A cysteine endopeptidase from tick (Rhipicephalus (Boophilus) microplus) larvae with vitellin digestion activity

The hard tick Rhipicephalus (Boophilus) microplus is a blood-sucking ectoparasite. R. microplus free-living stage comprises egg development, hatching, and subsequent larval development until encountering a host. In order to complete the embryological development, this tick relies on yolk reserve substances, mainly vitellin (Vt), which is still present in the larval stage. The present study demonstrates presence and digestion of Vt in unfed R. microplus larvae. An increasing proteolytic activity is observed in larval development, as well as a decrease in total protein and in Vt content. Partial purification and characterization of a R. microplus larval cysteine endopeptidase (RmLCE) with Vt-degrading activity is also described. RmLCE has optimal activity at 37 °C at pH 5.0, being unstable at pH ≥ 7.5. This enzyme is active upon fluorogenic peptide substrates and is able to degrade Vt, its putative natural substrate. These results indicate that RmLCE has a role in supporting the nutritional needs of unfed R. microplus larva through Vt proteolysis, allowing survival until the first blood meal.     

Megaselia scalaris reared on Rhipicephalus (Boophilus) microplus laboratory cultures

Different laboratory cultures of the acarine tick Rhipicephalus (Boophilus) microplus (Canestrini, 1888) (Ixodida: Ixodidae) were infested by small Megaselia scalaris (Loew, 1866) (Diptera: Phoridae) flies. Larvae of this species exhibited opportunistic parasitism predominantly on engorged female ticks, causing severe damage to their cuticle through which the flies were able to reach R. microplus internal organs, on which they fed until developing into pupae in the tick's remains. The flies were kept by continuous propagation on fresh ticks over six generations during which the same parasitoid behaviour was observed. Here we report on an ixodid tick laboratory culture used for rearing M. scalaris.     

Resistance of the cattle tick Rhipicephalus (Boophilus) microplus to fluazuron in Argentina

The aim of this work is to report the presence of resistance to fluazuron in a population of Rhipicephalus microplus in Argentina. The evidence was obtained from field and in vitro trials. In the field trial, cattle infested with ticks was treated with two commercial formulations of fluazuron. The in vitro trial (adult immersion test, AIT) was performed by using technical grade fluazuron. In the field trial, there were no significant differences between the treated and control groups between days 2 and 34 post-treatment. The only exceptions (treated group I in day 14 post-treatment, treated group II in days 23 and 29 post-treatment) had a significantly lower tick load than the untreated group, but the efficacy was not higher than 70%. Viable engorged females were collected on both groups of treated bovines in all counts, and the production of viable larvae was not precluded with the application of the two commercial formulations of fluazuron evaluated in this study. The results obtained with the in vitro assay (AIT) also indicate that the R. microplus population tested in this work has a higher level of resistance to fluazuron than another susceptible field strain. The integrated analysis of the field and in vitro trials clearly reveals the emergence of resistance to fluazuron in a R. microplus population from Argentina. This diagnosis of resistance does not imply that the fluazuron has lost its functionality at a regional scale, but it highlights the need to establish control strategies that minimize the use of this drug in order to preserve its functionality as an acaricide.

     

A Rhipicephalus (Boophilus) microplus cathepsin with dual peptidase and antimicrobial activity

The cattle tick, Rhipicephalus (Boophilus) microplus, is a haematophagous arthropod responsible for considerable losses in the livestock industry. Immunological control with vaccines is a promising alternative to replace chemical acaricides. Due to their importance in parasite physiology, cysteine endopeptidases are potential targets. In a previous study, native Vitellin Degrading Cysteine Endopeptidase (VTDCE) was successfully tested as a vaccine antigen for bovines against R. microplus. In this work, nucleotide and amino acid VTDCE sequences were obtained from cDNA databanks, based on data from Edman sequencing and mass spectrometry. Subsequently, cloning and expression, purification, immunological and biochemical characterisation of the recombinant protein were performed to determine the biological importance of VTDCE. By Western blot, polyclonal antibodies produced against recombinant VTDCE recognised native VTDCE. Interestingly, molecular analysis showed that the VTDCE sequence has similarity to antimicrobial peptides. Indeed, experimental results revealed that VTDCE has an antimicrobial activity which is independent of endopeptidase activity. We believe that this is the first known study to show that an arthropod enzyme has antimicrobial activity.     

