The red flour beetle Tribolium castaneum is a destructive insect pest of stored food and feed products, and a model organism for development, evolutionary biology and immunity. The insect innate immune system includes antimicrobial peptides (AMPs) with a wide spectrum of targets including viruses, bacteria, fungi and parasites. Defensins are an evolutionarily-conserved class of AMPs and a potential new source of antimicrobial agents. In this context, we report the antimicrobial activity, phylogenetic and structural properties of three T. castaneum defensins (Def1, Def2 and Def3) and their relevance in the immunity of T. castaneum against bacterial pathogens. All three recombinant defensins showed bactericidal activity against Micrococcus luteus and Bacillus thuringiensis serovar tolworthi, but only Def1 and Def2 showed a bacteriostatic effect against Staphylococcus epidermidis. None of the defensins showed activity against the Gram-negative bacteria Escherichia coli and Pseudomonas entomophila or against the yeast Saccharomyces cerevisiae. All three defensins were transcriptionally upregulated following a bacterial challenge, suggesting a key role in the immunity of T. castaneum against bacterial pathogens. Phylogenetic analysis showed that defensins from T. castaneum, mealworms, Udo longhorn beetle and houseflies cluster within a well-defined clade of insect defensins. We conclude that T. castaneum defensins are primarily active against Gram-positive bacteria and that other AMPs may play a more prominent role against Gram-negative species.

Mycobacteria of the Mycobacterium tuberculosis complex (MTBC) greatly affect humans and animals worldwide. The life cycle of mycobacteria is complex and the mechanisms resulting in pathogen infection and survival in host cells are not fully understood. Recently, comparative genomics analyses have provided new insights into the evolution and adaptation of the MTBC to survive inside the host. However, most of this information has been obtained using M. tuberculosis but not other members of the MTBC such as M. bovis and M. caprae. In this study, the genome of three M. bovis (MB1, MB3, MB4) and one M. caprae (MB2) field isolates with different lesion score, prevalence and host distribution phenotypes were sequenced. Genome sequence information was used for whole-genome and protein-targeted comparative genomics analysis with the aim of finding correlates with phenotypic variation with potential implications for tuberculosis (TB) disease risk assessment and control. At the whole-genome level the results of the first comparative genomics study of field isolates of M. bovis including M. caprae showed that as previously reported for M. tuberculosis, sequential chromosomal nucleotide substitutions were the main driver of the M. bovis genome evolution. The phylogenetic analysis provided a strong support for the M. bovis/M. caprae clade, but supported M. caprae as a separate species. The comparison of the MB1 and MB4 isolates revealed differences in genome sequence, including gene families that are important for bacterial infection and transmission, thus highlighting differences with functional implications between isolates otherwise classified with the same spoligotype. Strategic protein-targeted analysis using the ESX or type VII secretion system, proteins linking stress response with lipid metabolism, host T cell epitopes of mycobacteria, antigens and peptidoglycan assembly protein identified new genetic markers and candidate vaccine antigens that warrant further study to develop tools to evaluate risks for TB disease caused by M. bovis/M.caprae and for TB control in humans and animals.

Antimicrobial peptides are ubiquitous components of eukaryotic innate immunity. Defensins are a well-known family of antimicrobial peptides, widely distributed in ticks, insects, plants and mammals, showing activity against bacteria, viruses, fungi, yeast and protozoan parasites. Ixodes ricinus is the most common tick species in Europe and is a vector of pathogens affecting human and animal health. Recently, six defensins (including two isoforms) were identified in I. ricinus. We investigated the evolution of the antimicrobial activity of I. ricinus defensins. Among the five unique defensins, only DefMT3, DefMT5 and DefMT6 showed in vitro antimicrobial activity. Each defensin was active against rather distantly-related bacteria (P < 0.05), significantly among Gram-negative species (P < 0.0001). These three defensins represent different clades within the family of tick defensins, suggesting that the last common ancestor of tick defensins may have had comparable antimicrobial activity. Differences in electrostatic potential, and amino acid substitutions in the β-hairpin and the loop bridging the α-helix and β-sheet may affect the antimicrobial activity in DefMT2 and DefMT7, which needs to be addressed. Additionally, the antimicrobial activity of the γ-core motif of selected defensins (DefMT3, DefMT6, and DefMT7) was also tested. Interestingly, compared to full length peptides, the γ-core motifs of these defensins were effective against less species of bacteria. However, the antifungal activity of the γ-core was higher than full peptides. Our results broaden the scope of research in the field of antimicrobial peptides highlighting the overlooked ability of arthropod defensins to act against distantly-related microorganisms.

