The discovery of insect fossils that are approximately 400 million years old, such as those of Rhyniella praecursor and Rhyniognatha hirsti, demonstrates that insects were among the first animals to conquer the terrestrial habitat (Scourfield, 1940; Ross, 2017). Their evolution represents a successful process represented by their earth-wide distribution, inhabiting every environment except marine habitats.

These evolutionary achievements are mainly due to some basic defense lines which have evolved over time. The immune system of these animals is frequently a subject of study and well described, whereas the adaptive immunity still relies on theories and only some molecules have been described (Kurtz and Franz, 2003; Little et al., 2005; Watson et al., 2005; Sadd and Schimdt-Hempel, 2006).

The innate immune system is divided into cellular and humoral responses that act together in three interconnected cascades (Hoffman et al., 1996). The first is composed of enzymatic activation, the second is the fast and temporary production of antimicrobial peptides (AMPs), both belonging to the humoral response, and the third refers to phagocytic and encapsulation defenses, mediated specially by haemocytes (Hoffmann, 1995; Strand and Pech, 1995; Gillespie et al., 1997; Blandin and Levashina, 2004; Cerenius and Soderhall, 2004; Theopold et al., 2004; Irving et al., 2005; Strand, 2008; Pasupuleti et al., 2012).

Among the soluble molecules related to the humoral immunity, a growing interest in the AMP class emerged as several molecules with unique properties were discovered (Stephens, 1962), such as cecropins (Steiner et al., 2009) and defensins (Ganz et al., 1985). AMPs are generally amphipathic and cationic and have high hydrophobic properties at physiological pH. Due to these physical characteristics, AMPs are more likely to form alpha helixes. This property may help with peptide penetration and disruption of negatively charged microbial membranes. As this is a charge-based interaction, AMPs may act in an independent protein-binding manner (De Simone and Souza, 2000), which makes them more likely to evade resistance mechanisms. The specificity of AMPs toward target cell membranes correlates not only with prokaryotic membrane composition but also with the topological arrangement of their lipids (Matsuzaki, 1999; Pushpanathan et al., 2013). Bacterial membranes contain large amounts of negatively charged phospholipids, whereas eukaryotic cells, specifically mammalian cell membranes, are composed almost exclusively of electrically neutral lipids (Matsuzaki, 1999). These features suggest why AMPs tend to be less toxic to eukaryotic cells.

Recent studies have demonstrated an increase in antimicrobial drug resistance. In 2000, bacteria resistant to one or more antibiotic classes started being described more commonly (Tavares, 2000; Santos Filho et al., 2002; Figueiredo et al., 2009; Zanol et al., 2010; Neves et al., 2011). The increasing rate of resistance and the diversity of resistant bacteria are two of the main justifications for research into the production of new antimicrobial drugs (Ferreira et al., 2001; Santos, 2004; Brito and Cordeiro, 2012).

The AMPs represent an interesting alternative to commercial antibiotics due to factors such as their low toxicity to eukaryotic cells, specificity for bacterial cell membranes, potential antifungal, antiparasitic, and antitumour activities, impact on cell differentiation, as well as vasculogenesis, antiobesity, antiviral, wound healing, and cell recruitment properties (Liang and Kim, 1999; Yi et al., 2014; Mahlapuu et al., 2016; Marxer et al., 2016; Mylonakis et al., 2016; Tonk et al., 2016).

Owing to their medical relevance in South and Central America, triatomine insects represent the main target of several fields of research ranging from public health to host–pathogen evolution. However, few studies have shown the role of AMPs in triatomine–pathogen interactions.

The isolation of the prolixin AMP from Rhodnius prolixus, expression of three different defensins by Pyrrhocoris apterus (Cociancich et al., 1993), expression of two defensins (def3 and def4) in several tissues of the barber bug Triatoma brasiliensis (Waniek et al., 2009), a description of trialysin expression in the salivary glands of Triatoma infestans (Assumpção et al., 2008) and two different types of digestive tract lysozymes (Kollien et al., 2003; Balczun et al., 2008; Flores-Villegas et al., 2015) provide evidence for the role of AMPs in triatomine immune defense mechanisms.

