merck ® , Darmstadt, Germany) in conical glass tubes for 10 min, three times each group. To separate the hydrocarbons, the samples were filtered through a flash chromatography column, filled with Florisil ® (Sigma-Aldrich, São Paulo, SP, Brazil). GC-MS analyses were performed on a gas chromatograph coupled to a mass spectrometer (GCMS-QP 2010 Plus, Shimadzu, Kyoto, Japan). The analyses were carried out using a HP-5 capillary column of 30 mm × 0.25 mm × 0.25 µm, with helium as a carrier gas at 1.0 mL min −1 . The GC temperature was set as: 80 • C (1 min), 5 • C min −1 at 290 • C, then 290 • C for 17 min. Injector, ion source and interface temperatures were 290, 250 and 310 • C, respectively. The sample injection volume was 1.0 µL, all diluted with CH2Cl2. Data acquisition and analysis were performed by using Lab Solutions Software (CS 20A) and the NIST MS search database.

Activity of Lipid Extracts to Mycelial Growth and Conidial Viability
The antifungal activity of lipid extracts was tested by spreading 50 µL of M. robertsii IP 146 or B. bassiana s.l. IP 361 conidial suspensions on PDA medium supplemented with chloramphenicol (500 mg L −1 ) in Petri plates (90 × 10 mm). Three 6 mm-diameter paper disks (Qualy ® , 80/m 2 , 12.5 cm) were impregnated by immersion in 50 µL of lipids extracted from tick cuticles (the same extracts used for the GC analysis reported above). Lipid-impregnated paper disks were placed on the culture medium inoculated with fungi. Control plates received filter paper disks treated with hexane right after evaporation. Treated or control plates were incubated at 27 ± 1 • C and RH > 80% for 48 h; the fungicidal activity of a tested compound was demonstrated by the inhibition of fungal growth around the impregnated filter paper. The size of the halos was measured by using a vernier caliper (Mitutoyo, São Paulo, SP, Brazil), in its greater diameter.
The activity of lipids on the viability of conidia was also investigated. Two microliters of each conidial suspension were inoculated (without spreading) in Petri dishes containing 4 mL of PDA plus chloramphenicol. At the same inoculation point, 3 µL of the lipids extract were also inoculated; in the control group, 3 µL of hexane (solvent) were inoculated instead.
The plates were incubated at 27 ± 1 • C for 24 h. A minimum of 300 conidia per plate was evaluated and counted, and the percent relative germination was determined [19,41]. Conidia were considered germinated when the germ tube was longer than the maximum conidial diameter. The experiments were conducted three times on different days.

Cytotoxicity of Cuticle Extracts
The cytotoxic effect of cuticle extracts to fungal conidia was assessed. Conidia of IP 146 or IP 361 were scraped from culture plates and suspended in 500 µL of tick cuticle extracts (the same used for the GC analysis reported above) and incubated overnight at room temperature (20 to 25 • C). For the control group, conidia of IP 146 or IP 361 were suspended with hexane for the same period; hexane was also used for the blank group. After exposure to the cuticle extracts or hexane (control), the conidia were resuspended in 500 µL PBS solution and propidium iodide (PI 3 µL/mL) (Sigma-Aldrich ® , São Paulo, SP, Brazil). The viability of conidia was examined in a FACSCanto II flow cytometer (Becton Dickinson, San Jose, CA, USA) by the acquisition of 10,000 events, and the data were analyzed using Version 6.0 of the FACSDiva software.

Germination of Conidia on Tick Cuticle
The germination of conidia of M. robertsii IP 146 and B. bassiana IP 361 was assessed on the cuticle of A. sculptum, D. nitens, R. microplus or R. sanguineus engorged females by Scanning Electron Microscopy (SEM). Three engorged females of each tick species were fixed with adhesive tape in Petri plates and treated topically on the dorsal side by applying 50 µL of IP 146 or IP 361 conidial suspension (1.0 × 10 8 conidia mL −1 ). Each treated engorged female was incubated at 27 ± 1 • C and RH > 90% for 24, 48 or 72 h, and processed for SEM according to Barreto [42]. Briefly, after drying with hexamethyldisilane (Electron Microscopy Sciences, Hatfield, PA, USA), the samples were placed on a stub, and coated with gold in a sputter-applicator (Denton Vacuum, Desk V); conidial germination and appressorium formation were then qualitatively assessed by using a Jeol JSM 6610 microscope, at an accelerating voltage of 5 kV.

