A Novel Receptor Binding Progesterone, a Possible Transregulation Mechanism in the Rhipicephalus microplus-Host Interaction.

BACKGROUND: Hormone receptors exert their function through binding with their ligands, which results in cellular signaling activation mediated by genomic or non-genomic mechanisms. The intrinsic molecular communication of tick Rhipicephalus microplus and its host Bos taurus comprises an endocrine regulation involving hormones. In the present study, we performed a molecular and in silico analysis of a Membrane Associated Progesterone Receptor in R. microplus (RmMAPRC). METHODS: The RmMAPRC protein sequence was analyzed with bioinformatics tools, and its structure was characterized by three-dimensional (3D) modeling and molecular docking. A semi-quantitative reverse transcription and polymerase chain reaction (sqRT-PCR) assessed the RmMAPRC gene presence and relative expression in tick organs and embryonic cells. RESULTS: RmMAPRC relative expression in salivary glands, ovaries, and embryonic cells showed overexpression of 3%, 13%, and 24%, respectively. Bioinformatic analysis revealed that RmMAPRC corresponded to a Progesterone Receptor Membrane Component 1 (RmPGRMC1) of ~23.7 kDa, with an N-terminal transmembrane domain and a C-terminal Cytochrome b5-like heme/steroid binding domain. The docking results suggest that RmPGRMC1 could bind to progesterone (P4), some progestins, and P4 antagonists. The phylogenetic reconstruction showed that Rhipicephalus spp. MAPRC receptors were clustered in a clade that includes R. appendiculatus, R. sanguineus, and R. microplus (RmMAPRC), and mammals and helminths MAPRC receptors clustered in two separated clades away from ticks. CONCLUSIONS: The presence of RmPGRMC1 highlights the importance of transregulation as a conserved adaptive mechanism that has succeeded for arthropod parasites, making it a target for tick control.

Differential paired stage-specific expression of Babesia bovis cysteine-rich GCC2/GCC3 domain family proteins (BboGDP) during development within Rhipicephalus microplus.

BACKGROUND: Babesia bovis, an intra-erythrocytic apicomplexan parasite, is one of the causative agents of bovine babesiosis, the most important tick-borne disease of cattle in tropical and subtropical regions. Babesia bovis has a complex life-cycle that includes sexual development within the tick vector. The development of a transmission blocking vaccine to control bovine babesiosis requires the identification of antigens displayed on the surface of the parasite during its development within tick vectors. Four B. bovis cysteine-rich GCC2/GCC3 domain protein (BboGDP) family members were previously identified and are differentially expressed as discrete pairs by either blood stages or kinetes. In this study we focused on two family members, BboGDP1 and -3, that are expressed by Babesia parasites during tick infection. METHODS AND RESULTS: Transcription analysis using quantitative PCR demonstrated that BboGDP1 and -3 were upregulated in in vitro-induced sexual stage parasites and during parasite development in the tick midgut. Moreover, protein expression analysis of BboGDP1 and -3 during the development of sexual stages in in vitro culture was consistent with their transcription profile. Live immunofluorescence analysis using polyclonal antibodies confirmed surface expression of BboGDP1 and -3 on in vitro-induced sexual stage parasites. In addition, fixed immunofluorescence analysis showed reactivity of anti-BboGDP1 and -3 polyclonal antibodies to kinetes. CONCLUSIONS: The collective data indicate that BboGDP1 and -3 are expressed by kinetes and on the surface of sexual stages of the parasites. The identified parasite surface membrane proteins BboGDP1 and -3 are potential candidates for the development of a B. bovis transmission blocking vaccine.

Observing How Glutathione and S-Hexyl Glutathione Bind to Glutathione S-Transferase from Rhipicephalus (Boophilus) microplus.

