The anal pore route is efficient to infect Amblyomma spp. ticks with Rickettsia rickettsii and allows the assessment of the role played by infection control targets.

Adult Amblyomma sculptum and Amblyomma aureolatum ticks are partially refractory to Rickettsia rickettsii when fed on infected hosts, hindering the functional characterization of potentially protective targets in the bacterial acquisition. In the current study, we used the anal pore route to infect adult A. sculptum and A. aureolatum ticks with R. rickettsii and to assess the effects of the knockdown of microplusin in infection control. The anal pore route was efficient to infect both species, resulting in a prevalence of around 100% of infected ticks. Higher loads of R. rickettsii were detected in microplusin-silenced A. aureolatum in relation to the control, as previously obtained when microplusin-silenced ticks were fed on R. rickettsii-infected rabbits. This is the first report showing R. rickettsii infection through the anal pore in Amblyomma ticks, highlighting this route as a powerful tool to assess the role played by additional targets in the control of pathogens.

The survival of Amblyomma sculptum ticks upon blood-feeding depends on the expression of an inhibitor of apoptosis protein.

BACKGROUND: The tick Amblyomma sculptum is the major vector of Rickettsia rickettsii, the causative agent of the highly lethal Brazilian spotted fever. It has been shown that R. rickettsii inhibits apoptosis in both human endothelial cells and tick cells. Apoptosis is regulated by different factors, among which inhibitors of apoptosis proteins (IAPs) play a central role. In the study reported here, we selected an IAP of A. sculptum that has not yet been characterized to assess its role in cell death and to determine the effects of its gene silencing on tick fitness and R. rickettsii infection. METHODS: An A. sculptum cell line (IBU/ASE-16) was treated with specific double-stranded RNA (dsRNA) for either IAP (dsIAP) or green fluorescent protein (dsGFP; as a control). The activity of caspase-3 and the exposure of phosphatidylserine were determined in both groups. In addition, unfed adult ticks, infected or not infected with R. rickettsii, were treated with either dsIAP or dsGFP and allowed to feed on noninfected rabbits. In parallel, noninfected ticks were allowed to feed on an R. rickettsii-infected rabbit. Ticks (infected or not with R. rickettsii) that remained unfed were used as a control. RESULTS: Caspase-3 activity and the externalization of phosphatidylserine were significantly higher in IBU/ASE-16 cells treated with dsIAP than in those treated with dsGFP. The mortality rates of ticks in the dsIAP group were much higher than those in the dsGFP group when they were allowed to feed on rabbits, independent of the presence of R. rickettsii. Conversely, lower mortality rates were recorded in unfed ticks. CONCLUSIONS: Our results show that IAP negatively regulates apoptosis in A. sculptum cells. Moreover, IAP-silenced ticks experienced higher mortality rates following the acquisition of a blood meal, suggesting that feeding may trigger the activation of apoptosis in the absence of this physiological regulator. These findings indicate that IAP is a potential antigen for an anti-tick vaccine.

Tick Immune System: What Is Known, the Interconnections, the Gaps, and the Challenges.

Ticks are ectoparasitic arthropods that necessarily feed on the blood of their vertebrate hosts. The success of blood acquisition depends on the pharmacological properties of tick saliva, which is injected into the host during tick feeding. Saliva is also used as a vehicle by several types of pathogens to be transmitted to the host, making ticks versatile vectors of several diseases for humans and other animals. When a tick feeds on an infected host, the pathogen reaches the gut of the tick and must migrate to its salivary glands via hemolymph to be successfully transmitted to a subsequent host during the next stage of feeding. In addition, some pathogens can colonize the ovaries of the tick and be transovarially transmitted to progeny. The tick immune system, as well as the immune system of other invertebrates, is more rudimentary than the immune system of vertebrates, presenting only innate immune responses. Although simpler, the large number of tick species evidences the efficiency of their immune system. The factors of their immune system act in each tick organ that interacts with pathogens; therefore, these factors are potential targets for the development of new strategies for the control of ticks and tick-borne diseases. The objective of this review is to present the prevailing knowledge on the tick immune system and to discuss the challenges of studying tick immunity, especially regarding the gaps and interconnections. To this end, we use a comparative approach of the tick immune system with the immune system of other invertebrates, focusing on various components of humoral and cellular immunity, such as signaling pathways, antimicrobial peptides, redox metabolism, complement-like molecules and regulated cell death. In addition, the role of tick microbiota in vector competence is also discussed.