Malaria is a devastating infectious disease caused by Plasmodium parasites transmitted through the bites of infected Anopheles mosquitoes. Salivary glands are the only mosquito tissue invaded by Plasmodium sporozoites, being a key stage for the effective parasite transmission, making the study of Anopheles sialome highly relevant.

Anaplasma phagocytophilum is an emerging zoonotic pathogen transmitted by Ixodes scapularis that causes human granulocytic anaplasmosis. Here, a high throughput quantitative proteomics approach was used to characterize A. phagocytophilum proteome during rickettsial multiplication and identify proteins involved in infection of the tick vector, I. scapularis. The first step in this research was focused on tick cells infected with A. phagocytophilum and sampled at two time points containing 10-15% and 65-71% infected cells, respectively to identify key bacterial proteins over-represented in high percentage infected cells. The second step was focused on adult female tick guts and salivary glands infected with A. phagocytophilum to compare in vitro results with those occurring during bacterial infection in vivo. The results showed differences in the proteome of A. phagocytophilum in infected ticks with higher impact on protein synthesis and processing than on bacterial replication in tick salivary glands. These results correlated well with the developmental cycle of A. phagocytophilum, in which cells convert from an intracellular reticulated, replicative form to the nondividing infectious dense-core form. The analysis of A. phagocytophilum differentially represented proteins identified stress response (GroEL, HSP70) and surface (MSP4) proteins that were over-represented in high percentage infected tick cells and salivary glands when compared to low percentage infected cells and guts, respectively. The results demonstrated that MSP4, GroEL and HSP70 interact and bind to tick cells, thus playing a role in rickettsia-tick interactions. The most important finding of these studies is the increase in the level of certain bacterial stress response and surface proteins in A. phagocytophilum-infected tick cells and salivary glands with functional implication in tick-pathogen interactions. These results gave a new dimension to the role of these stress response and surface proteins during A. phagocytophilum infection in ticks. Characterization of Anaplasma proteome contributes information on host-pathogen interactions and provides targets for development of novel control strategies for pathogen infection and transmission.

Ticks are blood-sucking arthropods and a primary function of tick salivary proteins is to counteract the host's immune response. Tick salivary Kunitz-domain proteins perform multiple functions within the feeding lesion and have been classified as venoms; thereby, constituting them as one of the important elements in the arms race with the host. The two main mechanisms advocated to explain the functional heterogeneity of tick salivary Kunitz-domain proteins are gene sharing and gene duplication. Both do not, however, elucidate the evolution of the Kunitz family in ticks from a structural dynamic point of view. The Red Queen hypothesis offers a fruitful theoretical framework to give a dynamic explanation for host-parasite interactions. Using the recent salivary gland Ixodes ricinus transcriptome we analyze, for the first time, single Kunitz-domain encoding transcripts by means of computational, structural bioinformatics and phylogenetic approaches to improve our understanding of the structural evolution of this important multigenic protein family.

Ticks transmit zoonotic pathogens worldwide. Nevertheless, very little information is available on their genome, transcriptome and proteome. Herein, we characterized the proteome of Amblyomma americanum adults and nymphs because of their role in pathogen transmission and compared the proteome of A. americanum, A. cajennense and A. variegatum adult ticks. We also used de novo sequencing proteomics data for the analysis of the phylogenetic relationships between the three Amblyomma spp. in a proof of concept for phyloproteomics. The results showed that host and tick proteins involved in blood digestion, heme detoxification, development and innate immunity were differentially represented between adults and nymphs. Although these ticks were unfed, over-represented host proteins may supply nutrients during off-host periods. Tick proteins involved in tick attachment, feeding, heat shock response, protease inhibition and heme detoxification were differentially represented between Amblyomma spp., suggesting adaptation processes to biotic and abiotic factors. These results suggested that phyloproteomics might be a useful tool for the phylogenetic analysis of tick species in which sequence data is a limiting factor and demonstrate the possibilities of proteomics studies for the characterization of relevant tick vector species and provide new relevant information to understand the physiology, development and evolution of these tick species.

