Hexapod Assassins' Potion: Venom Composition and Bioactivity from the Eurasian Assassin Bug Rhynocoris iracundus.

Assassin bug venoms are potent and exert diverse biological functions, making them potential biomedical goldmines. Besides feeding functions on arthropods, assassin bugs also use their venom for defense purposes causing localized and systemic reactions in vertebrates. However, assassin bug venoms remain poorly characterized. We collected the venom from the assassin bug Rhynocoris iracundus and investigated its composition and bioactivity in vitro and in vivo. It caused lysis of murine neuroblastoma, hepatoma cells, and healthy murine myoblasts. We demonstrated, for the first time, that assassin bug venom induces neurolysis and suggest that it counteracts paralysis locally via the destruction of neural networks, contributing to tissue digestion. Furthermore, the venom caused paralysis and melanization of Galleria mellonella larvae and pupae, whilst also possessing specific antibacterial activity against Escherichia coli, but not Listeria grayi and Pseudomonas aeruginosa. A combinatorial proteo-transcriptomic approach was performed to identify potential toxins responsible for the observed effects. We identified neurotoxic Ptu1, an inhibitory cystin knot (ICK) toxin homologous to ω-conotoxins from cone snails, cytolytic redulysins homologous to trialysins from hematophagous kissing bugs, and pore-forming hemolysins. Additionally, chitinases and kininogens were found and may be responsible for insecticidal and cytolytic activities. We demonstrate the multifunctionality and complexity of assassin bug venom, which renders its molecular components interesting for potential biomedical applications.

Potent Activity of Hybrid Arthropod Antimicrobial Peptides Linked by Glycine Spacers.

Arthropod antimicrobial peptides (AMPs) offer a promising source of new leads to address the declining number of novel antibiotics and the increasing prevalence of multidrug-resistant bacterial pathogens. AMPs with potent activity against Gram-negative bacteria and distinct modes of action have been identified in insects and scorpions, allowing the discovery of AMP combinations with additive and/or synergistic effects. Here, we tested the synergistic activity of two AMPs, from the dung beetle Copris tripartitus (CopA3) and the scorpion Heterometrus petersii (Hp1090), against two strains of Escherichia coli. We also tested the antibacterial activity of two hybrid peptides generated by joining CopA3 and Hp1090 with linkers comprising two (InSco2) or six (InSco6) glycine residues. We found that CopA3 and Hp1090 acted synergistically against both bacterial strains, and the hybrid peptide InSco2 showed more potent bactericidal activity than the parental AMPs or InSco6. Molecular dynamics simulations revealed that the short linker stabilizes an N-terminal 310-helix in the hybrid peptide InSco2. This secondary structure forms from a coil region that interacts with phosphatidylethanolamine in the membrane bilayer model. The highest concentration of the hybrid peptides used in this study was associated with stronger hemolytic activity than equivalent concentrations of the parental AMPs. As observed for CopA3, the increasing concentration of InSco2 was also cytotoxic to BHK-21 cells. We conclude that AMP hybrids linked by glycine spacers display potent antibacterial activity and that the cytotoxic activity can be modulated by adjusting the nature of the linker peptide, thus offering a strategy to produce hybrid peptides as safe replacements or adjuncts for conventional antibiotic therapy.

Compelling Evidence for the Activity of Antiviral Peptides against SARS-CoV-2.