A physiologic overview of the organ-specific transcriptome of the cattle tick Rhipicephalus microplus.

To further obtain insights into the Rhipicephalus microplus transcriptome, we used RNA-seq to carry out a study of expression in (i) embryos; (ii) ovaries from partially and fully engorged females; (iii) salivary glands from partially engorged females; (iv) fat body from partially and fully engorged females; and (v) digestive cells from partially, and (vi) fully engorged females. We obtained > 500 million Illumina reads which were assembled de novo, producing > 190,000 contigs, identifying 18,857 coding sequences (CDS). Reads from each library were mapped back into the assembled transcriptome giving a view of gene expression in different tissues. Transcriptomic expression and pathway analysis showed that several genes related in blood digestion and host-parasite interaction were overexpressed in digestive cells compared with other tissues. Furthermore, essential genes for the cell development and embryogenesis were overexpressed in ovaries. Taken altogether, these data offer novel insights into the physiology of production and role of saliva, blood digestion, energy metabolism, and development with submission of 10,932 novel tissue/cell specific CDS to the NCBI database for this important tick species.

Comparative Analysis of Infection by Rickettsia rickettsii Sheila Smith and Taiaçu Strains in a Murine Model.

Rocky Mountain spotted fever (RMSF) is a life-threatening tick-borne disease caused by Rickettsia rickettsii, which is widely distributed throughout the Americas. Over 4000 cases of RMSF are recorded annually in the United States, while only around 100 cases are reported in Brazil. Conversely, while case fatality rates in the United States oscillate around 5%, in Brazil they can surpass 70%, suggesting that differences in tick vectoring capacity, population sensitivity, and/or variability in virulence of the rickettsial strains may exist. In this study, we compared the susceptibility of C3H/HeN mice to two highly virulent strains of R. rickettsii, one from the United States (Sheila Smith) and the other from Brazil (Taiaçu). Animals inoculated with the Taiaçu strain succumbed to infection earlier and exhibited severe histological lesions in both liver and spleen sooner than mice infected with the Sheila Smith strain. These differences in survival and signs of the disease are not related to a greater proliferation of the Taiaçu strain, as there were no significant differences in the rickettsial load in mice tissues inoculated with either strain. The present study is the first step to experimentally assess differences in fatality rates of RMSF in two different regions of the American continent.

Comparative analysis of the midgut microbiota of two natural tick vectors of Rickettsia rickettsii.

Although the ticks Amblyomma sculptum and Amblyomma aureolatum are important vectors of Rickettsia rickettsii, causative agent of the life-threatening Rocky Mountain spotted fever, A. aureolatum is considerably more susceptible to infection than A. sculptum. As the microbiota can interfere with the colonization of arthropod midgut (MG) by pathogens, in the current study we analyzed the MG microbiota of both tick species. Our results revealed that the MG of A. aureolatum harbors a prominent microbiota, while A. sculptum does not. Remarkably, a significant reduction of the bacterial load was recorded in R. rickettsii-infected A. aureolatum. In addition, the taxonomy analysis of the MG bacterial community of A. aureolatum revealed a dominance of the genus Francisella, suggesting an endosymbiosis. This study is the first step in getting insights into the mechanisms underlying the interactions among Amblyomma species, their microbiota and R. rickettsii. Additional studies to better understand these mechanisms are required and may help the development of novel alternatives to block rickettsial transmission.

The Transcriptome of the Salivary Glands of Amblyomma aureolatum Reveals the Antimicrobial Peptide Microplusin as an Important Factor for the Tick Protection Against Rickettsia rickettsii Infection.