Rhipicephalus (Boophilus) microplus is one of the most widespread ticks causing a massive loss to livestock production. The long-term use of acaracides rapidly develops acaracide resistance. In R. microplus, enhancing the metabolic activity of glutathione S-transferase (RmGST) is one of the mechanisms underlying acaracide resistance. RmGST catalyzes the conjugation of glutathione (GSH) to insecticides causing an easy-to-excrete conjugate. The active RmGST dimer contains two active sites (hydrophobic co-substrate binding site (H-site) and GSH binding site (G-site)) in each monomer. To preserve the insecticide efficacy, s-hexyl glutathione (GTX), a GST inhibitor, has been used as a synergist. To date, no molecular information on the RmGST-GSH/GTX complex is available. The insight is important for developing a novel RmGST inhibitor. Therefore, in this work, molecular dynamics simulations (MD) were performed to explore the binding of GTX and GSH to RmGST. GSH binds tighter and sits rigidly inside the G-site, while flexible GTX occupies both active sites. In GSH, the backbone mainly interacts with W8, R43, W46, K50, N59, L60, Q72, and S73, while its thiol group directs to Y7. In contrast, the aliphatic hexyl of GTX protrudes into the H-site and allows a flexible peptide core to form various interactions. Such high GTX flexibility and the protrusion of its hexyl moiety to the H-site suggest the dual role of GTX in preventing the conjugation reaction and the binding of acaracide. This insight can provide a better understanding of an important insecticide-resistance mechanism, which may in turn facilitate the development of novel approaches to tick control.

Neuropeptides in Rhipicephalus microplus and other hard ticks.

The synganglion is the central nervous system of ticks and, as such, controls tick physiology. It does so through the production and release of signaling molecules, many of which are neuropeptides. These peptides can function as neurotransmitters, neuromodulators and/or neurohormones, although in most cases their functions remain to be established. We identified and performed in silico characterization of neuropeptides present in different life stages and organs of Rhipicephalus microplus, generating transcriptomes from ovary, salivary glands, fat body, midgut and embryo. Annotation of synganglion transcripts led to the identification of 32 functional categories of proteins, of which the most abundant were: secreted, energetic metabolism and oxidant metabolism/detoxification. Neuropeptide precursors are among the sequences over-represented in R. microplus synganglion, with at least 5-fold higher transcription compared with other stages/organs. A total of 52 neuropeptide precursors were identified: ACP, achatin, allatostatins A, CC and CCC, allatotropin, bursicon A/B, calcitonin A and B, CCAP, CCHamide, CCRFamide, CCH/ITP, corazonin, DH31, DH44, eclosion hormone, EFLamide, EFLGGPamide, elevenin, ETH, FMRFamide myosuppressin-like, glycoprotein A2/B5, gonadulin, IGF, inotocin, insulin-like peptides, iPTH, leucokinin, myoinhibitory peptide, NPF 1 and 2, orcokinin, proctolin, pyrokinin/periviscerokinin, relaxin, RYamide, SIFamide, sNPF, sulfakinin, tachykinin and trissin. Several of these neuropeptides have not been previously reported in ticks, as the presence of ETH that was first clearly identified in Parasitiformes, which include ticks and mites. Prediction of the mature neuropeptides from precursor sequences was performed using available information about these peptides from other species, conserved domains and motifs. Almost all neuropeptides identified are also present in other tick species. Characterizing the role of neuropeptides and their respective receptors in tick physiology can aid the evaluation of their potential as drug targets.

Redox imbalance induces remodeling of glucose metabolism in Rhipicephalus microplus embryonic cell line.

Carbohydrate metabolism not only functions in supplying cellular energy but also has an important role in maintaining physiological homeostasis and in preventing oxidative damage caused by reactive oxygen species. Previously, we showed that arthropod embryonic cell lines have high tolerance to H2O2 exposure. Here, we describe that Rhipicephalus microplus tick embryonic cell line (BME26) employs an adaptive glucose metabolism mechanism that confers tolerance to hydrogen peroxide at concentrations too high for other organisms. This adaptive mechanism sustained by glucose metabolism remodeling promotes cell survival and redox balance in BME26 cell line after millimolar H2O2 exposure. The present work shows that this tick cell line could tolerate high H2O2 concentrations by initiating a carbohydrate-related adaptive response. We demonstrate that gluconeogenesis was induced as a compensation strategy that involved, among other molecules, the metabolic enzymes NADP-ICDH, G6PDH, and PEPCK. We also found that this phenomenon was coupled to glycogen accumulation and glucose uptake, supporting the pentose phosphate pathway to sustain NADPH production and leading to cell survival and proliferation. Our findings suggest that the described response is not atypical, being also observed in cancer cells, which highlights the importance of this model to all proliferative cells. We propose that these results will be useful in generating basic biological information to support the development of new strategies for disease treatment and parasite control.