As an ecological adaptation venoms have evolved independently in several species of Metazoa. As haematophagous arthropods ticks are mainly considered as ectoparasites due to directly feeding on the skin of animal hosts. Ticks are of major importance since they serve as vectors for several diseases affecting humans and livestock animals. Ticks are rarely considered as venomous animals despite that tick saliva contains several protein families present in venomous taxa and that many Ixodida genera can induce paralysis and other types of toxicoses. Tick saliva was previously proposed as a special kind of venom since tick venom is used for blood feeding that counteracts host defense mechanisms. As a result, the present study provides evidence to reconsider the venomous properties of tick saliva.

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.

Anaplasma phagocytophilum transmembrane and surface proteins play a role during infection and multiplication in host neutrophils and tick vector cells. Recently, A. phagocytophilum Major surface protein 4 (MSP4) and Heat shock protein 70 (HSP70) were shown to be localized on the bacterial membrane, with a possible role during pathogen infection in ticks. In this study, we hypothesized that A. phagocytophilum MSP4 and HSP70 have similar functions in tick-pathogen and host-pathogen interactions. To address this hypothesis, herein we characterized the role of these bacterial proteins in interaction and infection of vertebrate host cells. The results showed that A. phagocytophilum MSP4 and HSP70 are involved in host-pathogen interactions, with a role for HSP70 during pathogen infection. The analysis of the potential protective capacity of MSP4 and MSP4-HSP70 antigens in immunized sheep showed that MSP4-HSP70 was only partially protective against pathogen infection. This limited protection may be associated with several factors, including the recognition of non-protective epitopes by IgG in immunized lambs. Nevertheless, these antigens may be combined with other candidate protective antigens for the development of vaccines for the control of human and animal granulocytic anaplasmosis. Focusing on the characterization of host protective immune mechanisms and protein-protein interactions at the host-pathogen interface may lead to the discovery and design of new effective protective antigens.

Ticks are ectoparasites of animals and humans that serve as vectors of Anaplasma and other pathogens that affect humans and animals worldwide. Ticks and the pathogens that they transmit have coevolved molecular interactions involving genetic traits of both the tick and the pathogen that mediate their development and survival. In this paper, the expression of heat shock proteins (HSPs) and other stress response proteins (SRPs) was characterized in ticks and cultured tick cells by proteomics and transcriptomics analyses in response to Anaplasma spp. infection and heat shock. The results of these studies demonstrated that the stress response was activated in ticks and cultured tick cells after Anaplasma spp. infection and heat shock. However, in the natural vector-pathogen relationship, HSPs and other SRPs were not strongly activated, which likely resulted from tick-pathogen coevolution. These results also demonstrated pathogen- and tick-specific differences in the expression of HSPs and other SRPs in ticks and cultured tick cells infected with Anaplasma spp. and suggested the existence of post-transcriptional mechanisms induced by Anaplasma spp. to control tick response to infection. These results illustrated the complexity of the stress response in ticks and suggested a function for the HSPs and other SRPs during Anaplasma spp. infection.

Tick-borne infectious diseases and allergies are a growing problem worldwide. Tick bite allergy has been associated with the direct effect of immunoglobulin E (IgE) response to tick salivary antigens, or secondary to the induction of allergy to red meat consumption through IgE antibodies against the carbohydrate α-Gal (Gal α 1-3Gal β 1-(3)4GlcNAc-R). However, despite the growing burden of this pathology, the proteins associated with anaphylaxis to tick bite have not been characterized. To address this question, a comparative proteomics approach was used to characterize tick proteins producing an IgE antibody response in a healthy individual with record of tick bites, which had not resulted in any allergic reactions, and two patients with anaphylactic reactions to Rhipicephalus bursa or Hyalomma marginatum tick bites. Both patients and the healthy individual were red meat tolerant. The results supported a patient-specific IgE antibody response to tick species responsible for the anaphylaxis to tick bite. Both patients and the healthy individual serologically recognized tick proteins with and without α-Gal modifications, with proteins differentially recognized by patients but not control sera. These proteins could be used as potential antigens for diagnostics, treatment and prevention of tick bite-induced allergies.

