Ultrastructural characterization and quantification of hemocytes in engorged female Amblyomma sculptum ticks.

Amblyomma sculptum (formerly Amblyomma cajennense) ticks have been implicated in the transmission of pathogens that cause diseases in animals and humans. Their wide geographic distribution and high impact on animal health and zoonotic disease transmission highlight the importance of studying and implementing effective control measures to mitigate the risks associated with this tick species. The aim of this study was to quantify and characterize the morphology and the ultrastructure of different types of hemocytes in the hemolymph in engorged A. sculptum females fed on rabbits. The hemolymph samples were collected by perforation of the cuticle in the dorsal region. Hemocyte types, sizes, and differential counts were determined using light microscopy, while ultrastructural analysis of hemocytes was performed using transmission electron microscopy. The average number of total hemocytes in the hemolymph was 1024 ± 597.6 cells µL-1. Five morphologically distinct cell types were identified in A. sculptum females: prohemocytes (6 % ± 8.8), plasmatocytes (10 % ± 7.7), granulocytes (78 % ± 12.2), spherulocytes (5 % ± 4.48), and oenocytoids (1 % ± 1.6). In general, prohemocytes were the smallest hemocytes. The ultrastructural morphology of A. sculptum hemocytes described in the present study agrees with the findings for other hard ticks. This is the first study to investigate ultrastructural characteristics of hemocytes of female A. sculptum ticks.

Metarhizium pingshaense photolyase expression and virulence to Rhipicephalus microplus after UV-B exposure.

The current study examined the impact of ultraviolet (UV)-B radiation in Metarhizium pingshaense blastospores' photolyase expression and their virulence against Rhipicephalus microplus. Blastospores were exposed to UV under laboratory and field conditions. Ticks were treated topically with fungal suspension and exposed to UV-B in the laboratory for three consecutive days. The expression of cyclobutane pyrimidine dimmers (CPDs)-photolyase gene maphr1-2 in blastospores after UV exposure followed by white light exposure was accessed after 0, 8, 12, 24, 36, and 48 h. Average relative germination of blastospores 24 h after in vitro UV exposure was 8.4% lower than 48 h. Despite this, the relative germination of blastospores exposed to UV in the field 18 h (95.7 ± 0.3%) and 28 h (97.3 ± 0.8%) after exposure were not different (p > 0.05). Ticks treated with fungus and not exposed to UV exhibited 0% survival 10 days after the treatment, while fungus-treated ticks exposed to UV exhibited 50 ± 11.2% survival. Expression levels of maphr1-2 8, 12, and 24 h after UV-B exposure were not different from time zero. Maphr1-2 expression peak in M. pingshaense blastospores occurred 36 h after UV-B exposure, in the proposed conditions and times analyzed, suggesting repair mechanisms other than CPD-mediated-photoreactivation might be leading blastospores' germination from 0 to 24 h.

Inhibition of Dopamine Activity and Response of Rhipicephalus microplus Challenged with Metarhizium anisopliae.

Dopamine modulates ticks and insect hemocytes and links these arthropods' nervous and immune systems. For the first time, the present study analyzed the effect of a dopamine receptor antagonist on the survival, biological parameters, phagocytic index, and dopamine detection in the hemocytes of ticks challenged by Metarhizium anisopliae. The survival and egg production index of Rhipicephalus microplus were negatively impacted when ticks were inoculated with the antagonist and fungus. Five days after the treatment, the survival of ticks treated only with fungus was 2.2 times higher than ticks treated with the antagonist (highest concentration) and fungus. A reduction in the phagocytic index of hemocytes of 68.4% was observed in the group inoculated with the highest concentration of the antagonist and fungus compared to ticks treated only with fungus. No changes were detected in the R. microplus levels of intrahemocytic dopamine or hemocytic quantification. Our results support the hypothesis that dopamine is crucial for tick immune defense, changing the phagocytic capacity of hemocytes and the susceptibility of ticks to entomopathogenic fungi.

Characterization and manipulation of the bacterial community in the midgut of Ixodes ricinus.