Identification of the Bcl-2 and Bax homologs from Rhipicephalus haemaphysaloides and their function in the degeneration of tick salivary glands.

BACKGROUND: The salivary glands of female ticks degenerate rapidly by apoptosis and autophagy after feeding. Bcl-2 family proteins play an important role in the apoptosis pathways, but the functions of these proteins in ticks are unclear. We studied Bcl-2 and Bax homologs from Rhipicephalus haemaphysaloides and determined their functions in the degeneration of the salivary glands. METHODS: Two molecules containing conserved BH (Bcl-2 family homology) domains were identified and named RhBcl-2 and RhBax. After protein purification and mouse immunization, specific polyclonal antibodies (PcAb) were created in response to the recombinant proteins. Reverse transcription quantitative PCR (RT-qPCR) and western blot were used to detect the presence of RhBcl-2 and RhBax in ticks. TUNEL assays were used to determine the level of apoptosis in the salivary glands of female ticks at different feeding times after gene silencing. Co-transfection and GST pull-down assays were used to identify interactions between RhBcl-2 and RhBax. RESULTS: The RT-qPCR assay revealed that RhBax gene transcription increased significantly during feeding at all tick developmental stages (engorged larvae, nymphs, and adult females). Transcriptional levels of RhBcl-2 and RhBax increased more significantly in the female salivary glands than in other tissues post engorgement. RhBcl-2 silencing significantly inhibited tick feeding. In contrast, RhBax interference had no effect on tick feeding. TUNEL staining showed that apoptosis levels were significantly reduced after interference with RhBcl-2 expression. Co-transfection and GST pull-down assays showed that RhBcl-2 and RhBax could interact but not combine in the absence of the BH3 domain. CONCLUSIONS: This study identified the roles of RhBcl-2 and RhBax in tick salivary gland degeneration and finds that the BH3 domain is a key factor in their interactions.

A novel type 1 cystatin involved in the regulation of Rhipicephalus microplus midgut cysteine proteases.

Rhipicephalus microplus is a cattle ectoparasite found in tropical and subtropical regions around the world with great impact on livestock production. R. microplus can also harbor pathogens, such as Babesia sp. and Anaplasma sp. which further compromise cattle production. Blood meal acquisition and digestion are key steps for tick development. In ticks, digestion takes place inside midgut cells and is mediated by aspartic and cysteine peptidases and, therefore, regulated by their inhibitors. Cystatins are a family of cysteine peptidases inhibitors found in several organisms and have been associated in ticks with blood acquisition, blood digestion, modulation of host immune response and tick immunity. In this work, we characterized a novel R. microplus type 1 cystatin, named Rmcystatin-1b. The inhibitor transcripts were found to be highly expressed in the midgut of partially and fully engorged females and they appear to be modulated at different days post-detachment. Purified recombinant Rmcystatin-1b displayed inhibitory activity towards typical cysteine peptidases with high affinity. Moreover, rRmcystatin-1b was able to inhibit native R. microplus cysteine peptidases and RNAi-mediated knockdown of the cystatin transcripts resulted in increased proteolytic activity. Moreover, rRmcystatin-1b was able to interfere with B. bovis growth in vitro. Taken together our data strongly suggest that Rmcystatin-1b is a regulator of blood digestion in R. microplus midgut.

Rhipicephalus microplus cystatin as a potential cross-protective tick vaccine against Rhipicephalus appendiculatus.

