In vivo and in vitro apoptosis induced by new acaricidal ethyl-carbamates in Rhipicephalus microplus.

The ability of ethyl-4-bromophenylcarbamate (LQM 919) and ethyl-4-chlorophenylcarbamate (LQM 996) to induce in vivo apoptosis of Rhipicephalus microplus ovarian cells and in vitro apoptosis of tick and mammalian cell culture was evaluated. The ovaries of engorged females treated with 1 mg mL-1 LQM 919 or LQM 996 presented more (p < 0.001) peroxidase-TUNEL-positive labeled cells (apoptotic cells) in situ than their respective control groups, and this increase was time-dependent (p < 0.001). The majority of apoptotic cells were observed in the epithelium and ovarian pedicel. HepG2, Vero and Rm-sus cells, as well as cells from primary cultures of R. microplus salivary glands, intestine and ovaries were exposed to different concentrations of the ethyl-carbamates. Both ethyl-carbamates induced a concentration-dependent reduction in the viability of all cell types (p < 0.001). Exposure to the ethyl-carbamates increased caspase 3 activity (p < 0.01) in primary cultures and cell lines, except in HepG2 cells. Fluorescent TUNEL-positive cells were observed in all cell types treated with 600 µM LQM 919 or LQM 996. These results indicate that both ethyl-carbamates induce apoptosis of the ovarian, intestinal and salivary glands cells in R. microplus and strongly suggest that this is their main mechanism of acaricidal action.

Rhipicephalus microplus serpins interfere with host immune responses by specifically modulating mast cells and lymphocytes.

Rhipicephalus microplus ticks feed on a bovine host for three weeks. At the attachment site, inflammatory and immune responses are triggered resulting in the recruitment of cells and production of a set of immunological mediators. To oppose the host's immune responses, ticks inoculate bioactive salivary molecules capable of interfering with these defense mechanisms. Serpins are among the most frequent molecules present in tick saliva and have been shown to negatively affect the host's anti-tick immunity. R. microplus has at least eighteen full-length serpins (RmS) and eleven are transcribed during blood feeding. Among them, RmS-3, RmS-6, and RmS-17 are present in the saliva of engorged females. Here, the effect of these serpins on the immune responses was evaluated in cells involved in innate/inflammatory (mast cells and macrophages) and adaptive (T cells) immunity. RmS-3 modulated mast cells due to its inhibitory activity on peritoneal rat chymase and on vascular permeability in acute inflammation. In addition, both RmS-6 and RmS-17 inhibited vascular permeability. Of the three serpins studied, neither affected activation nor inflammatory cytokine production by murine macrophages. On the other hand, RmS-3 and RmS-17 presented an inhibitory effect on the metabolic activity of lymphocytes, with the latter being the most potent, while RmS-6 had no effect on it. This activity was associated with a decrease in lymphocyte proliferation, but not with induction of cell death. The present study highlights the powerful modulatory role of tick salivary serpins in the host's immune system and inspire the discovery of targets for the treatment of inflammatory/immune disorders.

Comparison of the differential regulation of T and B-lymphocyte subsets in the skin and lymph nodes amongst three cattle breeds as potential mediators of immune-resistance to Rhipicephalus microplus.

Although varying natural resistance to ticks between highly resistant Brahman (Bos taurus indicus), resistant Bonsmara (5/8 B. t. indicus x 3/8 B. t. taurus) and susceptible Holstein-Friesian (B. t. taurus) breeds is documented in skin and blood, little information is available describing draining lymph nodes. To elucidate the cellular dynamics during Rhipicephalus microplus induced immune responses, this study analysed immune factors from these cattle breeds using histology, immunohistochemistry and flow cytometry. Following the collection of skin and lymph node samples before artificial tick infestation, cattle were infested with R. microplus larvae. Subsequent sampling coincided with the tick larvae and adult developmental stages. A significant influx of CD20+ B-lymphocytes in the dermis all cattle breeds was observed while CD3+ T-lymphocytes were significantly increased for more tick resistant breeds. Eosinophil infiltration in germinal centres of lymph nodes was significant for all cattle breeds while tingible body macrophages were significantly increased for adult infested Brahman animals. A negligible fluctuation in CD20+ and CD79α+ B-lymphocyte numbers was present in the lymph node of more resistant cattle breeds, while susceptible animals showed a decrease in B-lymphocytes after infestation, followed by an increase between larvae to adult infested time points. Increased variability of γd T-lymphocyte populations in lymph nodes was correlated with tick susceptibility. In addition, a more stable T helper lymphocyte population was identified in the lymph nodes for the Brahman cattle breed. Results suggest the association of tick susceptibility with differential B-lymphocyte regulation in lymph node tissues, increased variability of WC1+ γδ T-lymphocyte populations in the lymph node as well as a decrease in T helper lymphocytes in the lymph node.