Multiple outbreaks of epidemic and pandemic viral diseases have occurred in the last 20 years, including those caused by Ebola virus, Zika virus, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The emergence or re-emergence of such diseases has revealed the deficiency in our pipeline for the discovery and development of antiviral drugs. One promising solution is the extensive library of antimicrobial peptides (AMPs) produced by all eukaryotic organisms. AMPs are widely known for their activity against bacteria, but many possess additional antifungal, antiparasitic, insecticidal, anticancer, or antiviral activities. AMPs could therefore be suitable as leads for the development of new peptide-based antiviral drugs. Sixty therapeutic peptides had been approved by the end of 2018, with at least another 150 in preclinical or clinical development. Peptides undergoing clinical trials include analogs, mimetics, and natural AMPs. The advantages of AMPs include novel mechanisms of action that hinder the evolution of resistance, low molecular weight, low toxicity toward human cells but high specificity and efficacy, the latter enhanced by the optimization of AMP sequences. In this opinion article, we summarize the evidence supporting the efficacy of antiviral AMPs and discuss their potential to treat emerging viral diseases including COVID-19.

Tick defensin γ-core reduces Fusarium graminearum growth and abrogates mycotoxins production with high efficiency.

Fusarium graminearum is a major fungal pathogen affecting crops of worldwide importance. F. graminearum produces type B trichothecene mycotoxins (TCTB), which are not fully eliminated during food and feed processing. Therefore, the best way to minimize TCTB contamination is to develop prevention strategies. Herein we show that treatment with the reduced form of the γ-core of the tick defensin DefMT3, referred to as TickCore3 (TC3), decreases F. graminearum growth and abrogates TCTB production. The oxidized form of TC3 loses antifungal activity, but retains anti-mycotoxin activity. Molecular dynamics show that TC3 is recruited by specific membrane phospholipids in F. graminearum and that membrane binding of the oxidized form of TC3 is unstable. Capping each of the three cysteine residues of TC3 with methyl groups reduces its inhibitory efficacy. Substitutions of the positively-charged residues lysine (Lys) 6 or arginine 7 by threonine had the highest and the lesser impact, respectively, on the anti-mycotoxin activity of TC3. We conclude that the binding of linear TC3 to F. graminearum membrane phospholipids is required for the antifungal activity of the reduced peptide. Besides, Lys6 appears essential for the anti-mycotoxin activity of the reduced peptide. Our results provide foundation for developing novel and environment-friendly strategies for controlling F. graminearum.

Anthelminthic Activity of Assassin Bug Venom against the Blood Fluke Schistosoma mansoni.

Helminths such as the blood fluke Schistosoma mansoni represent a major global health challenge due to limited availability of drugs. Most anthelminthic drug candidates are derived from plants, whereas insect-derived compounds have received little attention. This includes venom from assassin bugs, which contains numerous bioactive compounds. Here, we investigated whether venom from the European predatory assassin bug Rhynocoris iracundus has antischistosomal activity. Venom concentrations of 10-50 µg/mL inhibited the motility and pairing of S. mansoni adult worms in vitro and their capacity to produce eggs. We used EdU-proliferation assays to measure the effect of venom against parasite stem cells, which are essential for survival and reproduction. We found that venom depleted proliferating stem cells in different tissues of the male parasite, including neoblasts in the parenchyma and gonadal stem cells. Certain insect venoms are known to lyse eukaryotic cells, thus limiting their therapeutic potential. We therefore carried out hemolytic activity assays using porcine red blood cells, revealing that the venom had no significant effect at a concentration of 43 µg/mL. The observed anthelminthic activity and absence of hemolytic side effects suggest that the components of R. iracundus venom should be investigated in more detail as potential antischistosomal leads.

The insect antimicrobial peptide cecropin A disrupts uropathogenic Escherichia coli biofilms.

Current antibiotics cannot eradicate uropathogenic Escherichia coli (UPEC) biofilms, leading to recurrent urinary tract infections. Here, we show that the insect antimicrobial peptide cecropin A (CecA) can destroy planktonic and sessile biofilm-forming UPEC cells, either alone or when combined with the antibiotic nalidixic acid (NAL), synergistically clearing infection in vivo without off-target cytotoxicity. The multi-target mechanism of action involves outer membrane permeabilization followed by biofilm disruption triggered by the inhibition of efflux pump activity and interactions with extracellular and intracellular nucleic acids. These diverse targets ensure that resistance to the CecA + NAL combination emerges slowly. The antimicrobial mechanisms of CecA, thus, extend beyond pore-forming activity to include an unanticipated biofilm-eradication process, offering an alternative approach to combat antibiotic-resistant UPEC infections.

