Bacterial biofilms are increasing its tolerance to conventional antimicrobial treatments. In this context biofilm formation becomes an important target to control pathogenic bacteria. Arthropods are known to be an important source of natural products; however, investigations exploring the biological activities of arthropod eggs are scarce. So, in this study we investigated the antibiofilm activity of the Mediterranean flour moth Anagasta kuehniella compounds against Staphylococcus epidermidis. The antibiofilm activity of organic and aqueous extractions performed on the eggs was assessed using the crystal violet method and scanning electron microscopy (SEM). Egg's aqueous extract (AE) inhibits up to 65 % biofilm formation by S. epidermidis. Heat and enzymatic treatments indicated that activity was related to protein. A partial purified active fraction (F9) was obtained by size exclusion chromatography and SDS-PAGE were performed using AE and F9. Mass spectrometry was used to identify the proteins in the F9. SEM analysis showed this fraction inhibits bacterial aggregates in treated S. epidermidis biofilms without alteration on bacterial cell morphology. Our results showed that proteins present in A. kuehniella eggs inhibit biofilm formation without killing S. epidermidis and reinforces the idea that arthropods are a potential source of natural products useful for biotechnology.

Background: The host skin is the first line of defense against most microorganisms and parasites such as bacteria, fungi and ticks. The immune system present in skin takes part of a sophisticate defense mechanism, firstly as physical, cellular and chemical barriers, followed by a wide range of antimicrobial molecules and specialized immune cells. These cells are responsible for inflammatory processes, antigen uptake and presentation, allergic responses that untimely could control the pathogens. Review: Concerning tick parasitism, skin immunity has a paramount role during tick attachment and blood feeding through both the innate and adaptive responses. In recent years, an increasing number of discoveries in tick physiology revealed a more detailed picture of the role of immune cells and their mediators against tick parasitism. Therefore, a systematic review and summarization of this information can give a more comprehensive understanding of the orchestration of the diverse and complex host immune response mechanisms that reject at least part of infesting ticks and give clues to suggest potential applications to develop better methods for tick control.Conclusion: The local skin immune response to tick and other ectoparasite infestations is intricately influenced by the microenvironment created by parasite attachment components and secreted proteins, attracting and engaging local immune cells. Host immune status further contributes to this dynamic. This review discusses the major cellular responses, functional diversity, and host skin immunity mechanisms stimulated by ticks. However, more research is needed to fill existing gaps and fully understand how the skin responds to ticks and other parasites. For example, studying B-cell responses, their diversity, and exploring the full Th2 immune response could provide valuable insights for improving tick control strategies.Keywords: tick, skin, immunity, parasite, vaccine

Bothrops pubescens is a clinically significant viperid snake species in Uruguay, accounting for over 60 cases of human envenomation annually, alongside Bothrops alternatus. Formerly classified within the Bothrops neuwiedi complex, recent taxonomic revisions now restrict its distribution to Rio Grande do Sul (Brazil) and Uruguay. This species is not very robust, with a body length ranging from 22 to 92 cm and displays a distinctive dorsolateral pattern consisting of dark brown trapezoidal blotches on each side.The venom of B. pubescens exhibits toxicological properties typical of the Bothrops genus, including coagulopathy and severe local tissue damage, which may result in limb amputation in extreme cases. As in other Bothrops species, metalloproteinases are the most abundant toxins, accompanied by high levels of phospholipase A2, serine proteases, L-amino acid oxidases, and C-type lectins. Minor venom components include vascular endothelial growth factor (VEGF), nerve growth factor (NGF), hyaluronidases, and cysteine-rich secretory proteins (CRISPs).Recent studies suggest that B. pubescens and B. diporus share a common evolutionary lineage and display highly similar venom compositions. In our study, biochemical and proteomic analyses revealed a venom profile in Uruguayan B. pubescens closely resembling that of B. diporus from Argentina yet differing slightly from B. pubescens specimens in Brazil.