Caspase-1 participates in apoptosis of salivary glands in Rhipicephalus haemaphysaloides.

BACKGROUND: Ticks are among the most harmful vectors worldwide. Their salivary glands play essential roles in blood-feeding and pathogen transmission and undergo apoptosis after feeding. Although it was previously reported that salivary degeneration in ixodid ticks is in response to hormonal stimulation, questions still exist with the underlying mechanisms of salivary gland apoptosis. METHODS: Salivary glands of Rhipicephalus haemaphysaloides were collected from 1 to 7 days after attachment to the host. TUNEL and Annexin V assays were used to check apoptosis during this time. To confirm the role of caspase-1, RNA interference was used to silence its expression, and the dynamic changes of associated cysteine proteases were also shown by quantitative real time PCR and western blot, while TUNEL and Annexin V assays were used to confirm apoptosis. RESULTS: In the present study, apoptosis of salivary glands in R. haemaphysaloides occurred 3 or 4 days after attachment to the host as determined by TUNEL and Annexin V assays. The expression of caspase-1 increased at 5-7 days. When the latter was silenced by RNA interference, apoptosis in the salivary glands was delayed. While there seemed to be another form of cell death in salivary glands of ticks, such occurrence may be caused by compensatory autophagy which involved autophagy-related gene 4D. CONCLUSIONS: This study describes the apoptosis of salivary glands in R. haemaphysaloides and the dynamic changes in cysteine proteases in this activity. Cysteine proteases were involved in this process, especially caspase-1. Caspase-1 participated in the apoptosis of salivary glands.

In vivo efficacy of a biotherapic and eugenol formulation against Rhipicephalus microplus.

The control of Rhipicephalus microplus is essential to prevent cattle discomfort and economic losses. However, increased resistance and acaricides inefficiency lead producers to adopt strategies that could result in the accumulation of chemical residues in meat and milk with possibilities of poisoning in animals and people. This scenario demonstrates the necessity of research into the identification of novel, effective and environmentally safe therapeutic options for cattle tick control. The objectives of this study were to develop and assess the efficacy of R. microplus biotherapic and of 5% eugenol for the control of R. microplus in artificially infested calves. Eighteen male 6-month-old Holstein calves were divided into three groups of six animals. In Group 1, the animals did not receive medication (control group); in Group 2, the animals received 1 mL of R. microplus biotherapic at dilution 6CH (centesimal Hahnemannian), orally administered twice daily. And in Group 3, they received a single application of eugenol 5% in the pour-on formulation. The median efficacy for biotherapy and eugenol 5% was respectively 10.13 and 13.97%; however, upon analyzing reproductive efficiency, it is noteworthy that the biotherapic had 45.86% efficiency and was superior to the action of eugenol (12.03%) after 37 days of treatment. The ultrastructural study provided information about the effects of R. microplus biotherapic on the ovaries of engorged females and showed disorganization in the deposition of the oocyte exochorion. The results suggest hatchability inhibition of larvae, interference in R. microplus reproduction and future possibilities for eco-friendly control of R. microplus with biotherapic 6CH.

Differential sialotranscriptomes of unfed and fed Rhipicephalus haemaphysaloides, with particular regard to differentially expressed genes of cysteine proteases.

