Systems biology of tissue-specific response to Anaplasma phagocytophilum reveals differentiated apoptosis in the tick vector Ixodes scapularis.

Anaplasma phagocytophilum is an emerging pathogen that causes human granulocytic anaplasmosis. Infection with this zoonotic pathogen affects cell function in both vertebrate host and the tick vector, Ixodes scapularis. Global tissue-specific response and apoptosis signaling pathways were characterized in I. scapularis nymphs and adult female midguts and salivary glands infected with A. phagocytophilum using a systems biology approach combining transcriptomics and proteomics. Apoptosis was selected for pathway-focused analysis due to its role in bacterial infection of tick cells. The results showed tissue-specific differences in tick response to infection and revealed differentiated regulation of apoptosis pathways. The impact of bacterial infection was more pronounced in tick nymphs and midguts than in salivary glands, probably reflecting bacterial developmental cycle. All apoptosis pathways described in other organisms were identified in I. scapularis, except for the absence of the Perforin ortholog. Functional characterization using RNA interference showed that Porin knockdown significantly increases tick colonization by A. phagocytophilum. Infection with A. phagocytophilum produced complex tissue-specific alterations in transcript and protein levels. In tick nymphs, the results suggested a possible effect of bacterial infection on the inhibition of tick immune response. In tick midguts, the results suggested that A. phagocytophilum infection inhibited cell apoptosis to facilitate and establish infection through up-regulation of the JAK/STAT pathway. Bacterial infection inhibited the intrinsic apoptosis pathway in tick salivary glands by down-regulating Porin expression that resulted in the inhibition of Cytochrome c release as the anti-apoptotic mechanism to facilitate bacterial infection. However, tick salivary glands may promote apoptosis to limit bacterial infection through induction of the extrinsic apoptosis pathway. These dynamic changes in response to A. phagocytophilum in I. scapularis tissue-specific transcriptome and proteome demonstrated the complexity of the tick response to infection and will contribute to characterize gene regulation in ticks.

Oral re-vaccination of Eurasian wild boar with Mycobacterium bovis BCG yields a strong protective response against challenge with a field strain.

BACKGROUND: Field vaccination trials with Mycobacterium bovis BCG, an attenuated mutant of M. bovis, are ongoing in Spain, where the Eurasian wild boar (Sus scrofa) is regarded as the main driver of animal tuberculosis (TB). The oral baiting strategy consists in deploying vaccine baits twice each summer, in order to gain access to a high proportion of wild boar piglets. The aim of this study was to assess the response of wild boar to re-vaccination with BCG and to subsequent challenge with an M. bovis field strain. RESULTS: BCG re-vaccinated wild boar showed reductions of 75.8% in lesion score and 66.9% in culture score, as compared to unvaccinated controls. Only one of nine vaccinated wild boar had a culture-confirmed lung infection, as compared to seven of eight controls. Serum antibody levels were highly variable and did not differ significantly between BCG re-vaccinated wild boar and controls. Gamma IFN levels differed significantly between BCG re-vaccinated wild boar and controls. The mRNA levels for IL-1b, C3 and MUT were significantly higher in vaccinated wild boar when compared to controls after vaccination and decreased after mycobacterial challenge. CONCLUSIONS: Oral re-vaccination of wild boar with BCG yields a strong protective response against challenge with a field strain. Moreover, re-vaccination of wild boar with BCG is not counterproductive. These findings are relevant given that re-vaccination is likely to happen under real (field) conditions.

Anaplasma phagocytophilum inhibits apoptosis and promotes cytoskeleton rearrangement for infection of tick cells.