Staphylococcus saprophyticus is a pathogen of the cattle tick Rhipicephalus (Boophilus) microplus

During experimental Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) infestation on cattle, approximately 5% of engorged female ticks showed symptoms of bacterial infection. The affected ticks were unable to oviposit, secreted a distinctive yellow exudate through the genital orifice and eventually died. Microscopic analysis of tick exudate showed abundant clusters of Gram-positive cocci bacteria that were isolated and cultured on bacteriological medium. Biochemical phenotyping and 16S rRNA ribotyping analysis on cultured bacteria identified it as Staphylococcus saprophyticus. This species was also isolated from healthy tick larvae, indicating that S. saprophyticus is commonly found in ticks during different developmental stages. However, conspicuous symptoms are only found on fully engorged females. Cultured S. saprophyticus induced identical pathological symptoms when the bacteria were experimentally inoculated into healthy ticks, demonstrating it to be the causative agent of the R. microplus infectious lethal disease described in this work.     

BYC, an atypical aspartic endopeptidase from Rhipicephalus (Boophilus) microplus eggs

An aspartic endopeptidase was purified in our laboratory from Rhipicephalus (Boophilus) microplus eggs [Logullo, C., Vaz, I.S., Sorgine, M.H., Paiva-Silva, G.O., Faria, F.S., Zingali, R.B., De Lima, M.F., Abreu, L., Oliveira, E.F., Alves, E.W., Masuda, H., Gonzales, J.C., Masuda, A., and Oliveira, P.L., 1998. Isolation of an aspartic proteinase precursor from the egg of a hard tick, Rhipicephalus (Boophilus) microplus. Parasitology 116, 525–532]. Boophilus yolk cathepsin (BYC) was tested as component of a protective vaccine against the tick, inducing a significant immune response in cattle [da Silva, V.I., Jr., Logullo, C., Sorgine, M., Velloso, F.F., Rosa de Lima, M.F., Gonzales, J.C., Masuda, H., Oliveira, P.L., and Masuda, A., 1998. Immunization of bovines with an aspartic proteinase precursor isolated from Rhipicephalus (Boophilus) microplus eggs. Vet. Immunol. Immunopathol. 66, 331–341]. In this work, BYC was cloned and its primary sequence showed high similarity with other aspartic endopeptidases. In spite of this similarity, BYC sequence shows many important differences in relation to other aspartic peptidases, the most important being the lack of the second catalytic Asp residue, considered to be essential for the catalysis of this class of endopeptidases. When we determined BYC cleavage specificity by LC-MS, we found out that it presents a preference for hydrophobic residues in P1 and P1' in accordance to most aspartic endopeptidases. Also, when analyzed by circular dicroism, BYC presented high β sheet content, also a characteristic of aspartic endopeptidases. On the other hand, although both native and recombinant BYC are catalytically active, they present a very low specific activity, what seems to indicate that this peptidase will digest its natural substrate, vitellin, very slowly. We speculate that such a slow Vn degradative process might constitute an important strategy to preserve egg protein content to the hatching larvae.     

Structural and biochemical characterization of a recombinant triosephosphate isomerase from Rhipicephalus (Boophilus) microplus

Triosephosphate isomerase (TIM) is an enzyme with a role in glycolysis and gluconeogenesis by catalyzing the interconversion between glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. This enzyme has been used as a target in endoparasite drug development. In this work we cloned, expressed, purified and studied kinetic and structural characteristics of TIM from tick embryos, Rhipicephalus (Boophilus) microplus (BmTIM). The Km and Vmax of the recombinant BmTIM with glyceraldehyde 3-phosphate as substrate, were 0.47 mM and 6031 ??mol min⁻¹ mg protein⁻¹, respectively. The resolution of the diffracted crystal was estimated to be 2.4 Å and the overall data showed that BmTIM is similar to other reported dimeric TIMs. However, we found that, in comparison to other TIMs, BmTIM has the highest content of cysteine residues (nine cysteine residues per monomer). Only two cysteines could make disulfide bonds in monomers of BmTIM. Furthermore, BmTIM was highly sensitive to the action of the thiol reagents dithionitrobenzoic acid and methyl methane thiosulfonate, suggesting that there are five cysteines exposed in each dimer and that these residues could be employed in the development of species-specific inhibitors.     