BACKGROUND: Rhipicephalus haemaphysaloides, a hard tick, is a common ectoparasite and can be found in many countries. It is recognized as the primary vector of bovine babesiosis in the south of China. During blood feeding, the tick's salivary glands secret numerous essential multifunctional proteins. In this study, a R. haemaphysaloides salivary gland transcriptome was described following the production and analysis of the transcripts from the two cDNA libraries of unfed and fed female ticks. The study focused on the differentially expressed genes and cysteine proteases, which play essential roles in the tick life cycle, that were detected most commonly in the up-regulation libraries. METHODS: The sialotranscriptome was assembled and analyzed though bioinformatic tools and the cysteine protease which is differentially expressed form sialotranscriptome were confirmed by Real-time PCR in salivary glands and different developments of ticks. RESULTS: On the basis of sequence similarities with other species in various databases, we analyzed the unfed and fed sialotranscriptome of R. haemaphysaloides to identify the differentially expressed proteins secreted from the salivary glands during blood feeding and to investigate their biological functions. There were 25,113 transcripts (35 % of the total assembled transcripts) that showed significant similarity to known proteins with high BLAST from other species annotated. In total, 88 % and 89 % of the sequencing reads could be mapped back to assembled sequences in the unfed and fed library, respectively. Comparison of the abundance of transcripts from similar contigs of the two salivary gland cDNA libraries allowed the identification of differentially expressed genes. In total, there were 1179 up-regulated genes and 574 down-regulated genes found by comparing the two libraries. Twenty-five predicted cysteine proteases were screened from the transcript databases, whereas only six protein molecules were confirmed by gene cloning and molecular expression in E.coli which all belonged to the cysteine protease family. Bioinformatic evolutionary analysis showed the relationship of cysteine proteases in ticks with those of other species, suggesting the origin and conservation of these genes. Analysis of sequences from different tick species indicated the further relationships among the proteases, suggesting the closely related function of these genes. Thus, we confirmed their changes in unfed, fed and engorged ticks and salivary glands. The dynamic changes revealed their important roles in the tick life cycle. CONCLUSIONS: Our survey provided an insight into the R. haemaphysaloides sialotranscriptome. The dynamic changes of cysteine proteases in ticks will assist further study of these proteases, which may contribute to the development of anti-tick vaccines or drugs, as well as improving understanding of the roles of cysteine proteases in the tick life cycle.

ATP Binding Cassette Transporter Mediates Both Heme and Pesticide Detoxification in Tick Midgut Cells.

In ticks, the digestion of blood occurs intracellularly and proteolytic digestion of hemoglobin takes place in a dedicated type of lysosome, the digest vesicle, followed by transfer of the heme moiety of hemoglobin to a specialized organelle that accumulates large heme aggregates, called hemosomes. In the present work, we studied the uptake of fluorescent metalloporphyrins, used as heme analogs, and amitraz, one of the most regularly used acaricides to control cattle tick infestations, by Rhipicephalus (Boophilus) microplus midgut cells. Both compounds were taken up by midgut cells in vitro and accumulated inside the hemosomes. Transport of both molecules was sensitive to cyclosporine A (CsA), a well-known inhibitor of ATP binding cassette (ABC) transporters. Rhodamine 123, a fluorescent probe that is also a recognized ABC substrate, was similarly directed to the hemosome in a CsA-sensitive manner. Using an antibody against conserved domain of PgP-1-type ABC transporter, we were able to immunolocalize PgP-1 in the digest vesicle membranes. Comparison between two R. microplus strains that were resistant and susceptible to amitraz revealed that the resistant strain detoxified both amitraz and Sn-Pp IX more efficiently than the susceptible strain, a process that was also sensitive to CsA. A transcript containing an ABC transporter signature exhibited 2.5-fold increased expression in the amitraz-resistant strain when compared with the susceptible strain. RNAi-induced down-regulation of this ABC transporter led to the accumulation of metalloporphyrin in the digestive vacuole, interrupting heme traffic to the hemosome. This evidence further confirms that this transcript codes for a heme transporter. This is the first report of heme transport in a blood-feeding organism. While the primary physiological function of the hemosome is to detoxify heme and attenuate its toxicity, we suggest that the use of this acaricide detoxification pathway by ticks may represent a new molecular mechanism of resistance to pesticides.

High efficacy of a 20 amino acid peptide of the acidic ribosomal protein P0 against the cattle tick, Rhipicephalus microplus.

Current strategies to control cattle ticks use integrated control programs (ICP) that include vaccination. Reduction in the use of chemicals and in the cost of tick control, the delay or elimination of acaricide resistance and the decreasing of environmental pollution are the advantages of using these programs. This integrated program is potentially applicable to all genotypes of chemical resistant ticks. However, the problem here is to improve the efficacy of anti-tick vaccines. The P0 protein is a structural component of the ribosome of all organisms. We have identified an immunogenic region of ribosomal protein P0 from Rhipicephalus spp. ticks that is not very conserved compared to the orthologous protein in their hosts. A synthetic 20 amino acid peptide from this sequence was effective as a vaccine against Rhipicephalus sanguineus infestations in an immunization and challenge experiment using rabbits. In this paper, the same peptide used as vaccine against the cattle tick Rhipicephalus Boophilus microplus shows a significant diminution in the number of engorged females recovered, in the weight of females and the weight of egg masses. The number of eggs hatched was also significantly reduced for the vaccinated group, with an overall effectivity for the antigen pP0 of 96%. These results, together with the conserved sequence of the P0 peptide among ticks, suggest that this antigen could be a good broad spectrum vaccine candidate. It would be expected to be active against many species of ticks and thus has promise in an ICP for effective control of ticks and thereby to improve the efficiency and productivity of the livestock industry.