ABSTRACT: Rhipicephalus (Boophilus) microplus is a hard tick endemic in livestock-growing regions and causes economic losses in the largest beef-producing countries, including Brazil, Mexico, Argentina, Australia and Uruguay. The use of chemical acaricides is still the main strategy to control R. microplusinfestations. Nevertheless, immunological control of R. microplus with an anti-tick vaccine is a suitable alternative and has manifold advantages because it can avoid drug-resistance and the presence of acaricide residues in milk, beef and in the environment. Indeed, vaccines based on the Bm86 antigen have had relative commercial and technical success to control R. microplus in some regions. Although, the efficacy of such vaccines varies among tick populations and is insufficient to provide an acceptable level of protection. Therefore, the need to search for better antigens is impelling. This review focused on the restrictions imposed on the use of acaricides in Brazil and in the European Union, as well as on the impacts of Bm86-based vaccines on R. microplus control. The efficacy of experimental anti-tick vaccines (based on subolesin, glutathione S-transferase, ferritin 2; voltage-dependent anion channel; aquaporin, 60 S acidic ribosomal protein, metalloprotease and trypsin) that can elicit an immune response against the physiological functions of various ticks is discussed.

Background When feeding on a vertebrate host, ticks secrete saliva, which is a complex mixture of proteins, lipids, and other molecules. Tick saliva assists the vector in modulating host hemostasis, immunity, and tissue repair mechanisms. While helping the vector to feed, its saliva modifies the site where pathogens are inoculated and often facilitates the infection process. The objective of this study is to uncover the variation in protein composition of Rhipicephalus microplus saliva during blood feeding. Methods Ticks were fed on calves, and adult females were collected, weighed, and divided in nine weight groups, representing the slow and rapid feeding phases of blood feeding. Tick saliva was collected, and mass spectrometry analyses were used to identify differentially secreted proteins. Bioinformatic tools were employed to predict the structural and functional features of the salivary proteins. Reciprocal best hit analyses were used to identify conserved families of salivary proteins secreted by other tick species. Results Changes in the protein secretion profiles of R. microplus adult female saliva during the blood feeding were observed, characterizing the phenomenon known as “sialome switching.” This observation validates the idea that the switch in protein expression may serve as a mechanism for evading host responses against tick feeding. Cattle tick saliva is predominantly rich in heme-binding proteins, secreted conserved proteins, lipocalins, and protease inhibitors, many of which are conserved and present in the saliva of other tick species. Additionally, another remarkable observation was the identification of host-derived proteins as a component of tick saliva. Conclusions Overall, this study brings new insights to understanding the dynamics of the proteomic profile of tick saliva, which is an important component of tick feeding biology. The results presented here, along with the disclosed sequences, contribute to our understanding of tick feeding biology and might aid in the identification of new targets for the development of novel anti-tick methods. Graphical Abstract

Recent advancements in molecular biology, particularly regarding massively parallel sequencing technologies, have enabled scientists to gain more insight into the physiology of ticks. While there has been progress in identifying tick proteins and the pathways they are involved in, the specificities of tick-host interaction at the molecular level are not yet fully understood. Indeed, the development of effective commercial tick vaccines has been slower than expected. While omics studies have pointed to some potential vaccine immunogens, selecting suitable antigens for a multi-antigenic vaccine is very complex due to the participation of redundant molecules in biological pathways. The expansion of ticks and their pathogens into new territories and exposure to new hosts makes it necessary to evaluate vaccine efficacy in unusual and non-domestic host species. This situation makes ticks and tick-borne diseases an increasing threat to animal and human health globally, demanding an urgent availability of vaccines against multiple tick species and their pathogens. This review discusses the challenges and advancements in the search for universal tick vaccines, including promising new antigen candidates, and indicates future directions in this crucial research field.