Neutral Lipids from Tick Cuticle
The expression of cuticular neutral lipids from engorged females was investigated by Thin Layer Chromatography (TLC) before and after treating the tick cuticle with IP 146 or IP 361 (1.0 × 10 8 conidia mL −1 ), as mentioned above. We also evaluated lipids present in the conidial suspensions tested. The neutral lipids on the tick cuticle or fungal suspensions were determined according to Bligh and Dyer [43].
Each group with four engorged females was vortexed in a mixture of chloroform:water (2:1, v/v) in glass conical centrifuge tubes (Merck KGaA ® , Darmstadt, Germany) for one minute. Then, 0.5 mL of distilled water was added to the tubes containing the females, which were vortexed again for 1 min. A volume of 800 µL of this mixture was submitted to intermittent agitation every 5 min for 1 h; the solution was then centrifuged for 10 min at 3000 rotations per minute, and the supernatant, with the lipids, was separated from the precipitate. Two milliliters of distilled water and 1 mL of chloroform were added to the supernatants to separate the organic phase (which includes the lipids) from the aqueous phase; the solution was again centrifuged. The organic phase was separated into new tubes and dried by using N2. The neutral lipids from the entomopathogenic fungi, M. robertsii and B. bassiana, were extracted from 800 µL of conidial suspensions at 10 8 conidia mL −1 . The organic phase of both, tick cuticles and conidial suspensions, was resuspended in 30 µL of chloroform and applied to the silica to conduct the analysis. After running the analysis in a mixture of hexane, ethyl ether and acetic acid (60:40:1 by volume), the plate was revealed by using the Cherring compound (10% CuSO4 (w/v) and 8% H3PO4 (v/v)), and the silica was burned in a hot-air oven to mark the bands.
Densitometry was obtained by the number of pixels that the band (spot) presented using ImageMaster Total Lab software, version 1.11 (Healthcare ® Brazil Life Sciences, São Paulo, SP, Brazil), and the quantification of lipids per spot was determined according to Ruiz and Ochoa (1997) [44]. The quantity (µg) of lipids per engorged female was calculated by using the standard curve of each class of lipids: fatty acid (FA), cholesterol (CHO), esterified cholesterol (CHOE) and triacylglycerol (TG) (Sigma-Aldrich ® , São Paulo, SP, Brazil), where the total amount of NL was divided by the number of females per treatment. Markers of each class of lipids investigated were applied on the plates at increasing concentrations of 2 µg, 4 µg, 6 µg, 8 µg and 10 µg. Simultaneously with fungal suspension treatment, a 10 µL aliquot of the suspension was inoculated in Petri plates (60 × 15mm) with 4 mL of PDA medium plus 0.002% (w/v) Benomyl (benzimidazole fungicide) to assess the viability of conidia [41]. Five plates were analyzed on different occasions.

Statistical Analysis
All data were checked for normality with the Shapiro-Wilk test by using the Easyanova package [45] in the software R (R Core Team, 2016). The differences in the diameter of halos in the disk diffusion test, as well as the difference in relative conidial germination were determined by the Kruskal-Wallis test, followed by an adapted Student's t-test in the Easyanova package. Lipids quantities, i.e., CHOE, CHO, TG and FA detected in cuticular extracts from each tick species were also compared by the Kruskal-Wallis test, followed by an adapted Student's t-test. The differences in mean viability of conidia from the flow cytometry analysis were evaluated by one-way analysis of variance (ANOVA), followed by Student's t-test. Values of p less than 0.05 were considered significant.

Ethics Statement
The studies with R.