Rhipicephalus microplus and Ixodes ovatus cystatins in tick blood digestion and evasion of host immune response.

BACKGROUND: Cystatins are a group of cysteine protease inhibitors responsible for physiological proteolysis regulation and present in a wide range of organisms. Studies about this class of inhibitors in parasites have contributed to clarify their roles in important physiological processes, like blood digestion and modulation of host immune response during blood feeding. Thus, cystatins are a subject of research on the development of new parasite control methods. Additionally, the characterization of proteins shared by different parasite species represents a valuable strategy to find potential targets in multi-species control methods. However, cystatin functions in ticks remain undetermined, especially in Rhipicephalus microplus and Ixodes ovatus, two species that affect livestock and human health, respectively. METHODS: Here we report the inhibitory profile of two R. microplus (BrBmcys2b and BrBmcys2c) and one I. ovatus (JpIocys2a) cystatins to commercial cathepsins B, C, and L. The presence of native cystatins in R. microplus tissues was analyzed using sera against recombinant BrBmcys2b and BrBmcys2c. Also, a peptide from JpIocys2a was synthesized for rabbit immunization, and this serum was used to analyze the cross antigenicity between R. microplus and I. ovatus cystatins. RESULTS: Enzymatic inhibition profile of tick cystatins shows a distinct modulation for cathepsins related to tick blood digestion and evasion of host immune response. Furthermore, BrBmcys2b was detected in saliva and different tissues along tick stages, while BrBmcys2c was detected mainly in gut from partially engorged R. microplus females, demonstrating a distinct pattern of cystatin expression, secretion and traffic between tick tissues. Moreover, phylogenetic analysis suggests that JpIocys2a belongs to the group of tick gut secreted cystatins. Finally, cross-antigenicity assays revealed that antibodies against the JpIocys2a peptide recognize native and recombinant R. microplus cystatins. CONCLUSION: The presence of these proteins in different tissues and their ability to differentially inhibit cathepsins suggest distinct roles for JpIocys2a, BrBmcys2b, and BrBmcys2c in blood digestion, egg and larvae development, and modulation of host immune response in tick physiology. The cross-antigenicity between native and recombinant cystatins supports further experiments using JpIocys2a, BrBmcys2b, and BrBmcys2c as vaccine antigens.

Role of Rhipicephalus microplus cheliceral receptors in gustation and host differentiation.

Rhipicephalus microplus is considered the most economically important ectoparasite of cattle worldwide. It is known that zebuine breeds of cattle are less susceptible to tick infestation than taurine breeds. Contact chemoreceptors in the cheliceral pit sensilla of ticks respond selectively to phagostimulant compounds, however their role in blood feeding relative to host susceptibility to infestation remains to be fully understood. We addressed this topic by conducting taste electrophysiology experiments with cheliceral pit sensilla preparations of R. microplus females. Solutions of five known ixodid tick phagostimulants were tested at different concentrations: sodium (NaCl), and potassium chloride (KCl) (10(-3)-10(-1)M); glucose (10(-4)-10(-1)M); adenosine triphosphate (ATP) (10(-6)-10(-2)M); and reduced l-glutathione (GSH) (10(-6)-10(-2)M). Serum samples from six susceptible animals of the Girolando breed (5/8 Bos indicus×3/8 B. taurus) and six resistant Nelore bovines (pure B. indicus) were also tested. A dose-dependent response of gustatory neurons associated with the chelicerae sensillum to NaCl, glucose, GSH, and ATP were observed. Responses by the cheliceral inner digit pit sensilla of R. microplus to KCl and glucose were also observed and they are reported here for the first time. In addition to an electrophysiological response to known phagostimulants, chemoreceptors in the chelicera of R. microplus responded differently to serum from cattle susceptible and resistant to infestation. The cheliceral pit neurons were more responsive to serum of R. microplus resistant bovines with a higher mean spike frequency (53.5±2spikess(-1)) than to serum samples from susceptible cattle (40.3±2spikess(-1)). The implications of chemosensation during tick blood feeding are discussed.