Tick sialome is comprised of a rich cocktail of bioactive molecules that function as a tool to disarm host immunity, assist blood-feeding, and play a vibrant role in pathogen transmission. The adaptation of the tick’s blood-feeding behavior has lead to the evolution of bioactive molecules in its saliva to assist them to overwhelm hosts’ defense mechanisms. During a blood meal, a tick secretes different salivary molecules including vasodilators, platelet aggregation inhibitors, anticoagulants, anti-inflammatory proteins, and inhibitors of complement activation; the salivary repertoire changes to meet various needs such as tick attachment, feeding, and modulation or impairment of the local dynamic and vigorous host responses. For instance, the tick’s salivary immunomodulatory and cement proteins facilitate the tick’s attachment to the host to enhance prolonged blood-feeding and to modulate the host’s innate and adaptive immune responses. Recent advances implemented in the field of “omics” have substantially assisted our understanding of host immune modulation and immune inhibition against the molecular dynamics of tick salivary molecules in a crosstalk between the tick–host interface. A deep understanding of the tick salivary molecules, their substantial roles in multifactorial immunological cascades, variations in secretion, and host immune responses against these molecules is necessary to control these parasites. In this article, we reviewed updated knowledge about the molecular mechanisms underlying host responses to diverse elements in tick saliva throughout tick invasion, as well as host defense strategies. In conclusion, understanding the mechanisms involved in the complex interactions between the tick salivary components and host responses is essential to decipher the host defense mechanisms against the tick evasion strategies at tick-host interface which is promising in the development of effective anti-tick vaccines and drug therapeutics.

Cholesterol is a known precursor of arthropod molecules such as the hormone 20-hydroxyecdysone and the antimicrobial boophiline, a component of tick egg wax coat. Because the cholesterol biosynthetic pathway is absent in ticks, it is necessarily obtained from the blood meal, in a still poorly understood process. In contrast, dietary cholesterol absorption is better studied in insects, and many proteins are involved in its metabolism, including Niemann-Pick C (NPC) transporter and acyl-CoA:cholesterol acyltransferase (ACAT), as well as enzymes to convert between free cholesterol and esterified cholesterol. The present work addresses the hypothesis that tick viability can be impaired by interfering with cholesterol metabolism, proposing this route as a target for novel tick control methods. Two drugs, ezetimibe (NPC inhibitor) and avasimibe (ACAT inhibitor) were added to calf blood and used to artificially feed Rhipicephalus microplus females. Results show that, after ingesting avasimibe, tick reproductive ability and egg development are impaired. Also, eggs laid by females fed with avasimibe did not hatch and were susceptible to Pseudomonas aeruginosa adhesion and biofilm formation in their surfaces. The immunoprotective potential of ACAT against ticks was also accessed using two selected ACAT peptides. Antibodies against these peptides were used to artificially feed female ticks, but no deleterious effects were observed. Taken together, data presented here support the hypothesis that enzymes and other proteins involved in cholesterol metabolism are suitable as targets for tick control methods.

The aim of this study was to select and identify thermophilic bacteria from Caatinga biome (Brazil) able to produce thermoactive keratinases and characterize the keratinase produced by the selected isolate. After enrichment in keratin culture media, an Anoxybacillus caldiproteolyticus PC2 was isolated. This thermotolerant isolate presents a remarkable feature producing a thermostable keratinase at 60°C. The partially purified keratinase, identified as a thermolysin-like peptidase, was active at a pH range of 5.0-10.0 with maximal activity at a temperature range of 50-80°C. The optimal activity was observed at pH 7.0 and 50-60°C. These characteristics are potentially useful for biotechnological purposes such as processing and bioconversion of keratin.