Cuticular Hydrocarbons Identification
Only hydrocarbons with more than 2% abundance (compounds with 11 to 44 carbons) of the total peaks revealed in the gas chromatography were considered for analysis in this study. The chromatogram of the compounds extracted from the cuticle of A. sculptum demonstrated eight peaks of hydrocarbons in which octacosyl (6.63%) and tetradecan (6.38%) had the highest percentage of area at peak. Compounds from the cuticle of D. nitens revealed nine peaks, among which cholesta-3,5-diene (11.32%) and pentadecane (9.32%) were the most abundant compounds. R. microplus demonstrated the highest percentage of eicosene (16.27%) and benzene (12.88%) among the eight peaks revealed in the chromatogram. The lowest number of peaks in the chromatogram was revealed from the cuticle of R. sanguineus (six), of which the compounds 1,19-eicosadiene (12.68%) and E,E,Z-1,3,12-Nonadecatriene-5,14-diol (9.2%) were the most prevalent. All hydrocarbons identified from the cuticle of the four ixodid ticks studied are listed in Table 1.

Activity of Lipid Extracts to Mycelial Growth and Conidial Viability
Total extracts from the cuticle of A. sculptum inhibited the growth of IP 146 in disk diffusion assays; halos had a mean diameter of 4 ± 1 mm. Total extracts from D. nitens also inhibited the growth of IP 146 within 48 h (3 ± 1.73 mm). The growth of IP 146 was not inhibited by the extracts from the cuticle of R. sanguineus or R. microplus. The growth of B. bassiana was not inhibited by the extracts from the cuticle of any tick species investigated here ( Figure 1). The total extracts from the cuticle of D. nitens or A. sculptum significantly inhibited the mean conidial germination of B. bassiana IP 361 (71% and 62%, respectively), as well as of M. robertsii IP 146 (68% and 49%, respectively). Cuticular extracts from R. microplus or R. sanguineus did not inhibit the germination of either IP 146 and IP 361 after 24 h incubation, and mean germination reached exactly or approximately 100% (see Figure 1). In the control groups, mean conidial germination of fungal isolates reached virtually 100% in all replicates after 24 h incubation.

Cytotoxicity of Cuticle Extracts
Total cuticular hydrocarbons extracted from A. sculptum caused significant conidial death in IP 146 (81%) and IP 361 (36%). In addition, total cuticular hydrocarbons from D. nitens caused conidial death in IP 146 (64%) and IP 361 (66%). Cuticular hydrocarbons extracted from R. microplus or R. sanguineus, however, caused low (<8%) conidial death in both fungal isolates, IP 146 and IP 361, which did not differ significantly from those conidia treated with hexane (control group) (Figure 2). The total extracts from the cuticle of D. nitens or A. sculptum significantly inhibited the mean conidial germination of B. bassiana IP 361 (71% and 62%, respectively), as well as of M. robertsii IP 146 (68% and 49%, respectively). Cuticular extracts from R. microplus or R. sanguineus did not inhibit the germination of either IP 146 and IP 361 after 24 h incubation, and mean germination reached exactly or approximately 100% (see Figure 1). In the control groups, mean conidial germination of fungal isolates reached virtually 100% in all replicates after 24 h incubation.

Germination of Conidia on Tick Cuticle
Conidia of both M. robertsii or B. bassiana germinated on the cuticle of treated e gorged females. Conidia of IP 146 formed appressoria on the cuticle of A. sculptum at 7 h. On the cuticle of D. nitens and R. sanguineus, conidial germination was documented 2 h after treatment with IP 146, and appressorial formation at 48 h and 24 h, respectivel whereas IP 361 colonized the cuticle 48 h after treatment. Cuticles of R. microplus engorge females were fungal colonized after 24 h incubation after treatment with IP 146 or IP 36 ( Figure 3).