Rhipicephalus (Boophilus) microplus in the western-central region of Rio Grande do Sul, Brazil: multiresistant tick / Rhipicephalus (Boophilus) microplus na região centro-ocidental rio-grandense – Multirresistência acaricida

The aim of the present study was to assess the acaricide resistance of tick populations in the western-central region of Rio Grande do Sul (Brazil), which has not previously been reported. Fifty-four cattle farms were visited and specimens of Rhipicephalus (Boophilus) microplus were collected and subjected to the adult immersion test, using nine commercial acaricides in the amidine, pyrethroid and organophosphate groups. Climatic data, including monthly precipitation, were recorded. The results from the present study demonstrated that seven of the acaricides analyzed presented mean efficacy values of less than 95%, with large differences among the products tested. Nine of them exhibited satisfactory and unsatisfactory acaricide results on at least one farm. In conclusion, the farms located in the western-central region of Rio Grande do Sul, Brazil, exhibited populations of R. (Boophilus) microplus with variable degrees of susceptibility to different acaricides, thus suggesting that resistance to the active compounds exists. It is suggested that treatment protocols should be implemented at the beginning of winter and summer, using the acaricides that showed efficacy in the adult immersion test.

O objetivo deste estudo foi o de aferir a situação da resistência dos produtos acaricidas utilizados no controle de carrapatos de bovinos, na mesorregião centro-ocidental rio-grandense. Para isso, cinquenta e quatro propriedades foram visitadas, e as teleóginas de Rhipicephalus (Boophilus) microplus foram coletadas e submetidas ao teste de eficácia acaricida in vitro, sendo utilizados nove produtos comerciais, pertencentes às classes químicas das amidinas, piretroides e organofosforados. Além disso, os dados das temperaturas máximas e mínimas médias e da precipitação acumulada foram compilados no período estudado. Os resultados demonstraram que sete produtos analisados apresentaram eficácia inferior a 95%, com grande disparidade dos resultados. Nove produtos testados apresentaram satisfatória e não satisfatória eficácia em pelo menos uma propriedade analisada. Neste contexto, concluiu-se que as propriedades da mesorregião centro-ocidental rio-grandense apresentam cepas de R. (B.) microplus com graus variáveis de susceptibilidade a diferentes acaricidas, sugerindo, assim, que a resistência aos princípios ativos existe. Sugere-se a adoção de protocolos de tratamento acaricida no início do inverno e do verão, com a escolha do produto a ser utilizado baseado nos resultados do teste de eficácia acaricida.

Influence of tea tree oil (Melaleuca alternifolia) on the cattle tick Rhipicephalus microplus.

The aim of this study was to verify the influence of tea tree oil (TTO) (Melaleuca alternifolia) tested in its pure and nanostructured (TTO nanoparticles) forms on the reproduction of female Rhipicephalus microplus. For our purpose, female ticks were collected from naturally infected animals and treated in vitro with TTO (1, 5, and 10 %) and TTO nanoparticles (0.075, 0.375, and 0.75 %). In order to validate the tests, they were performed in triplicate using positive (amitraz) and negative (untreated) controls. It was possible to observe that pure TTO (5 and 10 %) and TTO nanoparticles (0.375 and 0.75 %) showed 100 % reproductive inhibition on female ticks. Additionally, pure TTO (1 %) also showed an acaricide effect (70 %), similarly to the positive control (78.3 %). This is the first study demonstrating the activity of pure TTO and TTO nanoparticles on female ticks. Therefore, based on these results, we were able to show that both forms and all concentrations of M. alternifolia affected tick reproduction by inhibiting egg laying and hatching. We were also able to show that TTO nanoparticles potentiated the inhibitor effect of pure TTO on the reproduction of R. microplus.

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.

Acaricide resistance mechanisms in Rhipicephalus (Boophilus) microplus / Mecanismos de resistência aos acaricidas em Rhipicephalus (Boophilus) microplus

Acaricide resistance has become widespread in countries where cattle ticks, Rhipicephalus (Boophilus) microplus, are a problem. Resistance arises through genetic changes in a cattle tick population that causes modifications to the target site, increased metabolism or sequestration of the acaricide, or reduced ability of the acaricide to penetrate through the outer protective layers of the tick’s body. We review the molecular and biochemical mechanisms of acaricide resistance that have been shown to be functional in R. (B.) microplus. From a mechanistic point of view, resistance to pyrethroids has been characterized to a greater degree than any other acaricide class. Although a great deal of research has gone into discovery of the mechanisms that cause organophosphate resistance, very little is defined at the molecular level and organophosphate resistance seems to be maintained through a complex and multifactorial process. The resistance mechanisms for other acaricides are less well understood. The target sites of fipronil and the macrocyclic lactones are known and resistance mechanism studies are in the early stages. The target site of amitraz has not been definitively identified and this is hampering mechanistic studies on this acaricide.