Ticks inject serine protease inhibitors (serpins) into their feeding sites to evade serine protease-mediated host defenses against tick-feeding. This study describes two highly identitical (97%) but functionally different Amblyomma americanum tick saliva serpins (AAS41 and 46) that are secreted at the inception of tick-feeding. We show that AAS41, which encodes a leucine at the P1 site inhibits inflammation system proteases: chymase (SI = 3.23, Ka = 5.6 +/- 3.7X10(3)M(-1) s(-1)) and alpha-chymotrypsin (SI = 3.18, Ka = 1.6 +/- 4.1 X10(4)M(-1) s(-1)), while AAS46, which encodes threonine has no inhibitory activity. Similary, rAAS41 inhibits rMCP-1 purified from rat peritonuem derived mast cells. Consistently, rAAS41 inhibits chymase-mediated inflammation induced by compound 48/80 in rat paw edema and vascular permeability models. Native AAS41/46 proteins are among tick saliva immunogens that provoke anti-tick immunity in repeatedly infested animals as revealed by specific reactivity with tick immune sera. Of significance, native AAS41/46 play critical tick-feeding functions in that RNAi-mediated silencing caused ticks to ingest significantly less blood. Importantly, monospecific antibodies to rAAS41 blocked inhibitory functions of rAAS41, suggesting potential for design of vaccine antigens that provokes immunity to neutralize functions of this protein at the tick-feeding site. We discuss our findings with reference to tick-feeding physiology and discovery of effective tick vaccine antigens. (C) 2020 Elsevier B.V. All rights reserved.

In parasites, cathepsins are implicated in mechanisms related to organism surveillance and host evasion. Some parasite cathepsins have fibrinogenolytic and fibrinolytic activity, suggesting that they may contribute to maintain blood meal fluidity for extended feeding periods. Here, it is shown that BmGTI (Rhipicephalus [Boophilus] microplus Gut Thrombin Inhibitor), a protein previously described as an inhibitor of fibrinogen hydrolysis and platelet aggregation by thrombin, and BmCL1 (Rhipicephalus [Boophilus] microplus Cathepsin-L like 1) are the same protein, hereinafter referred to using the earliest name (BmCL1). To further characterize BmCL1, Rhipicephalus microplus native and recombinant (rBmCL1) proteins were obtained. Native BmCL1 was isolated using thrombin-affinity chromatography, and it displays thrombin inhibition activity. We subsequently investigated rBmCL1 interaction with thrombin. We show that rBmCL1 and thrombin have a dissociation constant (ΚD) of 130.2 ± 11.2 nM, and this interaction likely occurs due to a more electronegative surface of BmCL1 at pH 7.5 than at pH 5.0, which may favor an electrostatic binding to positively charged thrombin exosites. During BmCL1-thrombin interaction, thrombin is not degraded or inhibited. rBmCL1 impairs thrombin-induced fibrinogen clotting via a fibrinogenolytic activity. Fibrinogen degradation by BmCL1 occurs by the hydrolysis of Aα- and Bβ-chains, generating products similar to those produced by fibrinogenolytic cathepsins from other organisms. In conclusion, BmCL1 likely has an additional role in R. microplus blood digestion, besides its role in hemoglobin degradation at acid pH. BmCL1 fibrinogenolytic activity indicates a proteolytic activity in the neutral lumen of tick midgut, contributing to maintain the fluidity of the ingested blood, which remains to be confirmed in vivo.

Lipids play key roles in arthropod metabolism. In ticks, these biomolecules are transported from fat body to other organs, such as ovary and Gené’s organ. Gené’s organ, an apparatus found exclusively in female ticks, secretes a protective wax coat onto the egg surface, increasing egg viability in the environment due to waterproof, cohesive, and antimicrobial properties. In this work, a combined transcriptomic and proteomic approach shows that Gené’s organ not solely secrets compounds taken up from the hemolymph, but is actively engaged in synthesis, modification, and oxidation of lipids. Gené’s organ was analyzed at two distinct stages: 1) when ticks detach from host by the end of hematophagous phase, and 2) during egg-laying. Data show that Gené’s organ undergoes a maturation process before the onset of oviposition, in preparation for its role during egg-laying. Because it deals with a wax-secreting organ, the study focused on lipid metabolism, examining a full machinery to synthesize, modify, and oxidize fatty acids. Proteins involved in sterol modification, transport, and degradation were also addressed. In addition to highlighting Gené’s organ importance in tick reproductive physiology, the results reveal proteins and pathways crucial to egg wax secretion, and consequently, egg development in the environment. Tools targeting these molecules and pathways would impair egg viability in the environment, and therefore have the potential to be developed into novel tick control methods.