Assessment of bacterial diversity in the cattle tick Rhipicephalus (Boophilus) microplus through tag-encoded pyrosequencing

Background  

Ticks are regarded as the most relevant vectors of disease-causing pathogens in domestic and wild animals. The cattle tick, Rhipicephalus (Boophilus) microplus, hinders livestock production in tropical and subtropical parts of the world where it is endemic. Tick microbiomes remain largely unexplored. The objective of this study was to explore the R. microplus microbiome by applying the bacterial 16S tag-encoded FLX-titanium amplicon pyrosequencing (bTEFAP) technique to characterize its bacterial diversity. Pyrosequencing was performed on adult males and females, eggs, and gut and ovary tissues from adult females derived from samples of R. microplus collected during outbreaks in southern Texas.     

Anti-tick monoclonal antibody applied by artificial capillary feeding in Rhipicephalus (Boophilus) microplus females

The tick Rhipicephalus microplus is an ectoparasite harmful to livestock, a vector of disease agents that affects meat and milk production. However, resistance to acaricides reflects the need for alternative tick control methods, among which vaccines have gained increasing relevance. In this scenario, monoclonal antibodies can be used to identify and characterize antigens that can be used as vaccine immunogens. Capillary tube artificial feeding of partially engorged R. microplus females with monoclonal antibodies against proteins from the gut of tick were used to test the effects of immunoglobulins in the physiology of the parasite. The results of artificial feeding showed that female ticks over 25mg and under 60 mg in weight performed better in the artificial feeding process, with a 94-168% weight increase after 24h of feeding. Results showed that artificial feeding of ticks proved to be a viable technique to study the effects of antibodies or drugs in the physiology of the parasite. One monoclonal antibody (BrBm2) induced decreased oviposition. Moreover, the antigen recognized by BrBm2 was identified as a 27-kDa protein and immunolabeled on digestive vesicles membranes of digestive cells of partially and fully engorged females.     

A mitochondrial exopolyphosphatase activity modulated by phosphate demand in Rhipicephalus (Boophilus) microplus embryo

This study describes Exopolyphosphatases (PPX) activity in mitochondria of Rhipicephalus microplus embryos. Mitochondria were isolated by differential centrifugation and PPX activity was analyzed through the hydrolysis of the substrate Polyphosphate (Poly P(15)). We investigated the influence of NADH, NAD+, Pi and ADP in a concentration range of 0.1-2.0 mM. Poly P hydrolysis was stimulated about two-fold by NADH and strongly inhibited by Pi. The PPX activity also increased in the presence of the respiratory substrates pyruvic and succinic acids, and this stimulatory effect disappeared upon addition of KCN. Mitochondrial respiration was activated by ADP using poly P as the only source of Pi. Endogenous poly P content changed following PPX activity during embryogenesis from the first up to 18th day of development. The data describe exopoly P as being modulated by Pi demand and related to energy supply during embryogenesis of hard ticks.     

Exopolyphosphatases in nuclear and mitochondrial fractions during embryogenesis of the hard tick Rhipicephalus (Boophilus) microplus

The present work evaluated polyphosphate (poly P) metabolism in nuclear and mitochondrial fractions during Rhipicephalus microplus embryogenesis. Nuclear poly P decreased and activity of exopolyphosphatase (PPX - polyphosphate-phosphohydrolases; EC 3.6.1.11) increased after embryo cellularization until the end of embryogenesis. The utilization of mitochondrial poly P content occurred between embryo cellularization and segmentation stages. Increasing amounts of total RNA extracted from eggs progressively enhanced nuclear PPX activity, whereas it exerted no effect on mitochondrial PPX activity. The decline in total poly P content after the 7th day of embryogenesis does not reflect the free P(i) increase and the total poly P chain length decrease after embryo cellularization. The Km(app) utilizing poly P(3), poly P(15) and poly P(65) as substrate was almost the same for the nuclear fraction (around 1muM), while the affinity for substrate in mitochondrial fraction was around 10 times higher for poly P(3) (Km(app) = 0.2muM) than for poly P(15) (Km(app) = 2.8muM) and poly P(65) (Km(app) = 3.6muM). PPX activity was stimulated by a factor of two by Mg2+ and Co2+ in the nuclear fraction and only by Mg2+ in the mitochondrial fraction. Heparin (20microg/mL) inhibited nuclear and mitochondrial PPX activity in about 90 and 95% respectively. Together, these data are consistent with the existence of two different PPX isoforms operating in the nuclei and mitochondria of the hard tick R. microplus with distinct metal dependence, inhibitor and activator sensitivities. The data also shed new light on poly P biochemistry during arthropod embryogenesis, opening new routes for future comparative studies on the physiological roles of different poly P pools distributed over cell compartments.     

