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In the past decade, metagenomics studies exploring tick microbiota have revealed widespread interactions between bacteria and arthropods, including symbiotic interactions. Functional studies showed that obligate endosymbionts contribute to tick biology, affecting reproductive fitness and molting. Understanding the molecular basis of the interaction between ticks and their mutualist endosymbionts may help to develop control methods based on microbiome manipulation. Previously, we showed that Rhipicephalus microplus larvae with reduced levels of Coxiella endosymbiont of R. microplus (CERM) were arrested at the metanymph life stage (partially engorged nymph) and did not molt into adults. In this study, we performed a transcriptomic differential analysis of the R. microplus metanymph in the presence and absence of its mutualist endosymbiont. The lack of CERM resulted in an altered expression profile of transcripts from several functional categories. Gene products such as DA-P36, protease inhibitors, metalloproteases, and evasins, which are involved in blood feeding capacity, were underexpressed in CERM-free metanymphs. Disregulation in genes related to extracellular matrix remodeling was also observed in the absence of the symbiont. Taken together, the observed alterations in gene expression may explain the blockage of development at the metanymph stage and reveal a novel physiological aspect of the symbiont-tick-vertebrate host interaction.

Understanding the molecular basis of how ticks adapt to feed on different animal hosts is central to understanding tick and tick-borne disease (TBD) epidemiology. There is evidence that ticks differentially express specific sets of genes when stimulated to start feeding. This study was initiated to investigate if ticks such as Ixodes scapularis and Amblyomma americanum that are adapted to feed on multiple hosts utilized the same sets of proteins to prepare for feeding. We exposed I. scapularis and A. americanum to feeding stimuli of different hosts (rabbit, human, and dog) by keeping unfed adult ticks enclosed in a perforated microfuge in close contact with host skin, but not allowing ticks to attach on host. Our data suggest that ticks of the same species differentially express tick saliva proteins (TSPs) when stimulated to start feeding on different hosts. SDS-PAGE and silver staining analysis revealed unique electrophoretic profiles in saliva of I. scapularis and A. americanum that were stimulated to feed on different hosts: rabbit, human, and dog. LC-MS/MS sequencing and pairwise analysis demonstrated that I. scapularis and A. americanum ticks expressed unique protein profiles in their saliva when stimulated to start feeding on different hosts: rabbit, dog, or human. Specifically, our data revealed TSPs that were unique to each treatment and those that were shared between treatments. Overall, we identified a total of 276 and 340 non-redundant I. scapularis and A. americanum TSPs, which we have classified into 28 functional classes including: secreted conserved proteins (unknown functions), proteinase inhibitors, lipocalins, extracellularmatrix/cell adhesion, heme/ironmetabolism, signal transduction and immunity-related proteins being the most predominant in saliva of unfed ticks. With exception of research on vaccines against Rhipicephalus microplus, which its natural host, cattle, research on vaccine against other ticks relies feeding ticks on laboratory animals. Data here suggest that relying on lab animal tick feeding data to select target antigens could result in prioritizing irrelevant anti-tick vaccine targets that are expressed when ticks feed on laboratory animals. This study provides the platform that could be utilized to identify relevant target anti-tick vaccine antigens, and will facilitate early stage tick feeding research.

Carrapatos são ectoparasitos hematófagos que causam grandes impactos na saúde pública e na produção animal devido à sua capacidade vetorial e também pelos distúrbios associados diretamente ao parasitismo. O carrapato Rhipicephalus microplus é considerado o parasito mais importante de bovinos na América Latina e Oceania. Os carrapatos permanecem fixados ao hospedeiro por um longo período de tempo, e a saliva representa um fator crucial na relação parasito-hospedeiro. Os carrapatos secretam na saliva um conjunto de moléculas com atividades que modulam as respostas de defesa do hospedeiro frente ao parasitismo, tais como as reações do sistema hemostático e do sistema imune inato e adaptativo. Considerando a importância econômica do carrapato R. microplus, e o papel da saliva no parastismo, pouco se sabe sobre a composição proteica da saliva desse parasito. Os objetivos desse trabalho são caracterizar o proteoma da saliva do carrapato R. microplus em diferentes momentos da fase de alimentação no hospedeiro bovino e caracterizar três serpinas salivares. Neste sentido, este estudo foi organizado em três partes. Primeiramente, fizemos uma análise proteômica da saliva de fêmeas adultas em dois estágios de ingurgitamento, compreendendo fêmeas parcialmente ingurgitadas e fêmeas totalmente ingurgitadas. Num segundo momento, analisamos mais a fundo a variação dos componentes proteicos salivares das fêmeas, expandindo a análise proteômica para nove fases de ingurgitamento do parasito. Esses dados mostraram que a saliva de R. microplus é uma amostra proteica complexa, com variação nos diferentes tempos de alimentação. Entre as classes de proteínas identificadas, podemos citar proteínas relacionadas com metabolismo de heme e ferro; proteínas relacionadas com processos de oxidação e detoxificação; enzimas como as serino-proteases, cisteíno-proteases, metaloproteases; inibidores de proteases da família das serpinas, cistatinas, TIL e Kunitz; peptídeos antimicrobianos; e famílias de proteínas específicas e conservadas em carrapatos. A terceira parte do trabalho objetivou a caracterização de três serpinas que foram identificadas na saliva (RmS-3, RmS-6 e RmS-17). Essas proteínas foram expressas em Pichia pastoris e foram utilizadas em ensaios de caracterização funcional contra um painel de 16 proteases relacionadas com os sistemas de defesa do hospedeiro, revelando um perfil de inibição sobre proteases pró-inflamatórias e prócoagulantes. Os dados apresentados neste trabalho contribuem para o entendimento do papel de proteínas salivares durante o parasitismo pelo carrapato R. microplus. Ticks are hematophagous ectoparasites with worldwide distribution impacting on public health and animal production due to their vectorial capacity and also for disorders associated directly to parasitism. The tick Rhipicephalus microplus is considered the most important parasite of cattle in Latin America and Oceania. As hematophagous ectoparasites which remain attached to the host for a long period of time, the saliva of ticks represent a crucial factor in host-parasite relationship. The tick saliva comprises an arsenal of molecules with activities modulating the host defense responses trigged against tick feeding, such as the reactions of the hemostatic system, the innate immune system and the adaptive immune system. Considering the economic importance of the tick R. microplus, little is known about the protein composition of the saliva of this parasite. The aims of this study were to characterize the R. microplus tick saliva proteome at different feeding times, and to characterize three salivary serpin that were identified in the saliva of this parasite. Thus, this study was organized into three parts. Firstly, we conducted a proteomic analysis of female saliva in two developmental stages of adult tick feeding, including partially engorged and fully engorged females. Secondly, we analyzed in depth the variation of salivary protein components, expanding proteomics analysis to nine stages of tick feeding. These data showed that the R. microplus saliva is a complex protein sample, with variations in different feeding times. Among the classes of identified proteins, we can mention proteins related to heme and iron metabolism; proteins related to oxidation and detoxification processes; enzymes such as serine proteases, cysteine proteases, metalloproteases; proteases inhibitors belonging to serpin family, cystatins, TIL, and Kunitz; antimicrobial peptides; and families of tick-specific and conserved proteins. The third part of the study aimed to characterize three serpins that have been identified in saliva (RmS-3, RmS-6 and RmS-17). These proteins were expressed in Pichia pastoris and were used for functional characterization against a panel of 16 host proteases related to defense systems. Results revealed an inhibition profile for pro-inflammatory and pro-clotting proteases. The data presented here contributes to understanding the role of salivary proteins into tick-host relationship.