Anaplasma phagocytophilum causes human granulocytic anaplasmosis. Infection with this zoonotic pathogen affects gene expression in both the vertebrate host and the tick vector, Ixodes scapularis. Here, we identified new genes, including spectrin alpha chain or alpha-fodrin (CG8) and voltage-dependent anion-selective channel or mitochondrial porin (T2), that are involved in A. phagocytophilum infection/multiplication and the tick cell response to infection. The pathogen downregulated the expression of CG8 in tick salivary glands and T2 in both the gut and salivary glands to inhibit apoptosis as a mechanism to subvert host cell defenses and increase infection. In the gut, the tick response to infection through CG8 upregulation was used by the pathogen to increase infection due to the cytoskeleton rearrangement that is required for pathogen infection. These results increase our understanding of the role of tick genes during A. phagocytophilum infection and multiplication and demonstrate that the pathogen uses similar strategies to establish infection in both vertebrate and invertebrate hosts.

Identification of microorganisms in partially fed female horn flies, Haematobia irritans.

The horn fly Haematobia irritans (Linnaeus, 1758) (Diptera: Muscidae) is one of the most important ectoparasites of cattle. The parasitism of horn flies interferes with cattle feeding, thus reducing weight gain and milk production. Additionally, horn flies are mechanical vectors of pathogens that cause disease in cattle. The aims of this study were to identify microorganisms in partially fed female horn flies through mining of expressed sequence tags (ESTs) and to characterize microorganism prevalence using real-time RT-PCR. Seven unigenes containing 24 ESTs were homologous to infectious agents. Microorganisms identified in partially fed female horn flies ESTs included Nora virus (3 unigenes; 8 ESTs), Wolbachia endosymbionts (3 unigenes; 3 ESTs), and Mycobacterium bovis (1 unigene; 13 ESTs). These results expanded the repertoire of microorganisms that could cause persistent infections or be mechanically transmitted by horn flies and support further studies on the role of horn flies in the epidemiology of these pathogens in Mexico.

Development of Peptide Targeted PLGA-PEGylated Nanoparticles Loading Licochalcone-A for Ocular Inflammation.

Licochalcone-A is a natural compound with anti-inflammatory properties. However, it possesses low water solubility, making its application for the treatment of ocular inflammation difficult. To overcome this drawback, biodegradable nanoparticles incorporating Licochalcone-A have been developed. Additionally, to avoid fast clearance and increase cellular internalization into the ocular tissues, PLGA nanoparticles have been functionalized using PEG and cell penetrating peptides (Tet-1 and B6). To optimize the formulations, a factorial design was carried out and short-term stability of the nanoparticles was studied. Moreover, morphology was also observed by transmission electron microcopy and in vitro drug release was carried out. Ocular tolerance of the formulations was ensured in vitro and in vivo and anti-inflammatory therapeutic efficacy was also assessed. Surface functionalized nanoparticles loading Licochalcone-A were developed with an average size below 200 nm, a positive surface charge, and a monodisperse population. The formulations were non-irritant and showed a prolonged Licochalcone-A release. Despite the fact that both Licochalcone-A Tet-1 and B6 functionalized nanoparticles demonstrated to be suitable for the treatment of ocular inflammation, B6 targeted nanoparticles provided greater therapeutic efficacy in in vivo assays.

Functional genomics of the horn fly, Haematobia irritans (Linnaeus, 1758).

BACKGROUND: The horn fly, Haematobia irritans (Linnaeus, 1758) (Diptera: Muscidae) is one of the most important ectoparasites of pastured cattle. Horn flies infestations reduce cattle weight gain and milk production. Additionally, horn flies are mechanical vectors of different pathogens that cause disease in cattle. The aim of this study was to conduct a functional genomics study in female horn flies using Expressed Sequence Tags (EST) analysis and RNA interference (RNAi). RESULTS: A cDNA library was made from whole abdominal tissues collected from partially fed adult female horn flies. High quality horn fly ESTs (2,160) were sequenced and assembled into 992 unigenes (178 contigs and 814 singlets) representing molecular functions such as serine proteases, cell metabolism, mitochondrial function, transcription and translation, transport, chromatin structure, vitellogenesis, cytoskeleton, DNA replication, cell response to stress and infection, cell proliferation and cell-cell interactions, intracellular trafficking and secretion, and development. Functional analyses were conducted using RNAi for the first time in horn flies. Gene knockdown by RNAi resulted in higher horn fly mortality (protease inhibitor functional group), reduced oviposition (vitellogenin, ferritin and vATPase groups) or both (immune response and 5'-NUC groups) when compared to controls. Silencing of ubiquitination ESTs did not affect horn fly mortality and oviposition while gene knockdown in the ferritin and vATPse functional groups reduced mortality when compared to controls. CONCLUSIONS: These results advanced the molecular characterization of this important ectoparasite and suggested candidate protective antigens for the development of vaccines for the control of horn fly infestations.