Natural occurrence of lethal aspergillosis in the cattle tick Rhipicephalus (Boophilus) microplus (Acari:Ixodidae)

The purpose of this study was to describe an unreported entomopathogenic fungus that naturally infects the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae). Engorged female ticks, showed symptoms of fungal infection after controlled tick infestation of cattle. Infected ticks developed a distinctive dark colour, a pale mould grew over the cuticle and the ticks eventually died covered with fungal conidiophores. The responsible fungus was isolated and cultured on mycological medium and submitted to microscopic morphology, biochemical phenotyping and 18S rRNA ribotyping analyses, which identified it as aflatoxin-producing Aspergillus flavus. Spores from the cultured fungus were experimentally sprayed over healthy engorged female ticks, obtaining an 80% prevalence of experimental infection of healthy ticks and their egg masses, the larval progeny after incubation under laboratory conditions was also infected. These results demonstrate that A. flavus is the causative agent of the natural fungal disease of the cattle tick R. microplus described here.     

Metarhizium anisopliae lipolytic activity plays a pivotal role in Rhipicephalus (Boophilus) microplus infection

Lipases secreted by Metarhizium anisopliae, an important biological control agent, could potentially be involved in the host infection process. Here, we present the activity profile during the host infection process and the effect of lipase activity inhibitor ebelactone B on infection. The previous treatment of spores with lipase activity inhibitor, ebelactone B, completely inhibited lipolytic activity and prevented the infection of the Rhipicephalus (Boophilus) microplus host. The results herein presented prove, for the first time, the importance of lipase activity in M. anisopliae host infection process. The filamentous fungus Metarhizium anisopliae is one of the most important and studied biological agents for the control of several arthropod pests, including the cattle tick Rhipicephalus (Boophilus) microplus. Lipases secreted by M. anisopliae could potentially be involved in the host infection process. This work presents the activity profile during the host infection process and the effect of lipase activity inhibitor ebelactone B on infection. During the course of tick exposure to spores (6-120 h) lipase activity increased from 0.03 ± 0.00 U to 0.312 ± 0.068 U using rho NP palmitate as substrate. In zymograms, bands of lipase activity were detected in ticks treated with spores without inhibitor. The previous treatment of spores with lipase activity inhibitor, ebelactone B, completely inhibited lipolytic activity, at all times specified, and prevented the infection of the R. microplus host. Spores treated with the inhibitor did not germinate on the tick, although this effect was not observed in the culture medium. The results herein presented prove, for the first time, the importance of lipase activity in M. anisopliae host infection process.     

A retrospective study of climatic suitability for the tick Rhipicephalus (Boophilus)microplus in the Americas

Aim Climate change has the potential to have an impact on the distribution of ticks and tick‐borne diseases. This paper identifies the changes in climate suitability for the tick Rhipicephalus (Boophilus) microplus in the Americas by analysing climate data for the period 1950–99. Location The model was applied to the American continent. Methods A model based on Environmental Niche Modelling was used on a gridded (0.5°) long‐term (1950–99) climate data set. A map of the core range of the species was constructed, and areas where habitat suitability (HS) changes suddenly over short time periods were identified as regions of high sensitivity. Tendency of climate in the continent was evaluated and scenarios constructed for 2025 and 2050. Results Regions of high sensitivity included the southern USA, Mexico and western and central Argentina. Analysis of climate variables in these regions identified water vapour pressure deficit and evaporation as underlying the high sensitivity of habitat suitability in the USA and Mexico, and showed that episodes of high variability are linked to the El Niño Southern Oscillation. Projections of the tendency of HS as observed for the 1950–99 period point to an increase in this value in parts of the southern USA and in central Argentina, a finding that can be attributed to the progressive increase in minimum and yearly averaged temperatures. Conclusions Short‐term changes in climate may drive the system into unstable situations with sudden changes in habitat suitability for the target tick in specific zones of the Americas. Results suggest an increased abiotic (climate) suitability for R. microplus in areas whose habitat is currently unsuitable for this species.     