Rhipicephalus appendiculatus, the brown ear tick, is an important disease vector of livestock in eastern, central and southern Africa. Rhipicephalus appendiculatus acaricide resistance requires the search for alternative methods for its control. Cystatins constitute a superfamily of cysteine peptidase inhibitors vital for tick blood feeding and development. These inhibitors were proposed as antigens in anti-tick vaccines. In this work, we applied structural and biochemical approaches to characterize a new cystatin named R. appendiculatus cystatin 2a (Racys2a). Structural modeling showed that this new protein possesses characteristic type 2 cystatin motifs, besides conservation of other structural patterns along the protein. Peptidase inhibitory assays with recombinant Racys2a showed modulation of tick and host cathepsins involved in blood digestion and immune system responses, respectively. A heterologous tick challenge with R. appendiculatus in rabbits immunized with recombinant Rhipicephalus microplus cystatin 2c (rBmcys2c) was performed to determine cross-reactivity. Histological staining showed that rBmcys2c vaccination caused damage to the gut, salivary gland and ovary tissues in R. appendiculatus. Furthermore, cystatin vaccine reduced the number of fully engorged adult females in 11.5 %. Consequently, strategies to increase the protection rate are necessary, including the selection of two or more antigens to compose a vaccine cocktail.

Gene Expression in the Salivary Gland of Rhipicephalus (Boophilus) microplus Fed on Tick-Susceptible and Tick-Resistant Hosts.

The success of cattle tick fixation largely depends on the secretion of substances that alter the immune response of the host. The majority of these substances are expressed by the parasite salivary gland and secreted in tick saliva. It is known that hosts can mount immune responses against ticks and bovine European breeds, and bovine industrial crossbreeds are more susceptible to infestations than are Bos indicus cattle. To identify candidates for the development of novel control strategies for the cattle tick Rhipicephalus (Boophilus) microplus, a salivary gland transcriptome analysis of engorged females fed on susceptible or resistant hosts was performed. Using RNA-Seq, transcriptomes were de novo assembled and produced a total of 235,451 contigs with 93.3% transcriptome completeness. Differential expression analysis identified 137 sequences as differentially expressed genes (DEGs) between ticks raised on tick-susceptible or tick-resistant cattle. DEGs predicted to be secreted proteins include innexins, which are transmembrane proteins that form gap junction channels; the transporters Na+/dicarboxylate, Na+/tricarboxylate, and phosphate transporter and a putative monocarboxylate transporter; a phosphoinositol 4-phosphate adaptor protein; a cysteine-rich protein containing a trypsin inhibitor-like (TIL) domain; a putative defense protein 3 containing a reeler domain; and an F-actin-uncapping protein LRRC16A with a CARMIL_C domain; these genes were upregulated in ticks fed on tick-susceptible cattle. DEGs predicted to be non-secreted proteins included a small heat shock protein and the negative elongation factor B-like, both acting in a coordinated manner to increase HSP transcript levels in the salivary glands of the ticks fed on tick-susceptible cattle; the 26S protease regulatory subunit 6B and another chaperone with similarity to calnexin, also upregulated in ticks fed on tick-susceptible cattle; an EF-hand calcium binding protein and a serine carboxypeptidase (SCP), both involved in the blood coagulation cascade and upregulated in ticks fed on tick-susceptible cattle; and two ribosomal proteins, the 60S acidic ribosomal protein P2 and the 60S ribosomal protein L19. These results help to characterize cattle tick salivary gland gene expression in tick-susceptible and tick-resistant hosts and suggest new putative targets for the control of tick infestations, as those genes involved in the mechanism of stress response during blood feeding.

The dynamics of energy metabolism in the tick embryo / A dinâmica do metabolismo energético em embriões de carrapato

Abstract The cattle tick Rhipicephalus (Boophilus) microplus is an ectoparasite capable of transmitting a large number of pathogens, causing considerable losses in the cattle industry, with substantial damage to livestock. Over the years, important stages of its life cycle, such as the embryo, have been largely ignored by researchers. Tick embryogenesis has been typically described as an energy-consuming process, sustaining cell proliferation, differentiation, and growth. During the embryonic stage of arthropods, there is mobilization of metabolites of maternal origin for the development of organs and tissues of the embryo. Glycogen resynthesis in late embryogenesis is considered as an effective indicator of embryonic integrity. In the cattle tick R.(B. (B.) microplus, glycogen resynthesis is sustained by protein degradation through the gluconeogenesis pathway at the end of the embryonic period. Despite recent advancements in research on tick energy metabolism at the molecular level, the dynamics of nutrient utilization during R. (B.) microplus embryogenesis is still poorly understood. The present review aims to describe the regulatory mechanisms of carbohydrate metabolism during maternal-zygotic transition and identify possible new targets for the development of novel drugs and other control measures against R. (B.) microplus infestations.