A resistência aos acaricidas tornou-se amplamente difundida nos países onde os carrapatos bovinos, Rhipicephalus .Boophilus. microplus, são um problema. A resistência surge por meio de alterações genéticas em umapopulação de carrapatos que causam modificações no local de ação, aumento do metabolismo ou sequestro do acaricida, ou ainda redução na capacidade do acaricida em penetrar através das camadas protetoras do corpo do carrapato. Neste artigo, foram revisados os mecanismos moleculares e bioquímicos da resistência aos acaricidas que ocorrem em R. (B.) microplus. A partir de um ponto de vista dos mecanismos envolvidos, a resistência aos piretróides tem sido caracterizada em maior grau do que em qualquer outra classe de acaricida. Embora uma grande quantidade de pesquisas têm sido direcionada para a descoberta de mecanismos que causam resistência aos organofosforados, muito pouco é conhecido ao nível molecular, e essa resistência parece ser mantida por intermédio de um processo multifatorial e complexo. Os mecanismos de resistência para os demais acaricidas são bem menos compreendidos. Os alvos de ação do fipronil e das lactonas macrocíclicas são conhecidos, e os estudos dos mecanismos de ação envolvidos estão ainda em estágios iniciais. O alvo de ação do amitraz ainda não foi definitivamente identificado, e isso é limitante aos estudos dos mecanismos envolvidos na resistência a esse acaricida.

Perceptions about the biology of Rhipicephalus (Boophilus) microplus among milk producers in Divinópolis, Minas Gerais / Percepção dos produtores de leite de Divinópolis, Minas Gerais, sobre a biologia do Rhipicephalus (Boophilus) microplus

One hundred semi-systematized interviews were applied with the aim of surveying the perceptions of milk producers in the municipality of Divinópolis, Minas Gerais regarding the biology of the tick Rhipicephalus (Boophilus) microplus. Content analysis was conducted on each of the variables surveyed and their descriptions, highlighting the higher frequencies in order to construct profiles of perceptions about each of the matters surveyed. In addition, each of the producers was categorized regarding their readiness to proceed with efficient control, from the assessment of their responses. Among the variables surveyed were the tick lifespan, duration of parasitic life, time of greatest incidence, survival in pastures and egg-laying volume. These questions are considered important for defining the knowledge needed for rational combat. It was concluded that the information needed for adopting effective practices to combat ticks was insufficient among the milk farm properties in Divinópolis. Moreover, the producers interviewed had a good perception of what they observed in their daily routine, but did not have complementary information about the tick life cycle.

Foram aplicadas 100 entrevistas semissistematizadas com o objetivo de levantar a percepção dos produtores de leite do município de Divinópolis, Minas Gerais, sobre a biologia do carrapato Rhipicephalus (Boophilus) microplus. Foi feita "análise de conteúdo" de cada uma das variáveis levantadas e sua descrição com destaque das maiores frequências para a construção de perfis de percepção sobre cada um dos aspectos levantados. Além disso, cada um dos produtores foram categorizados quanto a estar ou não apto a proceder um controle eficiente pela avaliação de suas respostas. Algumas das variáveis levantadas foram tempo de vida do carrapato, tempo de vida parasitária, época de maior incidência, sobrevivência nas pastagens e volume de postura, questões consideradas importantes para se definir o conhecimento necessário a um combate racional. Concluiu-se que as informações necessárias à adoção de práticas efetivas de combate aos carrapatos são insuficientes nas propriedades de leite de Divinópolis e, ainda, que os produtores entrevistados têm boa percepção no que se observa no dia a dia e não têm informações complementares sobre o ciclo de vida dos carrapatos.

Acaricidal activity of the essential oil from Tetradenia riparia (Lamiaceae) on the cattle tick Rhipicephalus (Boophilus) microplus (Acari; Ixodidae).