Antibacterial and antifungal activity of defensins from the Australian paralysis tick, Ixodes holocyclus.

Tick innate immunity involves humoral and cellular responses. Among the humoral effector molecules in ticks are the defensins which are a family of small peptides with a conserved γ-core motif that is crucial for their antimicrobial activity. Defensin families have been identified in several hard and soft tick species. However, little is known about the presence and antimicrobial activity of defensins from the Australian paralysis tick Ixodes holocyclus. In this study the I. holocyclus transcriptome was searched for the presence of defensins. Unique and non-redundant defensin sequences were identified and designated as holosins 1 - 5. The antimicrobial activity of holosins 2 and 3 and of the predicted γ-cores of holosins 1-4 (HoloTickCores 1-4), was assessed using Gram-negative and Gram-positive bacteria as well as the fungus Fusarium graminearum and the yeast Candida albicans. All holosins had molecular features that are conserved in other tick defensins. Furthermore holosins 2 and 3 were very active against the Gram-positive bacteria Staphylococcus aureus and Listeria grayi. Holosins 2 and 3 were also active against F. graminearum and C. albicans and 5 µM of peptide abrogate the growth of these microorganisms. The activity of the synthetic γ-cores was lower than that of the mature defensins apart from HoloTickCore 2 which had activity comparable to mature holosin 2 against the Gram-negative bacterium Escherichia coli. This study reveals the presence of a multigene defensin family in I. holocyclus with wide antimicrobial activity.

The Drosophila melanogaster antimicrobial peptides Mtk-1 and Mtk-2 are active against the malarial parasite Plasmodium falciparum.

Antimicrobial peptides (AMPs) are important components of the vertebrate and invertebrate innate immune systems. Although AMPs are widely recognized for their broad-spectrum activity against bacteria, fungi, and viruses, their activity against protozoan parasites has not been investigated in detail. In this study, we tested 10 AMPs from three different insect species: the greater wax moth Galleria mellonella (cecropin A-D), the fruit fly Drosophila melanogaster (drosocin, Mtk-1 and Mtk-2), and the blow fly Lucilia sericata (LSerPRP-2, LSerPRP-3 and stomoxyn). We tested each AMP against the protozoan parasite Plasmodium falciparum which is responsible for the most severe form of malaria in humans. We also evaluated the impact of these insect AMPs on mouse and pig erythrocytes. Whereas all AMPs showed low hemolytic effects towards mouse and pig erythrocytes, only D. melanogaster Mtk-1 and Mtk-2 significantly inhibited the growth of P. falciparum at low concentrations. Mtk-1 and Mtk-2 could therefore be considered as leads for the development of antiparasitic drugs targeting the clinically important asexual blood stage of P. falciparum.

Antiplasmodial activity of tick defensins in a mouse model of malaria.

Malaria is a mosquito-borne disease affecting millions of people mainly in Sub-Saharan Africa, Asia and some South American countries. Drug resistance to first-line antimalarial drugs (e.g. chloroquine, sulfadoxine-pyrimethamine and artemisinin) is a major constrain in malaria control. Antimicrobial peptides (AMPs) have shown promising results in controlling Plasmodium spp. parasitemia in in vitro and in vivo models of infection. Defensins are AMPs that act primarily by disrupting the integrity of cell membranes of invasive microbes. We previously showed that defensins from the tick Ixodes ricinus inhibited significantly the growth of P. falciparum in vitro, a property that was conserved during evolution. Here, we tested the activity of three I. ricinus defensins against P. chabaudi in mice. A single dose of defensin (120 µl of 1 mg/ml solution) was administered intravenously to P. chabaudi-infected mice, and the parasitemia was followed for 24 h post-treatment. Defensin treatment inhibited significantly the replication (measured as increases in parasitemia) of P. chabaudi after 1 h and 12 h of treatment. Furthermore, defensin injection was not associated with toxicity. These results agreed with the previous report of antiplasmodial activity of tick defensins against P. falciparum in vitro and justify further studies for the use of tick defensins to control malaria.