Ticks successfully feed and transmit pathogens by injecting pharmacological compounds in saliva to thwart host defenses. We have previously used LC-MS/MS to identify proteins that are present in saliva of unfed Amblyomma americanum ticks that were exposed to different hosts. Here we show that A. americanum serine protease inhibitor (serpin) 27 (AAS27) is an immunogenic saliva protein that is injected into the host within the first day of tick feeding and is an anti-inflammatory protein that might act by blocking plasmin and trypsin functions. Although AAS27 is injected into the host throughout tick feeding, qRT-PCR and western blotting analyses indicate that the respective transcript and protein are present in high amounts within the first 24 h of tick feeding. Biochemical screening of Pichia pastoris-expressed recombinant (r) AAS27 against mammalian proteases related to host defense shows it is an inhibitor of trypsin and plasmin, with stoichiometry of inhibition indices of 3.5 and 3.8, respectively. Consistent with typical inhibitory serpins, rAAS27 formed heat- and SDS-stable irreversible complexes with both proteases. We further demonstrate that rAAS27 inhibits trypsin with ka of 6.46 ± 1.24 x 104 M-1 s-1, comparable to serpins of other tick species. We show that native AAS27 is part of the repertoire of proteins responsible for the inhibitory activity against trypsin in crude tick saliva. AAS27 is likely utilized by the tick to evade the hosts inflammation defense since rAAS27 blocks both formalin and compound 48/80-induced inflammation in rats. Tick immune sera of rabbits that had acquired resistance against tick feeding following repeated infestations with A. americanum or Ixodes scapularis ticks reacts with rAAS27. Of significant interest, antibody to rAAS27 blocks this serpin inhibitory functions. Taken together, we conclude that AAS27 is an anti-inflammatory protein secreted into the host during feeding and may represent a potential candidate for development of an anti-tick vaccine.

Peptidase inhibitors regulate a wide range of physiological processes involved in the interaction between hematophagous parasites and their hosts, including tissue remodeling, the immune response and blood coagulation. In tick physiology, peptidase inhibitors have a crucial role in adaptation to improve parasitism mechanisms, facilitating blood feeding by interfering with defense-related host peptidases. Recently, a larger number of studies on this topic led to the description of several new tick inhibitors displaying interesting novel features, for example a role in pathogen transmission to the host. A comprehensive review discussing these emerging concepts can therefore shed light on peptidase inhibitor functions, their relevance to tick physiology and their potential applications. Here, we summarize and examine the general characteristics, functional diversity and action of tick peptidase inhibitors with known physiological roles in the tick-host-pathogen interaction.

Ticks are arthropod ectoparasites of importance for public and veterinary health. The understanding of tick oogenesis and embryogenesis could contribute to the development of novel control methods. However, to date, studies on the temporal dynamics of proteins during ovary development were not reported. In the present study we followed protein profile during ovary maturation. Proteomic analysis of ovary extracts was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using shotgun strategy, in addition to dimethyl labelling-based protein quantification. A total of 3,756 proteins were identified, which were functionally annotated into 30 categories. Circa 80% of the annotated proteins belong to categories related to basal metabolism, such as protein synthesis and modification machineries, nuclear regulation, cytoskeleton, proteasome machinery, transcriptional machinery, energetic metabolism, extracellular matrix/cell adhesion, immunity, oxidation/detoxification metabolism, signal transduction, and storage. The abundance of selected proteins involved in yolk uptake and degradation, as well as vitellin accumulation during ovary maturation, was assessed using dimethyl-labelling quantification. In conclusion, proteins identified in this study provide a framework for future studies to elucidate tick development and validate candidate targets for novel control methods.