A system biology approach was used to gain insight into tick biology and interactions between vector and pathogen. Rhipicephalus annulatus is one of the main vectors of Babesia bigemina which has a massive impact on animal health. It is vital to obtain more information about this relationship, to better understand tick and pathogen biology, pathogen transmission dynamics, and new potential control approaches. In ticks, salivary glands (SGs) play a key role during pathogen infection and transmission. RNA sequencing obtained from uninfected and B. bigemina infected SGs obtained from fed female ticks resulted in 6823 and 6475 unigenes, respectively. From these, 360 unigenes were found to be differentially expressed (p < 0.05). Reversed phase liquid chromatography-mass spectrometry identified a total of 3679 tick proteins. Among them 406 were differently represented in response to Babesia infection. The omics data obtained suggested that Babesia infection lead to a reduction in the levels of mRNA and proteins (n = 237 transcripts, n = 212 proteins) when compared to uninfected controls. Integrated transcriptomics and proteomics datasets suggested a key role for stress response and apoptosis pathways in response to infection. Thus, six genes coding for GP80, death-associated protein kinase (DAPK-1), bax inhibitor-1 related (BI-1), heat shock protein (HSP), heat shock transcription factor (PHSTF), and queuine trna-ribosyltransferase (QtRibosyl) were selected and RNA interference (RNAi) performed. Gene silencing was obtained for all genes except phstf. Knockdown of gp80, dapk-1, and bi-1 led to a significant increase in Babesia infection levels while hsp and QtRibosyl knockdown resulted in a non-significant decrease of infection levels when compared to the respective controls. Gene knockdown did not affect tick survival, but engorged female weight and egg production were affected in the gp80, dapk-1, and QtRibosyl-silenced groups in comparison to controls. These results advanced our understanding of tick-Babesia molecular interactions, and suggested new tick antigens as putative targets for vaccination to control tick infestations and pathogen infection/transmission.

Ticks are arthropod ectoparasite vectors of pathogens and the cause of allergic reactions affecting human health worldwide. In humans, tick bites can induce high levels of immunoglobulin E antibodies against the carbohydrate Galα1-3Galβ1-(3)4GlcNAc-R (α-Gal) present in glycoproteins and glycolipids from tick saliva that mediate anaphylactic reactions known as the alpha-Gal syndrome (AGS) or red meat allergy. In this study, a new animal model was developed using zebrafish for the study of allergic reactions and the immune mechanisms in response to tick salivary biogenic substances and red meat consumption. The results showed allergic hemorrhagic anaphylactic-type reactions and abnormal behavior patterns likely in response to tick salivary toxic and anticoagulant biogenic compounds different from α-Gal. However, the results showed that only zebrafish previously exposed to tick saliva developed allergic reactions to red meat consumption with rapid desensitization and tolerance. These allergic reactions were associated with tissue-specific Toll-like receptor-mediated responses in types 1 and 2 T helper cells (TH1 and TH2) with a possible role for basophils in response to tick saliva. These results support previously proposed immune mechanisms triggering the AGS and provided evidence for new mechanisms also potentially involved in the AGS. These results support the use of the zebrafish animal model for the study of the AGS and other tick-borne allergies.

Passive immunotherapy with convalescent COVID-19 plasma (CCP) is used as a therapeutic procedure in many countries, including Serbia. In this study, we analyzed the association between demographic factors, COVID-19 severity and the reactivity of anti-SARS-CoV-2 antibodies (Abs) in Serbian CCP donors. Individuals (n = 468) recovered from confirmed SARS-CoV-2 infection, and who were willing to donate their plasma for passive immunization of COVID-19 patients were enrolled in the study. Plasma samples were tested for the presence of IgG reactive to SARS-CoV-2 spike glycoprotein (S1) and nucleocapsid antigens. Individuals were characterized according to age, gender, comorbidities, COVID-19 severity, ABO blood type and RhD factor. Total of 420 candidates (420/468; 89.74%) reached the levels of anti-SARS-CoV-2 IgG that qualified them for inclusion in CCP donation program. Further statistical analysis showed that male individuals (p = 0.034), older age groups (p < 0.001), existence of hypertension (p = 0.008), and severe COVID-19 (p = 0.000) are linked with higher levels of anti-SARS-CoV-2 Abs. These findings will guide the selection of CCP donors in Serbia. Further studies need to be conducted to assess the neutralization potency and clinical efficiency of CCP collected from Serbian donors with high anti-SARS-CoV-2 IgG reactivity.