Resumo O carrapato bovino Rhipicephalus (B.) microplus é um ectoparasita capaz de transmitir diversos patógenos, sendo responsável por grandes perdas na pecuária pelos danos causados ao gado. Atualmente, muitos estudos têm negligenciado fases importantes do ciclo de vida deste parasita, como a fase embrionária. A embriogênese é classicamente descrita como um processo que demanda um consumo de energia, possibilitando a proliferação celular, diferenciação e crescimento. Além disso, em artrópodes, o estágio da embriogênese é caracterizado pela mobilização de metabolitos de origem materna para o desenvolvimento de novos tecidos e órgãos. A ressíntese de glicogênio no final da embriogênese tem sido descrita em diversas espécies de artrópodes, sendo considerada um indicador de integridade do embrião. No caso do R. (B.) microplus a ressíntese de glicogênio é sustentada pela degradação de proteínas durante a gliconeogênese, no terço final da embriogênese. Apesar dos recentes avanços, no estudo molecular e do metabolismo energético, os mecanismos envolvidos na dinâmica da utilização de diferentes substratos energéticos durante a embriogênese do carrapato R. (B.) microplus ainda é pouco entendido. Diante deste panorama, estudos que descrevam a regulação destes mecanismos e da associação do metabolismo de carboidratos com a transição materno zigótica, pode auxiliar na busca de novos alvos para o desenvolvimento de novos acaricidas e outras intervenções para o controle infestações de R. (B.) microplus.

A new lipid carrier protein in the cattle tick Rhipicephalus microplus.

Tick infestation in cattle reflects the main cause of economic loss to cattle producers. This is due to several reasons but mainly to their ability to feed on blood and generate a huge amount of eggs. Lipid transport in arthropods is achieved by highly specialized hemolymphatic lipoproteins, which resemble those described in vertebrate blood. Such lipoproteins continuously deliver lipids through the blood to growing eggs. The injection of radioactive [3H] palmitic acid into tick hemocoel showed that the gut, ovary, fat body and Gene's organ were the main organs of incorporation of this labeled fatty acid. The rate of [3H] palmitic acid incorporation by the organs was high up to 30 min after injection. The [3H] palmitic acid incorporated by these organs was later found in phospholipids and neutral lipids. Here, we describe the purification and characterization of a key player of lipid dynamics in tick hemolymph. The Rhipicephalus microplus lipid-apolipoprotein complex (RmLCP) is a new high-density lipoprotein (1.18 g/mL), which accounts for over 90% of [3H] palmitic acid present in the hemolymph. It has a native molecular weight of 420 kDa and is composed of one subunit of 122 kDa. Protein identification analysis of RmLPC subunit showed two better hits: vitellogenin 2 (23% protein coverage) and vitellogenin 5 (29% protein coverage), respectively and similarities with hemolymphatic apolipoproteins of arachnids such as the tick Ixodes scapularis (80%), the mite Galendromus occidentalis (44%) and the spider Parasteatoda tepidariorum (43%) and also for the insects Locusta migratoria (45%), Drosophila melanogaster (42%) and Manduca sexta (47%) to vitellogenin 2 and tick Ixodes scapularis (83%), the crab Limulus polyphemus (55%) and the oyster Crassostrea gigas (55%) to vitellogenin 5. Furthermore, it shows a distinct lipid composition from most arthropod lipoproteins, being composed of 40% free cholesterol, 27% phospholipids, 20% triacylglycerol and 15% hydrocarbons. In addition to binding most hemolymphatic fatty acids, this lipoprotein also binds and transports free cholesterol. In conclusion, the present study provides insight into the macromolecules involved in arachnid metabolism, which have significant potential for future use for the biological control of ticks.