BACKGROUND: Ticks are obligate hematophagous arthropods transmitting a wide range of pathogens to humans and animals. They also harbor a non-pathogenic microbiota, primarily in the ovaries and the midgut. In the previous study on Ixodes ricinus, we used a culture-independent approach and showed a diverse but quantitatively poor midgut bacterial microbiome. Our analysis also revealed the absence of a core microbiome, suggesting an environmental origin of the tick midgut microbiota. METHODS: A bacterial analysis of the midgut of adult females collected by flagging from two localities in the Czech Republic was performed. Using the culture-independent approach, we tested the hypothesis that the midgut microbiome is of the environmental origin. We also cultured indigenous bacteria from the tick midgut and used these to feed ticks artificially in an attempt to manipulate the midgut microbiome. RESULTS: The midgut showed a very low prevalence and abundance of culturable bacteria, with only 37% of ticks positive for bacteria. The culture-independent approach revealed the presence of Borrelia sp., Spiroplasma sp., Rickettsia sp., Midichloria sp. and various mainly environmental Gram-positive bacterial taxa. The comparison of ticks from two regions revealed that the habitat influenced the midgut bacterial diversity. In addition, the midgut of ticks capillary fed with the indigenous Micrococcus luteus (Gram-positive) and Pantoea sp. (Gram-negative) could not be colonized due to rapid and effective clearance of both bacterial taxa. CONCLUSIONS: The midgut microbiome of I. ricinus is diverse but low in abundance, with the exception of tick-borne pathogens and symbionts. The environment impacts the diversity of the tick midgut microbiome. Ingested extracellular environmental bacteria are rapidly eliminated and are not able to colonize the gut. We hypothesize that bacterial elimination triggered in the midgut of unfed adult females is critical to maintain low microbial levels during blood-feeding.

The role of complement in the tick cellular immune defense against the entomopathogenic fungus Metarhizium robertsii.

Entomopathogenic fungi (EPF) have been widely explored for their potential in the biological control of insect pests and as an environmentally friendly alternative to acaricides for limiting tick infestation in the field. The arthropod cuticle is the main barrier against fungal infection, however, an understanding of internal defense mechanisms after EPF intrusion into the invertebrate hemocoel is still rather limited. Using an infection model of the European Lyme borreliosis vector Ixodes ricinus with the EPF Metarhizium robertsii, we demonstrated that ticks are capable of protecting themselves to a certain extent against mild fungal infections. However, tick mortality dramatically increases when the capability of tick hemocytes to phagocytose fungal conidia is impaired. Using RNAi-mediated silencing of tick thioester-containing proteins (TEPs), followed by in vitro and/or in vivo phagocytic assays, we found that C3-like complement components and α2-macroglobulin pan-protease inhibitors secreted to the hemolymph play pivotal roles in M. robertsii phagocytosis.

How Dopamine Influences Survival and Cellular Immune Response of Rhipicephalus microplus Inoculated with Metarhizium anisopliae.

Dopamine (DA) is a biogenic monoamine reported to modulate insect hemocytes. Although the immune functions of DA are known in insects, there is a lack of knowledge of DA's role in the immune system of ticks. The use of Metarhizium anisopliae has been considered for tick control, driving studies on the immune response of these arthropods challenged with fungi. The present study evaluated the effect of DA on the cellular immune response and survival of Rhipicephalus microplus inoculated with M. anisopliae blastospores. Exogenous DA increased both ticks' survival 72 h after M. anisopliae inoculation and the number of circulating hemocytes compared to the control group, 24 h after the treatment. The phagocytic index of tick hemocytes challenged with M. anisopliae did not change upon injection of exogenous DA. Phenoloxidase activity in the hemolymph of ticks injected with DA and the fungus or exclusively with DA was higher than in untreated ticks or ticks inoculated with the fungus alone, 72 h after treatment. DA was detected in the hemocytes of fungus-treated and untreated ticks. Unveiling the cellular immune response in ticks challenged with entomopathogenic fungi is important to improve strategies for the biological control of these ectoparasites.

Assessment of lipid profile in fat body and eggs of Rhipicephalus microplus engorged females exposed to (E)-cinnamaldehyde and α-bisabolol, potential acaricide compounds.