Subolesin expression in response to pathogen infection in ticks.

BACKGROUND: Ticks (Acari: Ixodidae) are vectors of pathogens worldwide that cause diseases in humans and animals. Ticks and pathogens have co-evolved molecular mechanisms that contribute to their mutual development and survival. Subolesin was discovered as a tick protective antigen and was subsequently shown to be similar in structure and function to akirins, an evolutionarily conserved group of proteins in insects and vertebrates that controls NF-kB-dependent and independent expression of innate immune response genes. The objective of this study was to investigate subolesin expression in several tick species infected with a variety of pathogens and to determine the effect of subolesin gene knockdown on pathogen infection. In the first experiment, subolesin expression was characterized in ticks experimentally infected with the cattle pathogen, Anaplasma marginale. Subolesin expression was then characterized in questing or feeding adult ticks confirmed to be infected with Anaplasma, Ehrlichia, Rickettsia, Babesia or Theileria spp. Finally, the effect of subolesin knockdown by RNA interference (RNAi) on tick infection was analyzed in Dermacentor variabilis males exposed to various pathogens by capillary feeding (CF). RESULTS: Subolesin expression increased with pathogen infection in the salivary glands but not in the guts of tick vector species infected with A. marginale. When analyzed in whole ticks, subolesin expression varied between tick species and in response to different pathogens. As reported previously, subolesin knockdown in D. variabilis infected with A. marginale and other tick-borne pathogens resulted in lower infection levels, while infection with Francisella tularensis increased in ticks after RNAi. When non-tick-borne pathogens were fed to ticks by CF, subolesin RNAi did not affect or resulted in lower infection levels in ticks. However, subolesin expression was upregulated in D. variabilis exposed to Escherichia coli, suggesting that although this pathogen may induce subolesin expression in ticks, silencing of this molecule reduced bacterial multiplication by a presently unknown mechanism. CONCLUSIONS: Subolesin expression in infected ticks suggested that subolesin may be functionally important for tick innate immunity to pathogens, as has been reported for the akirins. However, subolesin expression and consequently subolesin-mediated innate immunity varied with the pathogen and tick tissue. Subolesin may plays a role in tick innate immunity in the salivary glands by limiting pathogen infection levels, but activates innate immunity only for some pathogen in the guts and other tissues. In addition, these results provided additional support for the role of subolesin in other molecular pathways including those required for tissue development and function and for pathogen infection and multiplication in ticks. Consequently, RNAi experiments demonstrated that subolesin knockdown in ticks may affect pathogen infection directly by reducing tick innate immunity that results in higher infection levels and indirectly by affecting tissue structure and function and the expression of genes that interfere with pathogen infection and multiplication. The impact of the direct or indirect effects of subolesin knockdown on pathogen infection may depend on several factors including specific tick-pathogen molecular interactions, pathogen life cycle in the tick and unknown mechanisms affected by subolesin function in the control of global gene expression in ticks.

Characterization of Anaplasma phagocytophilum and A. ovis infection in a naturally infected sheep flock with poor health condition.