Tetradenia riparia (Lamiaceae) is a well-known herbal medicine with a variety of useful properties, including its acaricidal effect. This experiment was carried out to study the bioacaricidal activity of T. riparia essential oil (EO) against engorged females of Rhipicephalus (Boophilus) microplus (Acari; Ixodidae). For this purpose, nine serial concentrations (12.50%, 6.25%, 3.75%, 1.80%, 0.90%, 0.45%, 0.22%, 0.11%, and 0.056% w/v) of T. riparia were used for the adult immersion test (AIT). For the larval packet test (LPT), we used 14 serial concentrations (100.00%, 50.00%, 25.00%, 12.50%, 6.25%, 3.65%, 1.82%, 0.91%, 0.45%, 0.228%, 0.114%, 0.057%, 0.028%, and 0.014% w/v). The results for AIT showed 100.00% and 2.05% mortality, 19.00 and 90.20% for the total number of eggs, egg-laying inhibition of 0.00% and 90.20%, hatchability inhibition of 0.00% and 70.23%, and product effectiveness of 100.00% and 2.89%, respectively. The AIT indicated that the LC(50) and LC(99.9), calculated using the Probit test, were for mortality (%) 0.534g/mL (0.436-0.632) and 1.552g/mL (1.183-1.92); for total number of eggs were 0.449g/mL (0.339-0.558) and 1.76g/mL (1.27-2.248); and for hatchability inhibition were 0.114g/mL (0.0-0.31) and 2.462g/mL (1.501-3.422), respectively. Larvae between 14 and 21days old were fasted and placed in each envelope. Bioassays were performed at 27°±1°C, RH⩾80%. Larval mortality was observed 24h after treatment and showed 10.60-100% mortality in the LPT bioassay. The LPT showed that the LC(50) and LC(99.9) were 1.222g/mL (0.655-1.788) and 11.382g/mL (7.84-14.91), respectively. A positive correlation between T. riparia EO concentration and tick control, was observed by the strong acaricidal effects against R. (B.) microplus, and the mortality rate of ticks was dose-dependent. Our results showed that T. riparia is a promising candidate as an acaricide against resistant strains of R. (B.) microplus.

Differential expression of genes in resistant versus susceptible Gyr x Holstein cattle challenged with the tick Rhipicephalus (Boophilus) microplus.

The bovine tick Rhipicephalus (Boophilus) microplus causes major losses in cattle production systems in tropical regions. Bos indicus breeds are more resistant to ticks than B. taurus breeds. Resistance genes could be an alternative to control this parasite. We examined the pattern of gene expression of three calcium-binding-protein genes: translationally controlled tumor protein 1 (TPT1), allergen Bos d3 (S100A7), calcium channel protein transient receptor potential vanilloid 6 (TRPV6), and the cysteine proteinase inhibitor gene (CST6). These genes were selected from cDNA libraries prepared from skin biopsies taken from resistant and susceptible Gyr x Holstein F2 animals. These biopsies were also used to study the expression level of these genes through real-time PCR analysis. The relative expression levels of the S100A7, TPT1, TRPV6, and CST6 genes were 2.01 ± 0.6, 1.32 ± 0.9, 1.53 ± 1.2, and 2.03 ± 0.7 times higher in the susceptible group, respectively. Skin lesion tissue from the susceptible animals showed significantly more mRNA transcripts of these genes in comparison with the resistant animals (P = 0.001). However, this hypersensitivity does not seem to protect the susceptible animals against tick infestation.

Localization and function of Rhipicephalus (Boophilus) microplus vitellin-degrading cysteine endopeptidase.

The tick Rhipicephalus (Boophilus) microplus is an important parasite of cattle in many areas of the tropics. Characterization of molecules involved in mechanisms such as vitellogenesis and embryo development may contribute to a better understanding of this parasite's physiology. The vitellin-degrading cysteine endopeptidase (VTDCE) is the most active enzyme involved in vitellin hydrolysis in R. microplus eggs. Here we show an association between VTDCE and vitellin in an additional site, apart from the active site. Our data also demonstrate cysteine endopeptidase activity in different tissues such as ovary, gut, fat body, salivary gland and female haemolymph, where it is controlled by a physiological inhibitor. In R. microplus female gut, VTDCE is localized in areas of protein synthesis and trafficking with the underlying haemolymph. VTDCE is also localized in the ovary basal region, in vesicle membranes of ovary pedicel cells and in oocyte cytosol. These results suggest that VTDCE plays a role in vitellin digestion during tick development.

Ecdysis triggering hormone signaling in arthropods.