Bioactivity of Natural and Engineered Antimicrobial Peptides from Venom of the Scorpions Urodacus yaschenkoi and U. manicatus.

The spread of multidrug-resistant human pathogens has drawn attention towards antimicrobial peptides (AMPs), which are major players in the innate immune systems of many organisms, including vertebrates, invertebrates, plants and microbes. Scorpion venom is an abundant source of novel and potent AMPs. Here, we investigated natural and engineered AMPs from the scorpions Urodacus yaschenkoi and U. manicatus to determine their antimicrobial spectra as well as their hemolytic/cytotoxic activity. None of the AMPs were active against fungi, but many of them were active at low concentrations (0.25-30 µM) against seven different bacteria. Hemolytic and cytotoxic activities were determined using pig erythrocytes and baby hamster kidney cells, respectively. The amino acid substitutions in the engineered AMPs did not inhibit cytotoxicity, but reduced hemolysis and therefore increased the therapeutic indices. The phylogenetic analysis of scorpion AMPs revealed they are closely related and the GXK motif is highly conserved. The engineered scorpion AMPs offer a promising alternative for the treatment of multidrug-resistant bacterial infections and could be modified further to reduce their hemolytic/cytotoxic activity.

The Medical Potential of Antimicrobial Peptides from Insects.

Antimicrobial peptides (AMPs) are peptide-based effector molecules produced by the innate immune system to combat microbes. Insects produce the broadest repertoire of AMPs, and their potent antimicrobial activity in vitro and in vivo has promoted their development as alternatives to conventional antibiotics, in an attempt to address the threat of multidrug-resistant pathogens. Here we discuss current obstacles that hinder the therapeutic development of novel insect-derived AMPs, including potential cytotoxic, immunogenic and allergenic side effects, and the high costs of large-scale production. These challenges may be overcome by the falling costs of synthetic peptide analogs and the heterologous production of recombinant peptides in insect cells or plants (molecular pharming). Insect AMPs offer a promising alternative for the treatment of skin, eye and lung infections, and could also restore the susceptibility of multidrug-resistant pathogens to conventional antibiotics when used as combinatorial treatments. Insect AMPs can also be used as templates for the rational design of peptide mimetics to overcome the drawbacks of natural therapeutic peptides.

Antiplasmodial Activity Is an Ancient and Conserved Feature of Tick Defensins.

Ancestral sequence reconstruction has been widely used to test evolution-based hypotheses. The genome of the European tick vector, Ixodes ricinus, encodes for defensin peptides with diverse antimicrobial activities against distantly related pathogens. These pathogens include fungi, Gram-negative, and Gram-positive bacteria, i.e., a wide antimicrobial spectrum. Ticks do not transmit these pathogens, suggesting that these defensins may act against a wide range of microbes encountered by ticks during blood feeding or off-host periods. As demonstrated here, these I. ricinus defensins are also effective against the apicomplexan parasite Plasmodium falciparum. To study the general evolution of antimicrobial activity in tick defensins, the ancestral amino acid sequence of chelicerate defensins, which existed approximately 444 million years ago, was reconstructed using publicly available scorpion and tick defensin sequences (named Scorpions-Ticks Defensins Ancestor, STiDA). The activity of STiDA was tested against P. falciparum and the same Gram-negative and Gram-positive bacteria that were used for the I. ricinus defensins. While some extant tick defensins exhibit a wide antimicrobial spectrum, the ancestral defensin showed moderate activity against one of the tested microbes, P. falciparum. This study suggests that amino acid variability and defensin family expansion increased the antimicrobial spectrum of ancestral tick defensins.