The salivary glands (SG) of ixodid ticks play a pivotal role in blood feeding, producing both the cement and the saliva. The cement is an adhesive substance that helps the attachment of the tick to the host skin, while the saliva contains a rich mixture of antihemostatic, anti-inflammatory, and immunomodulatory substances that allow ticks to properly acquire the blood meal. The tick saliva is also a vehicle used by several pathogens to be transmitted to the vertebrate host, including various bacterial species from the genus Rickettsia. Rickettsia rickettsii is a tick-borne obligate intracellular bacterium that causes the severe Rocky Mountain spotted fever. In Brazil, the dog yellow tick Amblyomma aureolatum is a vector of R. rickettsii. In the current study, the effects of an experimental infection with R. rickettsii on the global gene expression profile of A. aureolatum SG was determined by next-generation RNA sequencing. A total of 260 coding sequences (CDSs) were modulated by infection, among which 161 were upregulated and 99 were downregulated. Regarding CDSs in the immunity category, we highlight one sequence encoding one microplusin-like antimicrobial peptide (AMP) (Ambaur-69859). AMPs are important effectors of the arthropod immune system, which lack the adaptive response of the immune system of vertebrates. The expression of microplusin was confirmed to be significantly upregulated in the SG as well as in the midgut (MG) of infected A. aureolatum by a quantitative polymerase chain reaction preceded by reverse transcription. The knockdown of the microplusin expression by RNA interference caused a significant increase in the prevalence of infected ticks in relation to the control. In addition, a higher rickettsial load of one order of magnitude was recorded in both the MG and SG of ticks that received microplusin-specific dsRNA. No effect of microplusin knockdown was observed on the R. rickettsii transmission to rabbits. Moreover, no significant differences in tick engorgement and oviposition were recorded in ticks that received dsMicroplusin, demonstrating that microplusin knockdown has no effect on tick fitness. Further studies must be performed to determine the mechanism of action of this AMP against R. rickettsii.

Analysis of the Salivary Gland Transcriptome of Unfed and Partially Fed Amblyomma sculptum Ticks and Descriptive Proteome of the Saliva.

Ticks are obligate blood feeding ectoparasites that transmit a wide variety of pathogenic microorganisms to their vertebrate hosts. Amblyomma sculptum is vector of Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever (RMSF), the most lethal rickettsiosis that affects humans. It is known that the transmission of pathogens by ticks is mainly associated with the physiology of the feeding process. Pathogens that are acquired with the blood meal must first colonize the tick gut and later the salivary glands (SG) in order to be transmitted during a subsequent blood feeding via saliva. Tick saliva contains a complex mixture of bioactive molecules with anticlotting, antiplatelet aggregation, vasodilatory, anti-inflammatory, and immunomodulatory properties to counteract both the hemostasis and defense mechanisms of the host. Besides facilitating tick feeding, the properties of saliva may also benefits survival and establishment of pathogens in the host. In the current study, we compared the sialotranscriptome of unfed A. sculptum ticks and those fed for 72 h on rabbits using next generation RNA sequencing (RNA-seq). The total of reads obtained were assembled in 9,560 coding sequences (CDSs) distributed in different functional classes. CDSs encoding secreted proteins, including lipocalins, mucins, protease inhibitors, glycine-rich proteins, metalloproteases, 8.9 kDa superfamily members, and immunity-related proteins were mostly upregulated by blood feeding. Selected CDSs were analyzed by real-time quantitative polymerase chain reaction preceded by reverse transcription (RT-qPCR), corroborating the transcriptional profile obtained by RNA-seq. Finally, high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis revealed 124 proteins in saliva of ticks fed for 96-120 h. The corresponding CDSs of 59 of these proteins were upregulated in SG of fed ticks. To the best of our knowledge, this is the first report on the proteome of A. sculptum saliva. The functional characterization of the identified proteins might reveal potential targets to develop vaccines for tick control and/or blocking of R. rickettsii transmission as well as pharmacological bioproducts with antihemostatic, anti-inflammatory and antibacterial activities.

The Distinct Transcriptional Response of the Midgut of Amblyomma sculptum and Amblyomma aureolatum Ticks to Rickettsia rickettsii Correlates to Their Differences in Susceptibility to Infection.

Rickettsia rickettsii is a tick-borne obligate intracellular bacterium that causes Rocky Mountain Spotted Fever (RMSF). In Brazil, two species of ticks in the genus Amblyomma, A. sculptum and A. aureolatum, are incriminated as vectors of this bacterium. Importantly, these two species present remarkable differences in susceptibility to R. rickettsii infection, where A. aureolatum is more susceptible than A. sculptum. In the current study, A. aureolatum and A. sculptum ticks were fed on suitable hosts previously inoculated with R. rickettsii, mimicking a natural infection. As control, ticks were fed on non-infected animals. Both midgut and salivary glands of all positively infected ticks were colonized by R. rickettsii. We did not observe ticks with infection restricted to midgut, suggesting that important factors for controlling rickettsial colonization were produced in this organ. In order to identify such factors, the total RNA extracted from the midgut (MG) was submitted to next generation RNA sequencing (RNA-seq). The majority of the coding sequences (CDSs) of A. sculptum differentially expressed by infection were upregulated, whereas most of modulated CDSs of A. aureolatum were downregulated. The functional categories that comprise upregulated CDSs of A. sculptum, for instance, metabolism, signal transduction, protein modification, extracellular matrix, and immunity also include CDSs of A. aureolatum that were downregulated by infection. This is the first study that reports the effects of an experimental infection with the highly virulent R. rickettsii on the gene expression of two natural tick vectors. The distinct transcriptional profiles of MG of A. sculptum and A. aureolatum upon infection stimulus strongly suggest that molecular factors in this organ are responsible for delineating the susceptibility to R. rickettsii. Functional studies to determine the role played by proteins encoded by differentially expressed CDSs in the acquisition of R. rickettsii are warranted and may be considered as targets for the development of strategies to control the tick-borne pathogens as well as to control the tick vectors.