A soluble inorganic pyrophosphatase from the cattle tick Rhipicephalus microplus capable of hydrolysing polyphosphates.

Polyphosphates have been found in all cell types examined to date and play diverse roles depending on the cell type. In eukaryotic organisms, polyphosphates have been investigated mainly in mammalian cells, and only a few studies have addressed arthropods. Pyrophosphatases have been shown to regulate polyphosphate metabolism. However, these studies were restricted to trypanosomatids. Here we focus on the tick Rhipicephalus microplus, a haematophagous ectoparasite that is highly harmful to cattle. We produced a recombinant R. microplus pyrophosphatase (rRmPPase) with the aim of investigating its kinetic parameters using polyphosphates as substrate. Molecular docking assays of RmPPase with polyphosphates were also carried out. The kinetic and Hill coefficient parameters indicated that rRmPPase has a greater affinity, higher catalytic efficiency and increased cooperativity for sodium phosphate glass type 15 (polyP15 ) than for sodium tripolyphosphate (polyP3 ). Through molecular docking, we found that polyP3 binds close to the Mg2+ atoms in the catalytic region of the protein, participating in their coordination network, whereas polyP15 interactions involve negatively charged phosphate groups and basic amino acid residues, such as Lys56, Arg58 and Lys193; polyP15 has a more favourable theoretical binding affinity than polyP3 , thus supporting the kinetic data. This study shows, for the first time in arthropods, a pyrophosphatase with polyphosphatase activity, suggesting its participation in polyphosphate metabolism.

Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells.

Rhipicephalus microplus - the cattle tick - is the most significant ectoparasite in terms of economic impact on livestock as a vector of several pathogens. Efforts have been dedicated to the cattle tick control to diminish its deleterious effects, with focus on the discovery of vaccine candidates, such as BM86, located on the surface of the tick gut epithelial cells. Current research focuses upon the utilization of cDNA and genomic libraries, to screen for other vaccine candidates. The isolation of tick gut cells constitutes an important advantage in investigating the composition of surface proteins upon the tick gut cells membrane. This paper constitutes a novel and feasible method for the isolation of epithelial cells, from the tick gut contents of semi-engorged R. microplus. This protocol utilizes TCEP and EDTA to release the epithelial cells from the subepithelial support tissues and a discontinuous density centrifugation gradient to separate epithelial cells from other cell types. Cell surface proteins were biotinylated and isolated from the tick gut epithelial cells, using streptavidin-linked magnetic beads allowing for downstream applications in FACS or LC-MS/MS-analysis.

Characterization of a novel cystatin type 2 from Rhipicephalus microplus midgut.

The Rhipicephalus (Boophilus) microplus is an exclusive bovine ectoparasite responsible for the transmission of pathogens that decrease meat, leather and milk productions. Cattle vaccination is an alternative to control tick infestations, but the discovery of potential antigens is still a challenge for researchers. Recently, our group performed a midgut transcriptome of engorged R. microplus tick, and out of 800 ESTs sequences one cystatin-coding sequence was identified and named Rmcystatin-4. In order to understand the physiological role of Rmcystatin-4, the aim of this work was the expression, purification and functional characterization of a novel type 2 cystatin from the tick R. microplus. Rmcystatin-4 gene expression was identified mostly in tick midgut suggesting its possible role in blood digestion control. Our data showed that rRmcystatin-4 was successfully expressed in active form using Pichia pastoris system and the purified inhibitor presented high selectivity to BmCl-1 (Ki = 0.046 nM). Moreover, rRmcystatin-4 was able to impaired BmCl-1 activity towards bovine hemoglobin.

Molecular and structural characterization of novel cystatins from the taiga tick Ixodes persulcatus.