Insect antimicrobial peptides: potential tools for the prevention of skin cancer.

Antimicrobial peptides/proteins (AMPs) are biologically active molecules with diverse structural properties that are produced by mammals, plants, insects, ticks, and microorganisms. They have a range of antibacterial, antifungal, antiviral, and even anticancer activities, and their biological properties could therefore be exploited for therapeutic and prophylactic applications. Cancer and cancer drug resistance are significant current health challenges, so the development of innovative cancer drugs with minimal toxicity toward normal cells and novel modes of action that can evade resistance may provide a new direction for anticancer therapy. The skin is the first line of defense against heat, sunlight, injury, and infection, and skin cancer is thus the most common type of cancer. The skin that has been exposed to sunlight is particularly susceptible, but lesions can occur anywhere on the body. Skin cancer awareness and self-efficacy are necessary to improve sun protection behavior, but more effective preventative approaches are also required. AMPs may offer a new prophylactic approach against skin cancer. In this mini review, we draw attention to the potential use of insect AMPs for the prevention and treatment of skin cancer.

The potential of the Galleria mellonella innate immune system is maximized by the co-presentation of diverse antimicrobial peptides.

Antimicrobial peptides (AMPs) are ubiquitous components of the insect innate immune system. The model insect Galleria mellonella has at least 18 AMPs, some of which are still uncharacterized in terms of antimicrobial activity. To determine why G. mellonella secretes a repertoire of distinct AMPs following an immune challenge, we selected three different AMPs: cecropin A (CecA), gallerimycin and cobatoxin. We found that cobatoxin was active against Micrococcus luteus at a minimum inhibitory concentration (MIC) of 120 µm, but at 60 µm when co-presented with 4 µm CecA. In contrast, the MIC of gallerimycin presented alone was 60 µm and the co-presentation of CecA did not affect this value. Cobatoxin and gallerimycin were both inactive against Escherichia coli at physiological concentrations, however gallerimycin could potentiate the sublethal dose of CecA (0.25 µm) at a concentration of 30 µm resulting in 100% lethality. The ability of gallerimycin to potentiate the CecA was investigated by flow cytometry, revealing that 30 µm gallerimycin sensitized E. coli cells by inducing membrane depolarization, which intensified the otherwise negligible effects of 0.25 µm CecA. We therefore conclude that G. mellonella maximizes the potential of its innate immune response by the co-presentation of different AMPs that become more effective at lower concentrations when presented simultaneously.

Tribolium castaneum defensins are primarily active against Gram-positive bacteria.

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.

Ixodes ricinus defensins attack distantly-related pathogens.

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 ß-hairpin and the loop bridging the α-helix and ß-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.

Defensins from the tick Ixodes scapularis are effective against phytopathogenic fungi and the human bacterial pathogen Listeria grayi.