The transcription factor Relish controls Anaplasma marginale infection in the bovine tick Rhipicephalus microplus.

Rhipicephalus microplus is an important biological vector of Anaplasma marginale, the etiological agent of bovine anaplasmosis. The knowledge of tick immune responses to control bacterial infections remains limited. In this study, we demonstrate that transcription factor Relish from the IMD signaling pathway has an important role in the control of A. marginale infection in ticks. We found that RNA-mediated silencing of Relish caused a significant increase in the number of A. marginale in the midgut and salivary glands of R. microplus. In addition, the IMD pathway regulates the expression of the gene that encodes the antimicrobial peptide (AMP) microplusin. Moreover, microplusin expression was up-regulated in the midgut (2×) and salivary glands (8×) of A. marginale infected R. microplus. Therefore, it is plausible to hypothesize that microplusin may be involved in the A. marginale control. This study provides the first evidence of IMD signaling pathway participation on the A. marginale control in R. microplus.

Virulence genes of Rickettsia rickettsii are differentially modulated by either temperature upshift or blood-feeding in tick midgut and salivary glands.

BACKGROUND: Rickettsia rickettsii, the etiological agent of Rocky Mountain spotted fever, is transmitted to humans by ticks. During tick feeding, R. rickettsii is exposed to both temperature elevation and components of the blood meal, which have previously been associated with the reactivation of its virulence. These environmental stimuli were also reported to modulate virulence genes of R. rickettsii infecting a set of organs of adult females of its natural vector, Amblyomma aureolatum. METHODS: In this study, we determined the effects of a temperature upshift, blood-feeding, and both stimuli simultaneously on the expression of 85 selected genes of R. rickettsii infecting either the midgut (MG) or salivary glands (SG) of male and female A. aureolatum by microfluidic high-throughput RT-qPCR. These two organs are key for acquisition of this bacterium by the tick and transmission to the vertebrate host, respectively. RESULTS: Data showed that these environmental stimuli exert distinct effects on rickettsial transcription depending on the colonized organ and gender of the vector. Temperature upshift induced the majority of differentially expressed genes of R. rickettsii in tick SG, including tRNA synthetases encoding genes. On the contrary, blood-feeding downregulated most of differentially expressed genes in both organs, but induced type IV secretion system components and OmpB in tick MG. The combined effects of both stimuli resulted in a merged gene expression profile representing features of each stimulus analyzed independently, but was more similar to the profile induced by blood-feeding. CONCLUSION: The upregulation of the majority of differentially expressed genes in tick SG by temperature upshift suggests that this stimulus is important to prepare R. rickettsii for transmission to the vertebrate host. Blood-feeding, on the other hand, induced important virulence genes in the tick MG, which might be associated with colonization of the tick and transmission to the vertebrate host. The role of the proteins identified in this study must be addressed and might help to define future targets to block tick infection, thereby preventing RMSF. To our knowledge, this is the first transcriptional tissue-specific study of a virulent strain of R. rickettsii infecting a natural tick vector.

Exploring the immune signalling pathway-related genes of the cattle tick Rhipicephalus microplus: From molecular characterization to transcriptional profile upon microbial challenge.

In dipteran insects, invading pathogens are selectively recognized by four major pathways, namely Toll, IMD, JNK, and JAK/STAT, and trigger the activation of several immune effectors. Although substantial advances have been made in understanding the immunity of model insects such as Drosophila melanogaster, knowledge on the activation of immune responses in other arthropods such as ticks remains limited. Herein, we have deepened our understanding of the intracellular signalling pathways likely to be involved in tick immunity by combining a large-scale in silico approach with high-throughput gene expression analysis. Data from in silico analysis revealed that although both the Toll and JAK/STAT signalling pathways are evolutionarily conserved across arthropods, ticks lack central components of the D. melanogaster IMD pathway. Moreover, we show that tick immune signalling-associated genes are constitutively transcribed in BME26 cells (a cell lineage derived from embryos of the cattle tick Rhipicephalus microplus) and exhibit different transcriptional patterns in response to microbial challenge. Interestingly, Anaplasma marginale, a pathogen that is naturally transmitted by R. microplus, causes downregulation of immune-related genes, suggesting that this pathogen may manipulate the tick immune system, favouring its survival and vector colonization.