Cystatins are cysteine peptidase inhibitors that in ticks mediate processes such as blood feeding and digestion. The ixodid tick Ixodes persulcatus is endemic to the Eurasia, where it is the principal vector of Lyme borreliosis. To date, no I. persulcatus cystatin has been characterized. In the present work, we describe three novel cystatins from I. persulcatus, named JpIpcys2a, JpIpcys2b and JpIpcys2c. In addition, the potential of tick cystatins as cross-protective antigens was evaluated by vaccination of hamsters using BrBmcys2c, a cystatin from Rhipicephalus microplus, against I. persulcatus infestation. Sequence analysis showed that motifs that are characteristic of cystatins type 2 are fully conserved in JpIpcys2b, while mutations are present in both JpIpcys2a and JpIpcys2c. Protein-protein docking simulations further revealed that JpIpcys2a, JpIpcys2b and JpIpcys2c showed conserved binding sites to human cathepsins L, all of them covering the active site cleft. Cystatin transcripts were detected in different I. persulcatus tissues and instars, showing their ubiquitous expression during I. persulcatus development. Serological analysis showed that although hamsters immunized with BrBmcys2c developed a humoral immune response, this response was not adequate to protect against a heterologous challenge with I. persulcatus adult ticks. The lack of cross-protection provided by BrBmcys2c immunization is perhaps linked to the fact that cystatins cluster into multigene protein families that are expressed differentially and exhibit functional redundancy. How to target such small proteins that are secreted in low quantities remains a challenge in the development of suitable anti-tick vaccine antigens.

Alterations in the oxidative metabolism of Rhipicephalus (Boophilus) microplus ticks in response to exposure to the insect growth regulator fluazuron / Alterações no metabolismo oxidativo de carrapatos Rhipicephalus (Boophilus) microplus em resposta à exposição ao regulador de crescimento fluazuron

Abstract Aiming to characterize the potential off-target effects of fluazuron on ticks, biochemical analyses were conducted to evaluate changes in the carbohydrate metabolism of Rhipicephalus (Boophilus) microplus ticks after exposure to fluazuron. Hemolymph and fat body were collected from female ticks before and after (4, 8 and 15 days) exposure to fluazuron. Spectrophotometric analyses were done to quantify glucose concentration and lactate dehydrogenase (LDH) activity in the hemolymph and the concentration of glycogen in the tick’s fat body. High Performance Liquid Chromatography (HPLC) was employed to determine the concentration of carboxylic acids in the hemolymph and to evaluate changes in intermediary metabolic processes requiring oxygen consumption. Increases in the levels of LDH activity and lactic acid concentration indicated that fluazuron enhanced fermentative metabolism in ticks. Exposure to fluazuron was also found to increase glucose concentrations in the hemolymph over time, although no significant differences were noted daily. In addition to expanding the body of knowledge about the mode of action of fluazuron, investigations into these mechanisms may also be useful in discovering new and as yet unexplored secondary effects.

Resumo Com o objetivo de caracterizar os efeitos não-alvo da ação do fluazuron, foram realizados testes bioquímicos para analisar possíveis alterações no metabolismo de carboidratos em carrapatos Rhipicephalus (Boophilus) microplus após sua exposição ao composto. Foram coletados hemolinfa e corpo gorduroso de fêmeas ingurgitadas antes e após (4, 8 e 15 dias) a exposição ao fluazuron. Análises espectrofotométricas foram usadas para quantificar a concentração de glicose e a atividade da lactato desidrogenase (LDH) na hemolinfa e concentração de glicogênio no corpo gorduroso. Cromatografia Líquida de Alta Eficiência (CLAE) foi usada para determinação das concentrações de ácidos carboxílicos na hemolinfa e avaliar possíveis alterações em metabolismo intermediário em relação ao consumo de oxigênio. Aumento na atividade de LDH e concentração de ácido lático indicaram que o fluazuron pode regular o metabolismo fermentativo em carrapatos. A exposição ao fluazuron também aumentou a concentração de glicose na hemolinfa, apesar de não ter havido diferença significativa na comparação entre as médias no mesmo dia de avaliação. Além de aumentar o conhecimento sobre o modo de ação do fluazuron, investigações sobre tais mecanismos também são úteis no descobrimento de novos efeitos secundários ainda não explorados.

Characterization of a secreted cystatin from the tick Rhipicephalus haemaphysaloides.