In the present study, the lipid profile from the fat body and eggs of Rhipicephalus microplus was evaluated after exposure of engorged females to (E)-cinnamaldehyde and α-bisabolol, substances which have acaricide potential according to the literature. Engorged females collected from artificially infested cattle were immersed in a concentration of 10.0 mg/mL of each substance. Dissection of the female fat bodies was performed at different times (72 h and 120 h), for subsequent lipid extraction. In addition, on the fifth day of oviposition, were collected 50.0 ml50.0 mL aliquots of the egg mass of each treatment to perform the same lipid extraction procedure. To assess the lipid profiles, the samples were submitted to the thin layer chromatography (TLC) and gas chromatography-mass spectrometry (GCMS) analysis. Furthermore, an in silico analysis was performed using PASS online® software to predict the possible molecular targets of (E)-cinnamaldehyde and α-bisabolol. As result, the main lipids identified from the fat body were triacylglycerides, fatty acids, and cholesterol, whereas, triacylglycerides (TAG), fatty acids (FA), and cholesterol (CHO) and cholesterol esters (CHOE), were identified in the eggs. The results also showed a significant increase (p < 0.05) of CHO in the fat body in the group exposed to (E)-cinnamaldehyde at 72 h (0.12 µg/fat body) and 120 h (0.46 µg/fat body), in the eggs from females treated with this same substance, there was a significant reduction (p < 0.05) in the amount of CHO (0.21 µg), compared to the water control group (0.45 µg). In the GCMS technique, 5 chemical classes were found, and variations were observed between these substances, mainly hydrocarbons and steroids, in the different groups, and (E)-cinnamaldehyde promoted the greatest changes. From the predictions of the in silico study, 38 and 20 targets were selected, respectively, which are mainly related to alterations in lipid metabolism, immune system and nervous system. This study provides the first report of changes in lipid metabolism of R. microplus exposed to (E)-cinnamaldehyde and α-bisabolol, as well as presenting possible activity on the molecular targets of these substances, expanding knowledge for the potential use of these compounds in the development of botanical acaricides.

Exposure to a sublethal menadione concentration modifies the mycelial secretome and conidial enzyme activities of Metarhizium anisopliae sensu lato and increases its virulence against Rhipicephalus microplus.

Menadione (MND) is known to induce oxidative stress in fungal cells. Here, we explore how exposure to this molecule alters conidial enzyme activities, fungal efficacy against Rhipicephalus microplus, and mycelial secretion (secretome) of an isolate of Metarhizium anisopliae sensu lato. First, the fungus was exposed to different MND concentrations in potato-dextrose-agar (PDA) to determine the LC50 by evaluating conidia germination (38µM). To ensure high cell integrity, a sublethal dose of MND (half of LC50) was added to solid (PDA MND) and liquid media (MS MND). Changes in colony growth, a slight reduction in conidia production, decreases in conidial surface Pr1 and Pr2 activities as well as improvements in proteolytic and antioxidant (catalase, superoxide dismutase, and peroxidase) conidial intracellular activities were observed for PDA MND conidia. Additionally, PDA MND conidia had the best results for killing tick larvae, with the highest mortality rates until 15 days after treatment, which reduces both LC50 and LT50, particularly at 108 conidia mL-1. The diversity of secreted proteins after growth in liquid medium + R. microplus cuticle (supplemented or not with half of MND LC50), was evaluated by mass spectrometry-based proteomics. A total of 654 proteins were identified, 31 of which were differentially regulated (up or down) and mainly related to antioxidant activity (catalase), pathogenicity (Pr1B, Pr1D, and Pr1K), cell repair, and morphogenesis. In the exclusively MS MND profile, 48 proteins, mostly associated with cellular signaling, nutrition, and antioxidant functions, were distinguished. Finally, enzymatic assays were performed to validate some of these proteins. Overall, supplementation with MND in the solid medium made conidia more efficient at controlling R. microplus larvae, especially by increasing, inside the conidia, the activity of some infection-related enzymes. In the liquid medium (a consolidated study model that mimics some infection conditions), proteins were up- and/or exclusively-regulated in the presence of MND, which opens a spectrum of new targets for further study to improve biological control of ticks using Metarhizium species.