Culex quinquefasciatus storage proteins.

Insect storage proteins accumulate at high levels during larval development of holometabolous insects. During metamorphosis they are degraded, supplying energy and amino acids for the completion of adult development. The genome of Culex quinquefasciatus contains eleven storage protein-coding genes. Their transcripts are more abundant in larvae than in pupae and in adults. In fact, only four of these genes are transcribed in adults, two of which in blood-fed adult females but not in adult males. Transcripts corresponding to all Cx. quinquefasciatus storage proteins were detected by RT-PCR, while mass spectrometric analysis of larval and pupal proteins identified all storage proteins with the exception of one encoded by Cq LSP1.8. Our results indicate that the identified Cx. quinquefasciatus storage protein-coding genes are candidates for identifying regulatory sequences for the development of molecular tools for vector control.

Natural blood feeding and temperature shift modulate the global transcriptional profile of Rickettsia rickettsii infecting its tick vector.

Rickettsia rickettsii is an obligate intracellular tick-borne bacterium that causes Rocky Mountain Spotted Fever (RMSF), the most lethal spotted fever rickettsiosis. When an infected starving tick begins blood feeding from a vertebrate host, R. rickettsii is exposed to a temperature elevation and to components in the blood meal. These two environmental stimuli have been previously associated with the reactivation of rickettsial virulence in ticks, but the factors responsible for this phenotype conversion have not been completely elucidated. Using customized oligonucleotide microarrays and high-throughput microfluidic qRT-PCR, we analyzed the effects of a 10°C temperature elevation and of a blood meal on the transcriptional profile of R. rickettsii infecting the tick Amblyomma aureolatum. This is the first study of the transcriptome of a bacterium in the genus Rickettsia infecting a natural tick vector. Although both stimuli significantly increased bacterial load, blood feeding had a greater effect, modulating five-fold more genes than the temperature upshift. Certain components of the Type IV Secretion System (T4SS) were up-regulated by blood feeding. This suggests that this important bacterial transport system may be utilized to secrete effectors during the tick vector's blood meal. Blood feeding also up-regulated the expression of antioxidant enzymes, which might correspond to an attempt by R. rickettsii to protect itself against the deleterious effects of free radicals produced by fed ticks. The modulated genes identified in this study, including those encoding hypothetical proteins, require further functional analysis and may have potential as future targets for vaccine development.

Characterization of proteinases from the midgut of Rhipicephalus (Boophilus) microplus involved in the generation of antimicrobial peptides.

BACKGROUND: Hemoglobin is a rich source of biologically active peptides, some of which are potent antimicrobials (hemocidins). A few hemocidins have been purified from the midgut contents of ticks. Nonetheless, how antimicrobials are generated in the tick midgut and their role in immunity is still poorly understood. Here we report, for the first time, the contribution of two midgut proteinases to the generation of hemocidins. RESULTS: An aspartic proteinase, designated BmAP, was isolated from the midgut of Rhipicephalus (Boophilus) microplus using three chromatographic steps. Reverse transcription-quantitative polymerase chain reaction revealed that BmAP is restricted to the midgut. The other enzyme is a previously characterized midgut cathepsin L-like cysteine proteinase designated BmCL1. Substrate specificities of native BmAP and recombinant BmCL1 were mapped using a synthetic combinatorial peptide library and bovine hemoglobin. BmCL1 preferred substrates containing non-polar residues at P2 subsite and polar residues at P1, whereas BmAP hydrolysed substrates containing non-polar amino acids at P1 and P1'. CONCLUSIONS: BmAP and BmCL1 generate hemocidins from hemoglobin alpha and beta chains in vitro. We postulate that hemocidins may be important for the control of tick pathogens and midgut flora.

Effects of the antimicrobial peptide gomesin on the global gene expression profile, virulence and biofilm formation of Xylella fastidiosa.