Although there is evidence of AMP production by triatomines, there are no published descriptions of antimicrobial molecules isolated from T. infestans haemolymph yet. Four AMPs were characterized among ten isolated from T. infestans blood (Diniz, 2016—unpublished data). The most relevant isolated finding was the presence of human fibrinopeptide A (FbPA) with antimicrobial activity.

Regarding the relevance of the description of AMPs, elucidation of their role in the invertebrate immune system and, consequently, development of new AMP-dependent drugs, our aim was to identify and determine the origin of AMPs isolated from the Chagas disease-transmitting vector T. infestans haemolymph. By combining mass spectrometry approaches with functional assays, our results provide evidence that T. infestans is able to assimilate molecules through feeding and use them as part of their immune system, probably functioning as AMPs circulating in the haemolymph.

The experiments were performed under the exemption of the Animal Research Ethics Committee (CEUAIB—Comitê de ética no uso de animais do Instituto Butantan) n° I-1345/15.

Although several studies have been performed aimed at a wider comprehension of the immune system of invertebrates, there are no consistent data about their adaptive immunity. It is known that there is no cell-based immunological memory, but some molecules produced by invertebrates have been described, such as molecules related to the immunoglobulin G superfamily (Watson et al., 2005). Other studies were able to demonstrate a certain specificity of the immune response in a second pathogen exposure (Kurtz and Franz, 2003; Little et al., 2005; Sadd and Schimdt-Hempel, 2006).

As insects do not have a relevant immune memory, a rapid response mechanism is required. Therefore, AMPs play an important role on the immunological response of these animals.

Commonly, AMP levels in the insect's haemolymph without bacterial or fungal contamination is low, increasing only with the stimuli of invasion/infection. As insects lack immunological memory, there is the necessity to activate intracellular pathways to induce the production of AMPs against each microorganism invasion. The main AMP production pathways activated are the Spaetzle-Toll—activated by fungi and gram-positive bacteria—and Imd—activated by gram-negative bacteria (Schmid-Hempel, 2005). Representing rapid responses, the peptide production does not exceed 8 h (Dunn, 1986). Confirming that FbPA has antimicrobial activity represents a huge step because it reinforces the idea of evolutionary improvements of the T. infestans immune system.

FbPA was obtained from the T. infestans haemolymph in the presence or absence of bacterial challenge and was active against several microorganisms in both cases. Thus it is possible to infer that the presence of this molecule in the insect's haemolymph is relevant to the insect's protection, whereas it is a known fact that AMPs can act in synergy and potentialize their effect over a target cell (Aaron et al., 2000; Yan and Hancock, 2001; Pamma et al., 2012; Doern, 2014; Nuding et al., 2014; Zerweck et al., 2017). It is still unknown whether T. infestans internalizes other molecules from the human blood. Diniz (2016) isolated ten AMPs and identified four among them, but unlike FbPA, none of the AMPs described belonged to the blood ingested.

Few studies have been performed to identify the antimicrobial activities of fibrinopeptides. Although the work performed by Påhlman et al. (2013) was unable to prove FbPA's antimicrobial action, Tang et al. (2002) demonstrated that seven molecules derived from human platelets (including fibrinopeptides A and B) exhibited antimicrobial activity. The authors also confirmed that FbPA exhibited antimicrobial activity against E. coli, S. aureus, C. albicans, and C. neoformans, while our work demonstrated its activity against M. luteus, P. aeruginosa, E. coli, C. parapsilosis, C. neoformans, C. tropicalis, P. farinosus, Cladosporium sp., and P. expansum.

The native FbPA isolated has activity at a 0.002–0.005 mg/mL concentration, while the synthetic FbPA has activity at a 0.1–0.2 mg/mL concentration. This discrepancy can be explained by the fact that the synthetic peptide has an amide group in its C-terminal portion, whereas the original sequence has a carboxyl group in the C-terminal portion. This change to the structure may impact the peptide–microorganism interaction, leading to a higher peptide concentration required to obtain the same effect. It might also explain some differences found between our research and others demonstrating FbPA's antimicrobial activity (Tang et al., 2002).