Larvicidal activity, route of interaction and ultrastructural changes in Aedes aegypti exposed to entomopathogenic fungi.

Blastospores or conidia (formulated or not) of entomopathogenic fungi were assessed against Aedes aegypti larvae. Larvae (L2) were exposed to 105, 106, 107, and 108 propagules mL-1 water suspension. Mineral oil at 0.1%, 0.5%, or 1.0% (v/v) was employed to observe the effect on larval survival. The 0.1% mineral oil did not affect larval survival. Accordingly, 107 propagules mL-1 and 0.1% mineral oil were used to prepare all fungal emulsions. The fungal suspension or formulation was prepared as follows: 107 propagules mL-1 on 0.03% TweenⓇ 80 (v/v) aqueous solution or 107 propagules mL-1 on 0.03% TweenⓇ 80 plus 0.1% mineral oil; larval survival rates were evaluated for 7 days, and median survival time (S50) was also determined. The presence of fungi in larvae was examined both histologically and by scanning electron microscopy 24 h or 48 h after exposure. To evaluate the larval growth, larvae were exposed to 107 propagules mL-1 for 48 hours and their length measured using a digital caliper. Here, propagules had similar results in reducing the larvae survival rate and time. The treatment with Beauveria bassiana s.l. at 108 propagules mL-1 or with Metarhizium anisopliae s.l. at 108 blastopores mL-1 reduced the larval survival time to two days. M. anisopliae s.l. at 108 conidia mL-1 reduced the survival time to three days. The survival time of larvae submitted to the other treatments ranged from 6 days to over 7 days. M. anisopliae s.l. or B. bassiana s.l. oil-in-water emulsions at 107 propagules mL-1 yielded better results than the water suspensions, the larvae survival rate was 2 days for both propagules in oil-in-water emulsion. Larvae exposed to blastospores from both isolates or M. anisopliae conidia were longer than in the other treatments. Scanning electron microscopy and histology analyzes found fungi predominantly in the gut, mouthparts, and perispiracular lobes of larvae. Formulated fungus yielded better results than the aqueous suspensions for control of mosquito larvae. Thus, for the first time, the effect of mineral oil on the fungal interaction on A. aegypti larvae was observed as well as the effect of entomopathogenic fungi in the growth of larvae, supporting the search for strategies to control this arthropod.

In vitro efficacy of two commercial products of Metarhizium anisopliae s.l. for controlling the cattle tick Rhipicephalus microplus.

The effects of two different products - Metarril® SP Organic (dry conidia) and Metarril® SC Organic (emulsifiable concentrated conidia in vegetable oil) - on eggs, larvae and Rhipicephalus microplus engorged females were here explored. Three concentrations (108, 107, and 106 conidia mL-1) for both products were prepared in water + 0.1% Tween® 80 (v/v); afterward, bioassays were carried out for all R. microplus stages by immersion in suspensions (Metarril® SP) or formulations (Metarril® SC). Metarril® SP suspensions showed low efficacy and did not affect biological parameters of treated engorged females; for eggs and larvae, only slight decreases in hatchability and larvae population were observed. Despite a delay in germination, Metarril® SC presented better results; for females, reductions in Egg Mass Weight (EMW) and Egg Production Index (EPI) were reported. On eggs, 108 conidia mL-1 increased Incubation Period (IP), shortened Hatching Period (HP) and decreased hatchability by up to 61%; for larvae, 107 and 108 conidia mL-1 reached 99.6 and 100% larval mortality respectively, 10 days after fungal exposure. Thus, further studies involving the use of oil-based formulations for ticks such as Metarril® SC need to be performed, especially to control the most susceptible stages (eggs and larvae).

Ultrastructural and Cytotoxic Effects of Metarhizium robertsii Infection on Rhipicephalus microplus Hemocytes.