BACKGROUND: Ixodes scapularis is the most common tick species in North America and a vector of important pathogens that cause diseases in humans and animals including Lyme disease, anaplasmosis and babesiosis. Tick defensins have been identified as a new source of antimicrobial agents with putative medical applications due to their wide-ranging antimicrobial activities. Two multigene families of defensins were previously reported in I. scapularis. The objective of the present study was to characterise the potential antimicrobial activity of two defensins from I. scapularis with emphasis on human pathogenic bacterial strains and important phytopathogenic fungi. METHODS: Scapularisin-3 and Scapularisin-6 mature peptides were chemically synthesised. In vitro antimicrobial assays were performed to test the activity of these two defensins against species of different bacterial genera including Gram-positive bacteria Staphylococcus aureus, Staphylococcus epidermidis, and Listeria spp. as well as Gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa along with two plant-pathogenic fungi from the genus Fusarium. In addition, the tissue-specific expression patterns of Scapularisin-3 and Scapularisin-6 in I. scapularis midgut, salivary glands and embryo-derived cell lines were determined using PCR. Finally, tertiary structures of the two defensins were predicted and structural analyses were conducted. RESULTS: Scapularisin-6 efficiently killed L. grayi, and both Scapularisin-3 and Scapularisin-6 caused strong inhibition (IC50 value: ~1 µM) of the germination of plant-pathogenic fungi Fusarium culmorum and Fusarium graminearum. Scapularisin-6 gene expression was observed in I. scapularis salivary glands and midgut. However, Scapularisin-3 gene expression was only detected in the salivary glands. Transcripts from the two defensins were not found in the I. scapularis tick cell lines ISE6 and ISE18. CONCLUSION: Our results have two main implications. Firstly, the anti-Listeria and antifungal activities of Scapularisin-3 and Scapularisin-6 suggest that these peptides may be useful for (i) treatment of antibiotic-resistant L. grayi in humans and (ii) plant protection. Secondly, the antimicrobial properties of the two defensins described in this study may pave the way for further studies regarding pathogen invasion and innate immunity in I. scapularis.

Identification and partial characterisation of new members of the Ixodes ricinus defensin family.

The hard-bodied tick Ixodes ricinus (castor bean tick) is the most common tick species in Europe. I. ricinus is a vector of the causative agents of diseases that affect humans and animals including tick-borne encephalitis, borreliosis, tick-borne fever and babesiosis. The innate immune system provides ticks with quite an efficient defence against some pathogenic microorganisms in the event of their penetration into the tick body or through the blood meal. Antimicrobial peptides (AMPs) constitute an important feature of the tick immune system. Defensins are a well-known class of AMPs. Members of the defensin family of proteins have been reported in several tick species. So far, only two defensins had been identified from I. ricinus. In this study, we report the identification of six novel putative defensins from I. ricinus at the genomic and transcriptional levels. At the genomic level they show differences with one being intronless, while others contain two introns. The expression pattern of these molecules in the salivary glands, midgut, ovary, Malpighian tubules, haemolymph and the tick cell line IRE/CTVM19 was determined. Some of them are tissue specific while others seem to be ubiquitous. Molecular and phylogenetic analyses show that these novel members of the I. ricinus defensin family differ phylogenetically and structurally; nevertheless, the cysteine pattern is highly conserved among the family members. Finally, antimicrobial-peptide prediction tools were used to predict putative antimicrobial activity of our defensins. They show putative antimicrobial activity mainly against Gram-positive bacteria. This study displays the diversity of the defensin family in the tick I. ricinus.

Functional and immunological relevance of Anaplasma marginale major surface protein 1a sequence and structural analysis.

Bovine anaplasmosis is caused by cattle infection with the tick-borne bacterium, Anaplasma marginale. The major surface protein 1a (MSP1a) has been used as a genetic marker for identifying A. marginale strains based on N-terminal tandem repeats and a 5'-UTR microsatellite located in the msp1a gene. The MSP1a tandem repeats contain immune relevant elements and functional domains that bind to bovine erythrocytes and tick cells, thus providing information about the evolution of host-pathogen and vector-pathogen interactions. Here we propose one nomenclature for A. marginale strain classification based on MSP1a. All tandem repeats among A. marginale strains were classified and the amino acid variability/frequency in each position was determined. The sequence variation at immunodominant B cell epitopes was determined and the secondary (2D) structure of the tandem repeats was modeled. A total of 224 different strains of A. marginale were classified, showing 11 genotypes based on the 5'-UTR microsatellite and 193 different tandem repeats with high amino acid variability per position. Our results showed phylogenetic correlation between MSP1a sequence, secondary structure, B-cell epitope composition and tick transmissibility of A. marginale strains. The analysis of MSP1a sequences provides relevant information about the biology of A. marginale to design vaccines with a cross-protective capacity based on MSP1a B-cell epitopes.