Background : : : : Ticks are distributed worldwide, with impacts on human and animal health. The cattle tick Rhipicephalus (Boophilus) microplus is the main parasite that affects livestock in tropical and subtropical regions of the world, causing large economical losses. Tick control methods are based on the application of chemical acaricides, which has resulted in selection of resistant ticks and a potential risk of environmental pollution and food contamination. Vaccines have showed to be a feasible tick control method that offers a cost-effective, environmental friendly alternative to chemical control. However, more than ten years after the commercialization of the first vaccine against ticks, the identification of tick-protective antigens remains a limiting step in the development of an efficient formulation that would avoid the use of chemical acaricides. So, the study of parasite biology and understanding physiological mechanisms could be a good strategy to find new targets for an efficient vaccine. Review : It was reviewed the main insights about the reproductive process in ticks, emphasizing the hormonal control of vitellogenesis and enzymes involved in vitellin processing during embryogenesis. The processes of vitellogenesis and embryogenesis have been studied in various organisms, particularly in cockroaches, flies and ticks. Although the roles of 20-hydroxyecdysone (20E) and juvenile hormone have been well characterized for vitellogenesis in insects, we know much less about the hormonal control of vitellogenesis in ticks. Initially, it was hypothesized that juvenile hormone was involved in tick vitellogenin-synthesis. However, more critical studies uncovered no evidence for the occurrence of juvenile hormone or juvenile hormone-like molecules in several tick species. Current research shows that in ticks, it appears that ecdysteroids, and not juvenile hormone, regulate the expression of the vitellogenin gene and the synthesis and release of vitellogenin protein into the hemolymph. In general, the carbohydrate, lipid and amino acid composition of tick vitellogenin is similar to that of insect vitellogenin. Once in the hemolymph, oocytes uptake vitellogenin through receptor-mediated  endocytosys. However, there are different strategies to control vitellogenin synthesis and uptake by ovary in ixodide ticks. In the oocytes, vitellogenin is partially processed in the endosomal compartment and then stored as vitellin, the main reserve of protein for embryo development, in specialized organelles, the yolk granules. Embryo development depends on the availability of yolk material stored into oocytes. So, the characterization of molecules involved in vitellogenesis and embryo development contribute to a better understanding of the tick parasite physiology. During embryogesesis, acidic enzymes are responsible for the availability of this material and embryo nutrition. The Vitellin-Degrading Cysteine Endopeptidase (VTDCE), Boophilus Yolk Pro-Cathepsin (BYC) and Tick Heme Binding Aspartic Proteinase (THAP) are enzymes involved in vitellin hydrolysis in R. microplus eggs. These enzymes are produced by gut and fat body and transported through the hemolymph to be internalized into the oocytes and then play their role in tick embryo nutrition. As VTDCE, BYC and THAP are involved in an important physiological process, their potential as targets in an anti-tick vaccine is an attractive research topic. With this objective, various enzymes have been tested in native or recombinant forms as candidate immunogens to a multiantigenic anti-tick vaccine. Conclusion : Significant advancements have been made in recent years on understanding the tick reproductive process, and some molecules that can be possible targets for development of new tick control strategies have been characterized.

The vitellogenin receptor (VgR), which belongs to the low-density lipoprotein receptors (LDLR) family, regulates the absorption of yolk protein accumulated in developing oocytes during oogenesis. In the present study, the full sequence of Rhipicephalus microplus VgR (RmVgR) and the partial sequence of Rhipicephalus appendiculatus VgR (RaVgR) ORF were determined and cloned. The RmVgR amino acid sequence contains the five highly conserved structural motifs characteristic of LDLR superfamily members, the same overall structure as observed in other species. Phylogenetic analysis separated VgRs in two major groups, corresponding to receptors from acarines and insects. Consistent with observations from other arthropods, RmVgR was specifically expressed in the ovarian tissue and its peak of expression occurs in females that are detaching from the host. Silencing with RmVgR dsRNA reduced VgR expression, which resulted in reduced fertility, evidenced by a decrease in the number of larvae. The present study confirms RmVgR is a specific receptor involved in yolk protein uptake and oocyte maturation in R. microplus, playing an important role in tick reproduction.