Metarhizium is an entomopathogenic fungus widely employed in the biological control of arthropods. Hemocytes present in the hemolymph of invertebrates are the cells involved in the immune response of arthropods. Despite this, knowledge about Rhipicephalus microplus hemocytes morphological aspects as well as their role in response to the fungal infection is scarce. The present study aimed to analyze the hemocytes of R. microplus females after Metarhizium robertsii infection, using light and electron microscopy approaches associated with the cytotoxicity evaluation. Five types of hemocytes (prohemocytes, spherulocytes, plasmatocytes, granulocytes, and oenocytoids) were described in the hemolymph of uninfected ticks, while only prohemocytes, granulocytes, and plasmatocytes were observed in fungus-infected tick females. Twenty-four hours after the fungal infection, only granulocytes and plasmatocytes were detected in the transmission electron microscopy analysis. Hemocytes from fungus-infected tick females showed several cytoplasmic vacuoles with different electron densities, and lipid droplets in close contact to low electron density vacuoles, as well as the formation of autophagosomes and subcellular material in different stages of degradation could also be observed. M. robertsii propagules were more toxic to tick hemocytes in the highest concentration tested (1.0 × 108 conidia mL-1). Interestingly, the lowest fungus concentration did not affect significantly the cell viability. Microanalysis showed that cells granules from fungus-infected and uninfected ticks had similar composition. This study addressed the first report of fungal cytotoxicity analyzing ultrastructural effects on hemocytes of R. microplus infected with entomopathogenic fungi. These results open new perspectives for the comprehension of ticks physiology and pathology, allowing the identification of new targets for the biological control.

Disclosing Hemolymph Collection and Inoculation of Metarhizium Blastospores into Rhipicephalus Microplus Ticks Towards Invertebrate Pathology Studies.

Ticks are obligate hematophagous ectoparasites and Rhipicephalus microplus has great importance in veterinary medicine because it causes anemia, weight loss, depreciation of the animals' leather and also can act as a vector of several pathogens. Due to the exorbitant costs to control these parasites, damage to the environment caused by the inappropriate use of chemical acaricides, and the increased resistance against traditional parasiticides, alternative control of ticks, by the use of entomopathogenic fungi, for example, has been considered an interesting approach. Nevertheless, few studies have demonstrated how the tick's immune system acts to fight these entomopathogens. Therefore, this protocol demonstrates two methods used for entomopathogen inoculation into engorged females and two techniques used for hemolymph collection and hemocytes harvesting. Inoculation of pathogens at the leg insertion in the tick female's body allows evaluation of females biologic parameters unlike the inoculation between the scutum and capitulum, which frequently damages Gené's organ. Dorsal hemolymph collection yielded a higher volume recovery than collection through the legs. Some limitations of tick hemolymph collection and processing include i) high rates of hemocytes' disruption, ii) hemolymph contamination with disrupted midgut, and iii) low hemolymph volume recovery. When hemolymph is collected through the leg cutting, the hemolymph takes time to accumulate at the leg opening, favoring the clotting process. In addition, fewer hemocytes are obtained in the collection through the leg compared to the dorsal collection, even though the first method is considered easier to be performed. Understanding the immune response in ticks mediated by entomopathogenic agents helps to unveil their pathogenesis and develop new targets for tick control. The inoculation processes described here require very low technological resources and can be used not only to expose ticks to pathogenic microorganisms. Similarly, the collection of tick hemolymph may represent the first step for many physiological studies.

Ultrastructural and cytotoxic effects of Metarhizium robertsii Infection on Rhipicephalus microplus hemocytes