Anaplasma species are transmitted by ticks and cause diseases in humans and animals. These pathogens infect sheep, an economically important domestic animal worldwide. The current study was designed to characterize in 200 animals the infection with Anaplasma phagocytophilum and Anaplasma ovis and the genetic diversity of A. ovis strains collected from a naturally infected sheep flock with poor health condition. Sheep had 98% seroprevalence to Anaplasma spp. antibodies. PCR results confirmed the presence of A. phagocytophilum and A. ovis DNA in 11.5% and 37% of the sheep, respectively. Concurrent infections were detected in 6.5% of the sheep. Seventy-one adult ticks were collected from 45 sheep with infestations ranging from one to 15 ticks per animal. The analysis of A. ovis msp4 sequences demonstrated a previously unreported polymorphism for this pathogen with 17 different haplotypes in infected sheep. These results demonstrated that, although A. ovis msp4 haplotypes may be less variable when compared with Anaplasma marginale and A. phagocytophilum strains on a global scale, genetic polymorphisms occur in this locus in strains obtained from an infected sheep flock with poor health condition.

Current advances in the development of novel polymeric nanoparticles for the treatment of neurodegenerative diseases.

Effective intervention is essential to combat the coming epidemic of neurodegenerative (ND) diseases. Nanomedicine can overcome restrictions of CNS delivery imposed by the blood-brain barrier, and thus be instrumental in preclinical discovery and therapeutic intervention of ND diseases. Polymeric nanoparticles (PNPs) have shown great potential and versatility to encapsulate several compounds simultaneously in controlled drug-delivery systems and target them to the deepest brain regions. Here, we critically review recent advances in the development of drugs incorporated into PNPs and summarize the molecular changes and functional effects achieved in preclinical models of the most common ND disorders. We also briefly discuss the many challenges remaining to translate these findings and technological advances successfully to current clinical settings.

Metal-Based Nanoparticles as Antimicrobial Agents: An Overview.

Metal-based nanoparticles have been extensively investigated for a set of biomedical applications. According to the World Health Organization, in addition to their reduced size and selectivity for bacteria, metal-based nanoparticles have also proved to be effective against pathogens listed as a priority. Metal-based nanoparticles are known to have non-specific bacterial toxicity mechanisms (they do not bind to a specific receptor in the bacterial cell) which not only makes the development of resistance by bacteria difficult, but also broadens the spectrum of antibacterial activity. As a result, a large majority of metal-based nanoparticles efficacy studies performed so far have shown promising results in both Gram-positive and Gram-negative bacteria. The aim of this review has been a comprehensive discussion of the state of the art on the use of the most relevant types of metal nanoparticles employed as antimicrobial agents. A special emphasis to silver nanoparticles is given, while others (e.g., gold, zinc oxide, copper, and copper oxide nanoparticles) commonly used in antibiotherapy are also reviewed. The novelty of this review relies on the comparative discussion of the different types of metal nanoparticles, their production methods, physicochemical characterization, and pharmacokinetics together with the toxicological risk encountered with the use of different types of nanoparticles as antimicrobial agents. Their added-value in the development of alternative, more effective antibiotics against multi-resistant Gram-negative bacteria has been highlighted.

Expression of immunoregulatory genes in peripheral blood mononuclear cells of European wild boar immunized with BCG.

The objective of this study was to analyze the expression of immunoregulatory genes in European wild boar (Sus scrofa) immunized with BCG. Eighteen immunoregulatory genes were selected for expression analysis based on their role in host immune response during tuberculosis and/or for their association with resistance to bovine tuberculosis in European wild boar populations. Initially, mRNA levels were analyzed by quantitative real-time reverse transcription PCR (qRT-PCR) in spleen samples from Mycobacterium bovis-infected (N=18) and uninfected (N=22) European wild boar. Statistical analysis of qRT-PCR data revealed that four genes, complement component C3, IFN-gamma, IL-4 and RANTES were downregulated in infected animals (P<0.05). These genes were selected for analysis of mRNA levels in peripheral blood mononuclear cells (PBMCs) from seven wild boar experimentally immunized with BCG and seven non-immunized controls. Blood was collected at 0, 5, 13 and 25 weeks post-immunization (wpi). The mRNA levels of IFN-gamma and C3 showed a peak (>15-fold increase) at 5 wpi, whereas transcripts for RANTES and IL-4 showed a peak (>2-fold increase) at 13 wpi in BCG-immunized animals when compared to non-immunized controls. The pattern of expression of these genes over the time provides the first description of BCG specific immune response in European wild boar. These results provide new insights into the molecular basis of wild boar response to M. bovis infection and BCG vaccination and may be used to monitor BCG vaccination in this species.