Neutral Lipids from Tick Cuticle
Cuticular extracts from all four tick species investigated had esterified cholester (CHOE), triacylglycerol (TG), and cholesterol (CHO) profiles (Figure 4). Fatty acid (FA profiles were not detected in cuticular extracts from R. microplus engorged females, but was found in extracts from A. sculptum, D. nitens and R. sanguineus. Monoacylglycer (MG) profiles were visualized in extracts from females of R. microplus, R. sanguineus an D. nitens. In addition, six unidentified lipid classes were detected in the extracts from A sculptum, two from R. sanguineus, one from R. microplus and one from D. nitens. Percentag and distribution of lipid classes were calculated in consideration to the total classes with each sample. Altered lipid profiles were detected on tick cuticular extracts 24 h after trea ment with IP 146 or IP 361. Accordingly, TG was not detected in extracts from treated D

Neutral Lipids from Tick Cuticle
Cuticular extracts from all four tick species investigated had esterified cholesterol (CHOE), triacylglycerol (TG), and cholesterol (CHO) profiles (Figure 4). Fatty acid (FA) profiles were not detected in cuticular extracts from R. microplus engorged females, but it was found in extracts from A. sculptum, D. nitens and R. sanguineus. Monoacylglycerol (MG) profiles were visualized in extracts from females of R. microplus, R. sanguineus and D. nitens. In addition, six unidentified lipid classes were detected in the extracts from A. sculptum, two from R. sanguineus, one from R. microplus and one from D. nitens. Percentage and distribution of lipid classes were calculated in consideration to the total classes within each sample. Altered lipid profiles were detected on tick cuticular extracts 24 h after treatment with IP 146 or IP 361. Accordingly, TG was not detected in extracts from treated D. nitens, R. sanguineus or R. microplus, whereas this class of lipid was found in the control groups. In extracts from A. sculptum (10%) and D. nitens (7%), FA did not differ significantly after treatment, whereas in extracts from R. sanguineus FA was detected only in the control groups, with approximately 3% of the total lipids extracted.   The most abundant lipid class found in cuticular extracts from all tick species studied was CHOE: 47.9% in A. sculptum, 58.7% in D. nitens, 60% in R. microplus and 54.8% in R. sanguineus. TG represented 7% in A. sculptum, 11.2% in D. nitens, 40% in R. microplus and 8.3% in R. sanguineus. The percentage of CHO found was 10% in A. sculptum, 6% in D. nitens, 7% in R. microplus and 8.3% R. sanguineus; MG was, however, 8% in D. nitens, 11% in R. microplus and 20% in R. sanguineus. In addition, unidentified classes of lipids extracted from the cuticle of A. sculptum represented more than 20% of the total extract ( Figure 4). The amounts of CHOE, TG, CHO, and FA detected from each tick species were also calculated in µg per group of untreated females ( Figure 5). nitens, 7% in R. microplus and 8.3% R. sanguineus; MG was, however, 8% in D. niten in R. microplus and 20% in R. sanguineus. In addition, unidentified classes of lip tracted from the cuticle of A. sculptum represented more than 20% of the total extra ure 4). The amounts of CHOE, TG, CHO, and FA detected from each tick specie also calculated in µg per group of untreated females ( Figure 5).

Discussion
The viability of fungal propagules used for arthropod pest control is crucial an therefore, determine the success of the infection; this process begins, in general, w fungal germination and penetration through the host cuticle [30]. On the other han findings reveal that lipid compounds on the cuticle of ixodid ticks may reduce the v of at least some entomopathogenic fungi and limit the germination of their prop which may consequently limit the infection and the efficacy of biological control against ticks. In fact, the mean conidial germination of M. robertsii IP 146 and B. b IP 361 was considerably reduced after been exposed in the laboratory to the lipi pounds extracted from the cuticle of A. sculptum or D. nitens, whereas the expo conidia to the cuticular lipids from R. microplus or R. sanguineus did not affect their ity. These effects might be related to the presence of lipids with fungicidal activity