Metarhizium is an entomopathogenic fungus widely employed in the biological control of arthropods. Hemocytes present in the hemolymph of invertebrates are the cells involved in the immune response of arthropods. Despite this, knowledge about Rhipicephalus microplus hemocytes morphological aspects as well as their role in response to the fungal infection is scarce. The present study aimed to analyze the hemocytes of R. microplus females after Metarhizium robertsii infection, using light and electron microscopy approaches associated with the cytotoxicity evaluation. Five types of hemocytes (prohemocytes, spherulocytes, plasmatocytes, granulocytes, and oenocytoids) were described in the hemolymph of uninfected ticks, while only prohemocytes, granulocytes, and plasmatocytes were observed in fungus-infected tick females. Twenty-four hours after the fungal infection, only granulocytes and plasmatocytes were detected in the transmission electron microscopy analysis. Hemocytes from fungus-infected tick females showed several cytoplasmic vacuoles with different electron densities, and lipid droplets in close contact to low electron density vacuoles, as well as the formation of autophagosomes and subcellular material in different stages of degradation could also be observed. M. robertsii propagules were more toxic to tick hemocytes in the highest concentration tested (1.0 × 108 conidia mL-1). Interestingly, the lowest fungus concentration did not affect significantly the cell viability. Microanalysis showed that cells granules from fungus-infected and uninfected ticks had similar composition. This study addressed the first report of fungal cytotoxicity analyzing ultrastructural effects on hemocytes of R. microplus infected with entomopathogenic fungi. These results open new perspectives for the comprehension of ticks physiology and pathology, allowing the identification of new targets for the biological control.

Ultrastructural and cytotoxic effects of Metarhizium robertsii Infection on Rhipicephalus microplus hemocytes

Metarhizium is an entomopathogenic fungus widely employed in the biological control of arthropods. Hemocytes present in the hemolymph of invertebrates are the cells involved in the immune response of arthropods. Despite this, knowledge about Rhipicephalus microplus hemocytes morphological aspects as well as their role in response to the fungal infection is scarce. The present study aimed to analyze the hemocytes of R. microplus females after Metarhizium robertsii infection, using light and electron microscopy approaches associated with the cytotoxicity evaluation. Five types of hemocytes (prohemocytes, spherulocytes, plasmatocytes, granulocytes, and oenocytoids) were described in the hemolymph of uninfected ticks, while only prohemocytes, granulocytes, and plasmatocytes were observed in fungus-infected tick females. Twenty-four hours after the fungal infection, only granulocytes and plasmatocytes were detected in the transmission electron microscopy analysis. Hemocytes from fungus-infected tick females showed several cytoplasmic vacuoles with different electron densities, and lipid droplets in close contact to low electron density vacuoles, as well as the formation of autophagosomes and subcellular material in different stages of degradation could also be observed. M. robertsii propagules were more toxic to tick hemocytes in the highest concentration tested (1.0 × 108 conidia mL-1). Interestingly, the lowest fungus concentration did not affect significantly the cell viability. Microanalysis showed that cells granules from fungus-infected and uninfected ticks had similar composition. This study addressed the first report of fungal cytotoxicity analyzing ultrastructural effects on hemocytes of R. microplus infected with entomopathogenic fungi. These results open new perspectives for the comprehension of ticks physiology and pathology, allowing the identification of new targets for the biological control.

Metarhizium anisopliae s.l. modulation of lipid metabolism during tick infection is independent of AMPK and ERK pathways.

Despite the importance of fat body in metabolism of arthropods, studies in ticks are scarce. This study evaluated the lipid composition and activation of extracellular signal-regulated protein kinase (ERK) and AMP-activated protein kinase (AMPK) enzymes in Rhipicephalus microplus fat body after infection with different isolates of the fungus Metarhizium anisopliae sensu lato (Metschnikoff, 1879) Sorokin, 1883. The isolates CG 32, GC 112, GC 148, GC 347, and GC 629 were inoculated as viable or non-viable conidia in the ticks. The engorged females were dissected, and their fat bodies were collected 24 and 48 h after infection. The lipid composition was assessed by thin layer chromatography, and enzyme activation was detected by Western blotting with antibodies against p-AMPK and p-ERK. The study showed increased levels of triacylglycerol 24 and 48 h and fatty acid after 48 h after inoculation with different isolates of viable fungi in the tick's hemocoel. Detection of the active form of ERK was demonstrated only after inoculation with non-viable conidia of all isolates tested. The active form of AMPK, only isolate CG 112 was able to activate with viable or non-viable conidia, whereas isolates CG 32 and CG 629 were able to activate with non-viable conidia. This study provides the first report about changes in important metabolic pathways in ticks infected with entomopathogenic fungi and suggests that the lipid content is modulated by non-usual pathways. However, further studies may be necessary for a better elucidation of this interaction.