The synthetic FbPA exhibited the strongest antifungal activity against C. tropicalis at 5.2–10.5 μM in poor medium. Other fungi were incapable of growth after 72 h of incubation in either rich or poor medium at a higher peptide concentration (42–84 μM). A common recurrent infection that affects general insect species is induced by filamentous fungi and is described in several wild insects (Pagnocca et al., 2011; Biedermann et al., 2013; Bateman et al., 2015; Moubasher et al., 2017). These results indicate resistance specificity of the peptide against these microorganisms, suggesting that FbPA might play a role to help increase the efficiency of the insect's immunological barrier when facing these infections.

The inhibition assay of the FITC–FbPA conjugate indicates that it was active against M. luteus at a 0.02 mg/mL concentration. Although a higher concentration was necessary for it to be active, this result corroborates previous inhibition results: the native FbPA was active against the same M. luteus strain at 0.002 mg/mL and the synthetic variant at 0.2 mg/mL. This change might occur due to the coupling of the FITC to the peptide via its amino groups. As the sequence has three possible FITC binding sites, it could be covering one of the major active sites of the molecule, thus interfering with its action against the bacteria.

In humans, the cleavage of fibrinogen chains is via thrombin action. Thrombin cleaves a specific Arg-Gly at the C-terminus during the last part of the clot formation pathway, resulting in the release of FbPA and B (Riedel et al., 2011). Similarly, invertebrates produce molecules called fibrinogen-related peptides (FREP). Components of this class have been identified in ascidians, echinoderms, annelids, arthropods, nematodes, cnidarians, and molluscs (Wang et al., 2005; Fan et al., 2008; Sterba et al., 2011; Chai et al., 2012). Their function is related mainly to defense mechanisms such as agglutination and antimicrobial action (Hanington and Zhang, 2011). Although some similarities to specific portions of human fibrinogen have been identified, there are no known similarities between FREPs and human FbPA. Considered together with the fact that the T. infestans feeds on human blood, our results suggest that this insect can assimilate FbPA during feeding and internalize it in the midgut. In turn, it may use this peptide as an antimicrobial in its haemolymph. Similar strategies have already been described in different invertebrates (e.g., Fogaça et al., 1999; Riciluca et al., 2012). Beyond this capacity, some insects can absorb whole or partial molecules obtained during feeding (Jeffers and Roe, 2008). The presence of this internalization capability has been observed in Rhodnius prolixus (Wigglesworth, 1943), phylogenetically related to T. infestans that assimilates human hemoglobin.

The introduction of the insect proboscis into the host tissue causes local damage that activates immune responses as well as the clotting cascade, leading to thrombin activation and FbPA production (Scheraga, 2004). This pathway, however, is inhibited due to the release of thrombin inhibitors expressed within insect saliva (Zavalova et al., 2002). The presence of an anticoagulant buffer in the blood offered to the insects suggests that the cleavage can occur in the midgut, but this piece of information isolated does not represent a definite answer to this issue, because we analyzed only the blood with the presence of citrate buffer. It would be necessary to analyse the haemolymph of T. infestans after it feeds on a real organism instead of an in vitro system. Thus, it remains to be investigated whether FbPA internalized by T. infestans comes from endogenous cleavage within the host or another process.

Moreover, the role played by the peptide in the T. infestans haemolymph in vivo requires further investigation. Finally, further research is warranted as to the peptide's local action and its potential application as a therapeutic agent against infectious diseases.

Therefore, our results demonstrate the presence of antimicrobial active human FbPA in the haemolymph of the blood-sucking insect T. infestans. The hypothesis confirmed was that the presence of this molecule on the T. infestans haemolymph happens through intestinal absorption, through FITC–FbPA internalization experiments.

This discovery allows us to confirm that blood-sucking insects can gather different molecules from various resources as an attempt to defend themselves against pathogens. These results also contribute to a wider comprehension of the insect immune system, such as its role on an evolutive scale, and the results generate some necessary information to facilitate the discovery of new sources of antimicrobial peptides.

LD was mainly responsible for the development of all experiments and writing of the manuscript. PS participated during the purification experiments, mainly HPLC, and all of the internalization experiments. AM participated during the solid-phase synthesis of the peptide and mass spectrometry. All the authors contributed to the manuscript and development, and the entire group approves the entire manuscript content.