Gene expression profiles of European wild boar naturally infected with Mycobacterium bovis.

Global gene expression profiles were analyzed in European wild boar naturally infected with Mycobacterium bovis. Spleen RNA was extracted from 23 M. bovis-infected and 17 uninfected animals and analyzed using a Pigoligoarray representing 20,400 genes. Differentially expressed sequences (N=161) were identified affecting cellular processes such as apoptosis, cell communication and signal transduction, cell growth and/or maintenance, cytoskeleton organization and biogenesis, DNA repair, immune response, metabolism and energy pathways, protein metabolism, regulation of cell proliferation, regulation of gene expression, regulation of nucleic acid metabolism, regulation of physiological processes, and transport. Real-time RT-PCR analysis of mRNA levels was used to corroborate microarray results of selected genes. Immune response genes were among the most represented differentially expressed sequences and were selected for further discussion. Beta-defensin 129, T-cell surface glycoprotein CD8 and B-cell receptor-associated protein 29 were overexpressed in infected animals. Lower expression levels of the immune response genes galectin-1, complement component C1qB and certain HLA class I and class II histocompatibility antigens and immunoglobulin chains were found in infected animals. This study identified new mechanisms by which naturally infected European wild boar respond to M. bovis infection and how the pathogen circumvents host immune responses to establish infection. Gene expression studies in naturally infected wildlife reservoirs of bovine tuberculosis are important for functional genomics and vaccine studies to aid in disease control in wildlife.

Differential expression of inflammatory and immune response genes in rams experimentally infected with a rough virulent strain of Brucella ovis.

Infection of sheep with Brucella ovis results in ovine brucellosis, a disease characterized by infertility in rams, abortion in ewes and increased perinatal mortality in lambs. During the course of the infection both the ovine immune response and host cell gene expression are modified. The objective of this research was to conduct a preliminary characterization of differential gene expression in rams experimentally infected with B. ovis by microarray hybridization and real-time RT-PCR. Of the 600 ruminant inflammatory and immune response genes that were analyzed in the microarray, 20 and 14 genes displayed an expression fold change >1.75 with a P-value <0.05 at 15 and 60 days post-challenge (dpc), respectively. Of these genes, 16 were upregulated and 4 were downregulated in infected rams at 15 dpc. At 60 dpc, 11 and 3 genes were up- and down-regulated in infected rams, respectively. Only four genes, desmoglein, epithelial sodium channel, alpha subunit (ENaC-alpha), interleukin 18 binding protein (IL18BP) and macrophage migration inhibition factor (MIF) were found upregulated in infected rams at both 15 and 60 dpc. The analysis of differentially expressed genes demonstrated activation of inflammatory and innate immune pathways in infected animals. B. ovis infection also resulted in upregulation of genes involved in phagocytosis and downregulation of protective host defense mechanisms, both of which may contribute to the chronicity of B. ovis infection. The gene expression profiles differed between rams with severe and moderate B. ovis infection. This is the first analysis of differential gene expression in rough brucellae and particularly in B. ovis-infected rams. The characterization of the genes and their expression profiles in response to B. ovis infection further contributes to our understanding of the molecular mechanisms of infection and the pathogenesis of brucellosis.

Ocular penetration of fluorometholone-loaded PEG-PLGA nanoparticles functionalized with cell-penetrating peptides.

Aim: Development of fluorometholone-loaded PEG-PLGA nanoparticles (NPs) functionalized with cell-penetrating peptides (CPPs) for the treatment of ocular inflammatory disorders. Materials & methods: Synthesized polymers and peptides were used for elaboration of functionalized NPs, which were characterized physicochemically. Cytotoxicity and ability to modulate the expression of proinflammatory cytokines were evaluated in vitro using human corneal epithelial cells (HCE-2). NPs uptake was assayed in both in vitro and in vivo models. Results: NPs showed physicochemical characteristics suitable for ocular administration without evidence of cytotoxicity. TAT-NPs and G2-NPs were internalized and displayed anti-inflammatory activity in both HCE-2 cells and mouse eye. Conclusion: TAT-NPs and G2-NPs could be considered a novel strategy for the treatment of ocular inflammatory diseases of the anterior and posterior segment.