A novel cystatin, designated RHcyst-2, was isolated from the tick Rhipicephalus haemaphysaloides. The full-length cDNA of RHcyst-2 is 773 bp, including an intact open reading frame encoding an expected protein of 139 amino acids and consisting of a 23 amino acids signal peptide. Predicted RHcyst-2 mature protein molecular weight is about 13 kDa, isoelectric point is 4.96. A sequence analysis showed that it has significant homology with the known type 2 cystatins. The recombinant protein of RHcyst-2 was expressed in a glutathione S-transferase-fused soluble form in Escherichia coli, and its inhibitory activity against cathepsin L, B, C, H, and S, as well as papain, was identified by fluorogenic substrate analysis. The results showed that rRHcyst-2 can effectively inhibit the six cysteine proteases' enzyme activities. An investigation of the RHcyst-2 genes' expression profile by quantitative reverse transcription-PCR demonstrated that it was more richly transcribed in the embryo (egg) stage and mainly distributed in the mid-gut of adult ticks. Western blot analysis confirmed that RHcyst-2 was secreted into tick saliva.

Functional characterization of a cystatin from the tick Rhipicephalus haemaphysaloides.

BACKGROUND: Ticks and tick-borne diseases affect animal and human health worldwide and cause significant economic losses in the animal industry. Functional molecular research is important to understand the biological characteristics of ticks at the molecular level. Enzymes and enzyme inhibitory molecules play very important roles in tick physiology, and the cystatins are tight-binding inhibitors of papain-like cysteine proteases. To this end, a novel cystatin, designated RHcyst-1, was isolated from the tick Rhipicephalus haemaphysaloides. METHODS: The full-length gene of RHcyst-1 was cloning by RACE. The recombinant protein of RHcyst-1 was expressed in a glutathione S-transferase (GST)-fused soluble form in Escherichia coli, and its inhibitory activity against cathepsin L, B, C, H, and S, as well as papain, was identified by fluorogenic substrate analysis. Expression analysis of RHcyst-1 at different tick stages was performed by quantitative reverse transcription - PCR (qRT-PCR). An RNAi experiment for RHcyst-1 was performed to determine its function for tick physiology. RESULTS: The full-length cDNA of RHcyst-1 is 471 bp, including an intact open reading frame encoding an expected protein of 98 amino acids, without a signal peptide, having a predicted molecular weight of ~11 kDa and an isoelectric point of 5.66. A sequence analysis showed that it has significant homology with the known type 1 cystatins. The results of proteinase inhibition assays showed that rRHcyst-1 can effectively inhibit the six cysteine proteases' enzyme activities. An investigation of the RHcyst-1 genes' expression profile showed that it was more richly transcribed in the embryo (egg) stage. A disruption of the RHcyst-1 gene showed a significant decrease in the rate of tick hatching. CONCLUSIONS: Our results suggested that RHcyst-1 may be involved in the early embryonic development of ticks.

Molecular cloning and characterization of a glycine-like receptor gene from the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae).

The cattle tick Rhipicephalus (Boophilus) microplus is the most economically important ectoparasite affecting the cattle industry in tropical and subtropical areas around the world. The principal method of tick control has relied mainly on the use of chemical acaricides, including ivermectin; however, cattle tick populations resistant to ivermectin have recently been reported in Brazil, Mexico, and Uruguay. Currently, the molecular basis for ivermectin susceptibility and resistance are not well understood in R. microplus. This prompted us to search for potential molecular targets for ivermectin. Here, we report the cloning and molecular characterization of a R. microplus glycine-like receptor (RmGlyR) gene. The characterized mRNA encodes for a 464-amino acid polypeptide, which contains features common to ligand-gated ion channels, such as a large N-terminal extracellular domain, four transmembrane domains, a large intracellular loop and a short C-terminal extracellular domain. The deduced amino acid sequence showed around 30% identity to GlyRs from some invertebrate and vertebrate organisms. The polypeptide also contains the PAR motif, which is important for forming anion channels, and a conserved glycine residue at the third transmembrane domain, which is essential for high ivermectin sensitivity. PCR analyses showed that RmGlyR is expressed at egg, larval and adult developmental stages. Our findings suggest that the deduced receptor is an additional molecular target to ivermectin and it might be involved in ivermectin resistance in R. microplus.

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.