Discussion
The viability of fungal propagules used for arthropod pest control is crucial and may, therefore, determine the success of the infection; this process begins, in general, with the fungal germination and penetration through the host cuticle [30]. On the other hand, our findings reveal that lipid compounds on the cuticle of ixodid ticks may reduce the viability of at least some entomopathogenic fungi and limit the germination of their propagules, which may consequently limit the infection and the efficacy of biological control agents against ticks. In fact, the mean conidial germination of M. robertsii IP 146 and B. bassiana IP 361 was considerably reduced after been exposed in the laboratory to the lipid compounds extracted from the cuticle of A. sculptum or D. nitens, whereas the exposure of conidia to the cuticular lipids from R. microplus or R. sanguineus did not affect their viability. These effects might be related to the presence of lipids with fungicidal activity on the cuticle of ticks or to the ability of these fungi to use the cuticular lipids as an energy source [46,47].
The primary function of cuticular hydrocarbons is to protect arthropods against desiccation, but they also act importantly in chemical communication [48,49]. The role of hydrocarbons on the cuticular protection of ticks against invasion by pathogenic microorganisms is not well known, but this may be related to the antifungal activity of certain compounds [38,50,51]. In the present study, lipid compounds extracted directly from the cuticle of D. nitens or A. sculptum inhibited the mycelial growth of M. robertsii in solid medium diffusion assay. On the other hand, the total cuticular extract from R. microplus or R. sanguineus s.l. did not inhibit the growth of either IP 146 or IP 361. Squalene, a polyunsaturated hydrocarbon (C30), which corresponded to 25% of the secretory defense of the tick Dermacentor variabilis, did not protect it against predation by ants or caterpillars when tested individually [52]. Squalene hydrocarbon was also detected in extracts from the cuticles of D. nitens and R. microplus in the current study, but their direct effect against M. robertsii or B. bassiana was not investigated here.
On the cuticle of treated R. microplus and R. sanguineus, appressoria were detected on M. robertsii germ tubes 24 h after treatment. Similarly, appressoria on germinating M. anisopliae s.l. were observed on the R. microplus cuticle 24 h after treatment [53]. Evidence of penetration of M. anisopliae s.str. through the cuticle of R. sanguineus s.l., however, was not documented until 48 h after treatment [42]. In the present study, IP 146 appressoria were detected on the cuticle of D. nitens 48 h after treatment, whereas the formation of the appressoria of M. robertsii on A. sculptum was not seen earlier than 72 h after treatment. This longer period for detecting appressoria formation of IP 146 on A. sculptum (72 h) or on D. nitens (48 h) in comparison to R. microplus (24 h) or R. sanguineus s.l. (24 h), is possibly related to the antifungal activity of cuticular hydrocarbons on A. sculptum and D. nitens as documented by the conidial cytotoxicity (death) (see Figure 2) and by the mycelial growth inhibition of IP 146 in the disk diffusion assay (see Figure 1). This might also be the reason that geminated conidia of B. bassiana were not detected on the cuticle of A. sculptum 24 h after treatment, or may be the reason that late-germinated conidia (with a very short germ tube) were detected at 24 h on the cuticle of D. nitens (see Figure 2).
The ability of entomopathogenic fungi to overcome certain host antimicrobial defenses is associated with their production of enzymes that degrade the cuticle and assist fungal penetration, and to their capacity to use long-chain alkanes (between 16C and 27C) from the host cuticle as an exclusive source of carbon, which may favor the fungal infection [54,55]. In addition, cytochrome P450 monooxygenase belonging to the family CYP52 gene of M. robertsii was described as responsible for encoding an enzyme necessary for the efficient use of hydrocarbons from the host cuticle; once gene expression is disrupted, fungal growth is retarded. Although the M. robertsii genome encodes 123 cytochrome genes, only CYP52 is expressed and mediates the degradation of hydrocarbons [46].