Evidence of the role of tick subolesin in gene expression.

BACKGROUND: Subolesin is an evolutionary conserved protein that was discovered recently in Ixodes scapularis as a tick protective antigen and has a role in tick blood digestion, reproduction and development. In other organisms, subolesin orthologs may be involved in the control of developmental processes. Because of the profound effect of subolesin knockdown in ticks and other organisms, we hypothesized that subolesin plays a role in gene expression, and therefore affects multiple cellular processes. The objective of this study was to provide evidence for the role of subolesin in gene expression. RESULTS: Two subolesin-interacting proteins were identified and characterized by yeast two-hybrid screen, co-affinity purification and RNA interference (RNAi). The effect of subolesin knockdown on the tick gene expression pattern was characterized by microarray analysis and demonstrated that subolesin RNAi affects the expression of genes involved in multiple cellular pathways. The analysis of subolesin and interacting protein sequences identified regulatory motifs and predicted the presence of conserved protein kinase C (PKC) phosphorylation sites. CONCLUSION: Collectively, these results provide evidence that subolesin plays a role in gene expression in ticks.

Differential expression of inflammatory and immune response genes in sheep infected with Anaplasma phagocytophilum.

Anaplasma phagocytophilum infects a wide variety of host species and causes the diseases tick-borne fever (TBF) in ruminants and granulocytic anaplasmosis in humans, horses and dogs. TBF in sheep has become one of the more prevalent tick-borne diseases in some regions of Europe. A. phagocytophilum infection modifies host gene expression and immune response. The objective of this research was to characterize differential gene expression in sheep experimentally and naturally infected with A. phagocytophilum by microarray hybridization and real-time RT-PCR. The results of these studies demonstrated in sheep the activation of inflammatory and innate immune pathways and the impairment of adaptive immunity during A. phagocytophilum infection. The characterization of the genes and their expression profiles in sheep in response to A. phagocytophilum infection advances our understanding of the molecular mechanisms of pathogen infection and the pathogenesis of TBF. Collectively, these results expand current information on the mammalian host response to A. phagocytophilum infection.

Prospects for vaccination against the ticks of pets and the potential impact on pathogen transmission.

Diseases transmitted by arthropod vectors such as ticks greatly impact human and animal health. In particular, many diseases of dogs and cats are potentially transmissible to people by arthropod vectors and therefore their control is important for the eradication of vector-borne diseases (VBD). Vaccination is an environmentally friendly alternative for vector control that allows control of several VBD by targeting their common vector. Recent results have shown that it is possible to use vector protective antigens for the control of arthropod vector infestations and pathogen infection. However, as reviewed in this paper, very little progress has been made for the control of ectoparasite infestations and VBD in pets using vaccination with vector protective antigens. The growing interaction between pets and people underlines the importance of developing new interventions for the monitoring and control of VBD.

Nuclease Tudor-SN Is Involved in Tick dsRNA-Mediated RNA Interference and Feeding but Not in Defense against Flaviviral or Anaplasma phagocytophilum Rickettsial Infection.

Tudor staphylococcal nuclease (Tudor-SN) and Argonaute (Ago) are conserved components of the basic RNA interference (RNAi) machinery with a variety of functions including immune response and gene regulation. The RNAi machinery has been characterized in tick vectors of human and animal diseases but information is not available on the role of Tudor-SN in tick RNAi and other cellular processes. Our hypothesis is that tick Tudor-SN is part of the RNAi machinery and may be involved in innate immune response and other cellular processes. To address this hypothesis, Ixodes scapularis and I. ricinus ticks and/or cell lines were used to annotate and characterize the role of Tudor-SN in dsRNA-mediated RNAi, immune response to infection with the rickettsia Anaplasma phagocytophilum and the flaviviruses TBEV or LGTV and tick feeding. The results showed that Tudor-SN is conserved in ticks and involved in dsRNA-mediated RNAi and tick feeding but not in defense against infection with the examined viral and rickettsial pathogens. The effect of Tudor-SN gene knockdown on tick feeding could be due to down-regulation of genes that are required for protein processing and blood digestion through a mechanism that may involve selective degradation of dsRNAs enriched in G:U pairs that form as a result of adenosine-to-inosine RNA editing. These results demonstrated that Tudor-SN plays a role in tick RNAi pathway and feeding but no strong evidence for a role in innate immune responses to pathogen infection was found.