Mycobacteriosis affects wild fish and aquaculture worldwide, and alternatives to antibiotics are needed for an effective and environmentally sound control of infectious diseases. Probiotics have shown beneficial effects on fish growth, nutrient metabolism, immune responses, disease prevention and control, and gut microbiota with higher water quality. However, the identification and characterization of the molecules and mechanisms associated with probiotics is a challenge that requires investigation. To address this challenge, herein we used the zebrafish model for the study of the efficacy and mechanisms of probiotic interventions against tuberculosis. First, bacteria from fish gut microbiota were identified with high content of the surface glycotope Galα1-3Galβ1-(3)4GlcNAc-R (α-Gal) that has been shown to induce protective immune responses. The results showed that probiotics of selected bacteria with high α-Gal content, namely Aeromonas veronii and Pseudomonas entomophila, were biosafe and effective for the control of Mycobacterium marinum. Protective mechanisms regulating immunity and metabolism activated in response to α-Gal and probiotics with high α-Gal content included modification of gut microbiota composition, B-cell maturation, anti-α-Gal antibodies-mediated control of mycobacteria, induced innate immune responses, beneficial effects on nutrient metabolism and reduced oxidative stress. These results support the potential of probiotics with high -Gal content for the control of fish mycobacteriosis and suggested the possibility of exploring the development of combined probiotic treatments alone and in combination with -Gal for the control of infectious diseases.

The taxonomic composition and diversity of tick midgut microbiota have been extensively studied in different species of the genera Rhipicephalus, Ixodes, Amblyomma, Haemaphysalis, Hyalomma, Dermacentor, Argas and Ornithodoros, while the functional significance of bacterial diversity has been proportionally less explored. In this study, we used previously published 16S amplicon sequence data sets from three Ixodes scapularis cohorts, two of uninfected nymphs, and one of larvae experimentally infected with Borrelia burgdorferi, to test the functional redundancy of the tick microbiome. We predicted the metabolic profiling of each sample using the state-of-the-art metagenomics tool PICRUSt2. The results showed that the microbiomes of all I. scapularis samples share only 80 taxa (24.6%, total 324), while out of the 342 metabolic pathways predicted, 82.7%, were shared by all the ticks. Borrelia-infected larvae lack 15.4% of pathways found in the microbiome of uninfected nymphs. Taxa contribution analysis showed that the functional microbiome of uninfected ticks was highly redundant, with, in some cases, up to 198 bacterial taxa contributing to a single pathway. However, Borrelia-infected larvae had a smaller redundancy with 6.7% of pathways provided by more than 100 genera, while 15.7-19.2% of pathways were provided by more than 100 genera in the two cohorts of uninfected ticks. In addition, we compared the functional profiles of three microbial communities from each data set, identified through a network-based approach, and we observed functional similarity between them. Based on the functional redundancy and functional similarity of the microbiome of ticks in different developmental stages and infection status, we concluded that the tick gut microbiota is a self-regulating community of very diverse bacteria contributing to a defined set of metabolic pathways and functions with yet unexplored relevance for tick fitness and/or bacterial community stability. We propose a change of focus in which the tick microbiome must be analyzed in all dimensions, highlighting their functional traits, instead of the conventional taxonomic profiling.

Anaplasma marginale is an obligate intraerythrocytic bacterium of bovines, responsible for large economic losses worldwide. It is mainly transmitted by Rhipicephalus (Boophilus) microplus ticks and, despite mounting evidence suggesting transovarial transmission, the occurrence of this phenomenon remains controversial. We evaluated the vector competence of R. microplus larvae vertically infected with A. marginale to transmit the bacterium to a naïve bovine. A subgroup of engorged female ticks collected from an A. marginale-positive animal was dissected and the presence of the pathogen in its tissues was confirmed. A second subgroup of ticks was placed under controlled conditions for oviposition. After confirming the presence of A. marginale in the hatched larvae, an experimental infestation assay was conducted. Larvae were placed on an A. marginale-free splenectomized calf. The bacterium was detected in the experimentally infested bovine 22 days post-infestation. We analyzed the A. marginale diversity throughout the transmission cycle using the molecular marker MSP1a. Different genotypes were detected in the mammalian and arthropod hosts showing a reduction of strain diversity along the transmission process. Our results demonstrate the vertical transmission of A. marginale from R. microplus females to its larvae, their vector competence to transmit the pathogen, and a bottleneck in A. marginale strain diversity.

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.

The control of vector-borne diseases is important to improve human and animal health worldwide. Malaria is one of the world's deadliest diseases and is caused by protozoan parasites of the genus Plasmodium, which are transmitted by Anopheles spp. mosquitoes. Recent evidences using Subolesin (SUB) and Akirin (AKR) vaccines showed a reduction in the survival and/or fertility of blood-sucking ectoparasite vectors and the infection with vector-borne pathogens. These experiments suggested the possibility of using AKR for malaria control.