The toxicity of cuticular extracts from A. sculptum or D. nitens to M. robertsii and B. bassiana was verified by conidial death, revealed by the binding of propidium iodide to the DNA of conidia with damaged cellular membrane. Compounds extracted from the cuticle of either R. sanguineus s.l. or R. microplus, however, were not toxic to conidia of IP 146 or IP 361. The toxicity of total hydrocarbons extracted from A. sculptum or D. nitens may be related to the activity of one compound or a combination of compounds; while many extracted hydrocarbons have been identified, their toxicities to M. robertsii and B. bassiana conidia have not been tested individually in this study. The toxicity of several cuticular hydrocarbons was tested individually in various studies: (E)-2-Hexanol and (E)-2-decanol extracted from the soybean bug, Nezara viridula (Hemiptera: Pentatomidae), have fungicidal activity against the conidia of M. anisopliae s.l. [55][56][57]. In addition, (E)-2-hexenal, (E)-2-octenal, (E)-2-decenal extracted from Tibraca limbativentris (Hemiptera: Pentatomidae) totally inhibited the germination of M. anisopliae s.l., whereas a tetradecane compound did not completely inhibit fungal mycelial growth [58]. The number of cuticular hydrocarbons on arthropod species change depending on temperature, relative humidity, food, and developmental stage, etc. [59]; it is not feasible, however, to determine a precise relationship between these variables and the diversification of hydrocarbons [48]. In addition, the mechanism of synthesis of lipid profiles is regulated by genetic factors, indicating that the cuticular compounds reveal a chemical signature for each species [60,61]. In the current study, we characterized and compared the cuticular lipid compounds of four tick species most commonly found in Brazil and neighboring countries.
The compounds produced by the four ixodid tick species investigated here are the same as those often found in insects and various other animals: CHO, CHOE, TG and FA [31]. All tick species not treated with fungi (control) had similar amounts of CHOE (ca., 55%) and TG (ca., 18%), but this class of lipids was not detected on the cuticle of engorged females of D. nitens, R. sanguineus and R. microplus that had been treated with either B. bassiana or M. robertsii. In a previous study, an insignificant amount of TG was detected in the hemolymph of infected R. microplus; this class of lipid, however, is regularly found in low proportions (1%) in the hemolymph of R. microplus [62]. In the current study, the amount of CHO differed among the tick species investigated, but not between R. microplus and R. sanguineus. Free fatty acids were not detected in R. microplus engorged females regardless of whether they were in the treatment or control group; conversely, fatty acids were detected on extracts from A. sculptum and D. nitens, treated or not treated with fungi, and in R. sanguineus, this class of lipids was detected only in the control group, which suggests that these lipids play a role in defense against pathogens. We also found six unidentified lipid bands that were unique to A. sculptum; conversely, monoacylglycerol profiles detected in cuticular extracts from D. nitens, R. sanguineus and R. microplus were not detected in A. sculptum. Higher amounts of FA, as well as exclusive classes of lipids, were detected in cuticular extracts from Blatta orientalis (Blattodea: Blattidae) than in Blatella germanica (Blattodea: Ectobiidae), and these results might be related to their susceptibility to fungal infection. Further, M. anisopliae infects cockroaches, and B. germanica is more susceptible to fungal infection than B. orientalis [63].
The current study indicates a stronger relationship between the cuticular lipid components and the susceptibility to fungi for A. sculptum and D. nitens than for R. microplus and R. sanguineus s.l., although the mechanisms in which these lipids are used in the host defense remains uncertain. Further studies will determine the damage that cuticular lipid compounds extracted from ixodid ticks cause to entomopathogenic fungi; these results may suggest improved strategies to increase the efficacy of fungal biocontrol agents against ticks and other arthropod pests. Apart from the main objective of this study, our data also raise interest in the search for components with fungicidal activity that may be explored for industrial and pharmaceutical purposes.

Conclusions
The amount of CHO on the cuticle of A. sculptum or D. nitens is higher than that found on R. microplus cuticle extracts. FA was only detected on A. sculptum and D. nitens, whereas MG class was detected on R. microplus, R. sanguineus s.l. and D. nitens. The total lipid compounds extracted from the cuticle of A. sculptum or D. nitens inhibited the growth of M. robertsii. The growth of B. bassiana was not inhibited when exposed to hydrocarbons extracted from the cuticle of any ixodid tick species investigated here.

Institutional Review Board Statement:
The study was conducted in accordance with the regulations of the Ethics Committee on Animal Use (CEUA) from Universidade Federal de Goiás (UFG) and Universidade Federal Rural do Rio de Janeiro (UFRRJ) as described in Material and Methods, Section 2.9.
Data Availability Statement: Not applicable.