A systems biology approach to the characterization of stress response in Dermacentor reticulatus tick unfed larvae.

BACKGROUND: Dermacentor reticulatus (Fabricius, 1794) is distributed in Europe and Asia where it infests and transmits disease-causing pathogens to humans, pets and other domestic and wild animals. However, despite its role as a vector of emerging or re-emerging diseases, very little information is available on the genome, transcriptome and proteome of D. reticulatus. Tick larvae are the first developmental stage to infest hosts, acquire infection and transmit pathogens that are transovarially transmitted and are exposed to extremely stressing conditions. In this study, we used a systems biology approach to get an insight into the mechanisms active in D. reticulatus unfed larvae, with special emphasis on stress response. PRINCIPAL FINDINGS: The results support the use of paired end RNA sequencing and proteomics informed by transcriptomics (PIT) for the analysis of transcriptomics and proteomics data, particularly for organisms such as D. reticulatus with little sequence information available. The results showed that metabolic and cellular processes involved in protein synthesis were the most active in D. reticulatus unfed larvae, suggesting that ticks are very active during this life stage. The stress response was activated in D. reticulatus unfed larvae and a Rickettsia sp. similar to R. raoultii was identified in these ticks. SIGNIFICANCE: The activation of stress responses in D. reticulatus unfed larvae likely counteracts the negative effect of temperature and other stress conditions such as Rickettsia infection and favors tick adaptation to environmental conditions to increase tick survival. These results show mechanisms that have evolved in D. reticulatus ticks to survive under stress conditions and suggest that these mechanisms are conserved across hard tick species. Targeting some of these proteins by vaccination may increase tick susceptibility to natural stress conditions, which in turn reduce tick survival and reproduction, thus reducing tick populations and vector capacity for tick-borne pathogens.

Glutathione S-transferase affects permethrin detoxification in the brown dog tick, Rhipicephalus sanguineus.

Control of ticks on dogs is often done by application of repellents that contain permethrin as the active ingredient. In this research, we studied the role of a glutathione S-transferase (GST) gene in detoxification of permethrin by ticks using a gene silencing method RNA interference (RNAi). The brown dog tick, Rhipicephalus sanguineus, used in these studies, has a notable host preference for dogs, but also infests other mammals. In this research, R. sanguineus females were injected with gst double-stranded RNA (dsRNA) to effect gene silencing by RNAi and then exposed to sublethal doses of permethrin. Sixty hours after injection, the females were allowed to feed on sheep. The female ticks subjected to RNAi proved to be more susceptible to permethrin than the untreated controls. The effect of gene silencing was most notable in the highest dose group (50.3 ppm) in which all ticks died, while in the corresponding controls that were not subjected to RNAi this dose was not lethal. The acaricide treatment of the ticks resulted in a change in tick attachment behavior. Acaricide-treated ticks attached in a scattered pattern in contrast to the control ticks that attached and fed tightly clustered together. The time required for repletion for both the injected and non-injected females exposed to the higher permethrin level was shorter than that observed in the lower-dose groups and unexposed controls, and this more rapid attachment and feeding would likely favor more rapid transmission of pathogens. However, engorgement and egg mass weights were not significantly different among the experimental groups. This research demonstrated that the silencing of the gst gene increased the tick's susceptibility to permethrin. Overall, these results have contributed to our understanding of the detoxification mechanism of ticks and provide new considerations for the formulation of treatment strategies.