Ecdysis triggering hormones (ETHs) from endocrine Inka cells initiate the ecdysis sequence through action on central neurons expressing ETH receptors (ETHR) in model moth and dipteran species. We used various biochemical, molecular and BLAST search techniques to detect these signaling molecules in representatives of diverse arthropods. Using peptide isolation from tracheal extracts, cDNA cloning or homology searches, we identified ETHs in a variety of hemimetabolous and holometabolous insects. Most insects produce two related ETHs, but only a single active peptide was isolated from the cricket and one peptide is encoded by the eth gene of the honeybee, parasitic wasp and aphid. Immunohistochemical staining with antiserum to Manduca PETH revealed Inka cells on tracheal surface of diverse insects. In spite of conserved ETH sequences, comparison of natural and the ETH-induced ecdysis sequence in the honeybee and beetle revealed considerable species-specific differences in pre-ecdysis and ecdysis behaviors. DNA sequences coding for putative ETHR were deduced from available genomes of several hemimetabolous and holometabolous insects. In all insects examined, the ethr gene encodes two subtypes of the receptor (ETHR-A and ETHR-B). Phylogenetic analysis showed that these receptors fall into a family of closely related GPCRs. We report for the first time the presence of putative ETHs and ETHRs in genomes of other arthropods, including the tick (Arachnida) and water flea (Crustacea). The possible source of ETH in ticks was detected in paired cells located in all pedal segments. Our results provide further evidence of structural and functional conservation of ETH-ETHR signaling.

Comparison of efficiency and reproductive aptitude indexes between a reference and field strains of the cattle tick Rhipicephalus (Boophilus) microplus, in Sinaloa, Mexico / Comparação dos índices de eficiência e aptidão reprodutiva entre uma cepa de referência e outra de campo do carrapato do gado, Rhipicephalus (Boophilus) microplus, em Sinaloa, México

The objective of this work was to study the reproductive behavior of two strains of R. (B.) microplus, one wild-caught (Native) and one reference strain (Media Joya) for two years, measuring the reproductive efficiency index (REI) and reproductive aptitude index (RAI). Engorged ticks from each strain were collected monthly from February 2002 to February 2004. These were weighed and kept in the laboratory until egg-laying. Oogenic masses were individually weighed and incubated until larvae emergence. REI and RAI were calculated from each sample, grouping ticks by weight in 100 mg range classes. An analysis of variance and Duncan's multiple rank tests were carried out on the data (P < 0.05). Pearson's correlation coefficients were estimated (P < 0.01) in order to determine the relationship between engorgement weight and the weight and number of laid and hatched eggs. The Native strain showed a higher engorgement weight than Media Joya strain. In both strains REI and RAI were similar in all weight classes; with a quadratic tendency in Media Joya and linear-quadratic in the Native strain (P < 0.05). Significant differences where observed when REI and RAI of both strains were compared along the duration of the study (P < 0.01) with the Native strain performing better in both indexes, including better performance during the dry season. All parameters studied showed highly significant correlation; eggs laid and eggs hatched were notably highly correlated, 0.94 and 0.91, for Media Joya strain and Native strain respectively.

O objetivo deste trabalho foi estudar a conduta reprodutiva de duas cepas de R. (B.) microplus, uma coletada no campo (Nativa) e outra de referência (Média Jóia), por dois anos, calculando o índice de eficiência reprodutiva (REI) e o índice de aptidão reprodutiva (RAI). Foram coletados mensalmente carrapatos fêmeas engurgitadas de cada cepa no período de Fevereiro de 2002 a Fevereiro de 2004. Estas foram pesadas e conservadas no laboratório até ovipositar. As massas de ovos foram pesadas individualmente e incubadas até a eclosão das larvas. O REI e o RAI foram calculados para cada coleta, agrupando os carrapatos em classe de peso de 100 mg. Os dados foram submetidos a uma análise de variância e prova de classe múltipla de Duncan (P < 0,05). Foram calculados os coeficientes de correlação de Pearson (P < 0,01) para estimar a associação entre o peso dos carrapatos engurgitados e o peso e número de ovos postos e eclosionados. A cepa Nativa mostrou um peso ao engurgitamento mais alto do que a cepa Média Jóia. Os valores de REI e de RAI em ambas cepas foram similares em todos as classes de peso, mostrando tendência cuadrática na Média Jóia e linear-cuadrática na cepa Nativa (P < 0,05). Ambos os índices por classe de peso no engurgitamento foram sempre mais altos na cepa Nativa (P < 0,05). Os valores de REI e de RAI de ambas cepas ao longo do estudo mostraram diferenças (P < 0,01), sendo a cepa Nativa a de melhor desempenho em ambos índices, incluindo um melhor desenvolvimento durante a estação seca. Observou-se uma correlação altamente significativa em todos os parâmetros, sobressaindo o número de ovos postos com o número de ovos eclodidos, 0,94 e 0,91, para a cepa Média Jóia e a Nativa, respectivamente.