In the xylem vessels of susceptible hosts, such as citrus trees, Xylella fastidiosa forms biofilm-like colonies that can block water transport, which appears to correlate to disease symptoms. Besides aiding host colonization, bacterial biofilms play an important role in resistance against antimicrobial agents, for instance antimicrobial peptides (AMPs). Here, we show that gomesin, a potent AMP from a tarantula spider, modulates X. fastidiosa gene expression profile upon 60 min of treatment with a sublethal concentration. DNA microarray hybridizations revealed that among the upregulated coding sequences, some are related to biofilm production. In addition, we show that the biofilm formed by gomesin-treated bacteria is thicker than that formed by nontreated cells or cells exposed to streptomycin. We have also observed that the treatment of X. fastidiosa with a sublethal concentration of gomesin before inoculation in tobacco plants correlates with a reduction in foliar symptoms, an effect possibly due to the trapping of bacterial cells to fewer xylem vessels, given the enhancement in biofilm production. These results warrant further investigation of how X. fastidiosa would respond to the AMPs produced by citrus endophytes and by the insect vector, leading to a better understanding of the mechanism of action of these molecules on bacterial virulence.

The iron stimulon of Xylella fastidiosa includes genes for type IV pilus and colicin V-like bacteriocins.

Xylella fastidiosa is the etiologic agent of a wide range of plant diseases, including citrus variegated chlorosis (CVC), a major threat to citrus industry. The genomes of several strains of this phytopathogen were completely sequenced, enabling large-scale functional studies. DNA microarrays representing 2,608 (91.6%) coding sequences (CDS) of X. fastidiosa CVC strain 9a5c were used to investigate transcript levels during growth with different iron availabilities. When treated with the iron chelator 2,2'-dipyridyl, 193 CDS were considered up-regulated and 216 were considered down-regulated. Upon incubation with 100 microM ferric pyrophosphate, 218 and 256 CDS were considered up- and down-regulated, respectively. Differential expression for a subset of 44 CDS was further evaluated by reverse transcription-quantitative PCR. Several CDS involved with regulatory functions, pathogenicity, and cell structure were modulated under both conditions assayed, suggesting that major changes in cell architecture and metabolism occur when X. fastidiosa cells are exposed to extreme variations in iron concentration. Interestingly, the modulated CDS include those related to colicin V-like bacteriocin synthesis and secretion and to functions of pili/fimbriae. We also investigated the contribution of the ferric uptake regulator Fur to the iron stimulon of X. fastidiosa. The promoter regions of the strain 9a5c genome were screened for putative Fur boxes, and candidates were analyzed by electrophoretic mobility shift assays. Taken together, our data support the hypothesis that Fur is not solely responsible for the modulation of the iron stimulon of X. fastidiosa, and they present novel evidence for iron regulation of pathogenicity determinants.

Ixodidin, a novel antimicrobial peptide from the hemocytes of the cattle tick Boophilus microplus with inhibitory activity against serine proteinases.

The presence of an effective immune response in the hemocoel of arthropods is essential for survival as it prevents the invasion of pathogens throughout the animal body. Antimicrobial peptides (AMPs) play an important role in this response by rapidly killing invading microorganisms. In this study, a novel cysteine-rich AMP has been isolated and characterized from the hemocytes of the cattle tick, Boophilus microplus. In addition to growth inhibition of Escherichia coli and Micrococcus luteus, the newly described AMP, designated ixodidin (derived from the Family Ixodidae), was found to exert proteolytic inhibitory activity against two exogenous serine proteinases, elastase and chymotrypsin. This is the first report of a molecule of an arachnid that has been shown to inhibit bacterial growth and proteinase activity.

Cysteine-rich antimicrobial peptides of the cattle tick Boophilus microplus: isolation, structural characterization and tissue expression profile.

Antimicrobial peptides (AMPs) are components of the immune system of both vertebrate and invertebrate animals. This study describes the isolation, primary structure, cDNA cloning, and tissue expression profile of two cysteine-rich AMPs from the hemolymph of the cattle tick Boophilus microplus. A 10,204 Da polypeptide, with six cysteine residues and no sequence similarity to any known molecule, was isolated from the cell-free hemolymph. Because of its sequence originality, this peptide was named microplusin. The second AMP was isolated from the hemocytes of B. microplus. This peptide, with a molecular mass of 4285 Da and six cysteines, is a defensin with similarity to the insect defensin family members. The cDNA cloning established that microplusin is synthesized as a prepeptide while the tick defensin is synthesized as a prepromolecule. Interestingly, despite the fact that microplusin and defensin have been isolated from different compartments, their gene expression was found to have similar tissue distribution.