The effect of desiccation and low temperature on the viability of eggs and emerging larvae of the tick, Rhipicephalus (Boophilus) microplus (Canestrini) (Ixodidae)

Process-based population models need sound and comprehensive data on an animal's response to climatic factors if they are to function reliably under a wide range of climatic conditions. To this end, different aged egg masses of the livestock tick, Rhipicephalus (Boophilus) microplus, were either desiccated in atmospheres with saturation deficits of 5, 10, 15 or 20 mmHg at 20 or 26 degrees C, or chilled at temperatures of 5, 10 or 14 degrees C with a saturation deficit of 1 mmHg for varying periods. The survival rate of the eggs through to hatching was related to the initial age of the eggs, the severity of the treatments and the duration of exposure. We established a relationship between desiccation and weight loss of eggs and, secondarily between weight loss and mortality. Mortality increased with weight loss until it reached 100% when the weight loss was about 35%. Low temperatures were increasingly detrimental to eggs as they reduced from 14 to 5 degrees C. Freshly laid eggs were more susceptible to both low temperatures and desiccation than were older eggs. Larvae emerging from eggs that were stressed by either cold or desiccation lived for a shorter time under optimal conditions than did larvae from eggs incubated under optimal conditions. Larvae from eggs with the same hatching rate had the same viability, whether the stress was induced by desiccation or low temperatures. Models were developed to describe the dynamics of weight loss of eggs with desiccation, the accumulation of cold stress of the eggs, and their effects on egg survival and larval viability. These data provide a sound basis for the development of predictive models for use under field conditions, although the response of different aged eggs to low temperatures was too variable to allow us to develop an accurate model to describe that relationship. Field models will also need to take diurnal temperature fluctuations into account.     

Perturbation of tyraminergic/octopaminergic function inhibits oviposition in the cattle tick Rhipicephalus (Boophilus) microplus

The cattle tick Rhipicephalus microplus, is one of the most damaging livestock ectoparasites. Tropical tick infestation limits the introduction of high-yield bovine varieties because they do not have immunity to the diseases transmitted by these ectoparasites. This tick is usually controlled with chemical acaricides but their indiscriminate use has created resistant populations. The discovery of new molecules that can be used for tick control is urgent. Based on the knowledge that octopamine, a biogenic amine analog to epinephrine, is central to the regulation of oviposition in several studied arthropods and that an imbalance in octopamine release causes sterility in a Drosophila model. Tyramine, octopamine and epinastine and 83 adrenergic compounds classified by their effect in the vertebrate systems were screened for their ability to block oviposition in Rhipicephalus microplus. Of these molecules, we found that 10 alpha-agonists, 3 alpha-antagonists, 5 beta-adrenergic agonists, 7 beta-antagonists and Norepinephrine were able to inhibit oviposition in this tick at pharmacological concentrations. Surprisingly, tyramine appears to be more potent than octopamine. The probable physiological causes of this inhibition are discussed. Our results suggest that although there are alpha adrenergic-like receptors in the tick, they do not behave in a manner completely analogous to their vertebrate counterparts.     

Proteomic Analysis of Cattle Tick Rhipicephalus (Boophilus) microplus Saliva: A Comparison between Partially and Fully Engorged Females

The cattle tick Rhipicephalus (Boophilus) microplus is one of the most harmful parasites affecting bovines. Similarly to other hematophagous ectoparasites, R. microplus saliva contains a collection of bioactive compounds that inhibit host defenses against tick feeding activity. Thus, the study of tick salivary components offers opportunities for the development of immunological based tick control methods and medicinal applications. So far, only a few proteins have been identified in cattle tick saliva. The aim of this work was to identify proteins present in R. microplus female tick saliva at different feeding stages. Proteomic analysis of R. microplus saliva allowed identifying peptides corresponding to 187 and 68 tick and bovine proteins, respectively. Our data confirm that (i) R. microplus saliva is complex, and (ii) that there are remarkable differences in saliva composition between partially engorged and fully engorged female ticks. R. microplus saliva is rich mainly in (i) hemelipoproteins and other transporter proteins, (ii) secreted cross-tick species conserved proteins, (iii) lipocalins, (iv) peptidase inhibitors, (v) antimicrobial peptides, (vii) glycine-rich proteins, (viii) housekeeping proteins and (ix) host proteins. This investigation represents the first proteomic study about R. microplus saliva, and reports the most comprehensive Ixodidae tick saliva proteome published to date. Our results improve the understanding of tick salivary modulators of host defense to tick feeding, and provide novel information on the tick-host relationship.