Introduction
Insects have a complex immune machinery that has evolved as a response to their interaction in all their life-cycle stages with parasites and pathogens. Classic scholarly work has divided the insect immune response to invasive agents into humoral plasma-borne factors and cellular or haemocyte-linked molecules. These two types of response, which may act separately or in unison (Gillespie et al., Reference Gillespie, Kanost and Trenczek1997; Beckage, Reference Beckage2011), have received increasing attention among researchers in the last two decades.
One reason for the intense research on insect immunity is that insects are key players in diseases that directly or indirectly affect humans. For example, triatomines are vectors of Trypanosoma cruzi, the causative agent of Chagas disease (Rassi et al., Reference Rassi, Rassi and Neto2010). Triatomines include approximately 130 species that are haematophagous, feeding on vertebrate blood, especially that of small terrestrial and arboreal mammals (Schofield & Galvao, Reference Schofield and Galvao2009). This feeding habit and their preference for colonizing human dwellings make triatomines key agents in transmitting T. cruzi to humans and domestic animals.
As any other insect, triatomines are attacked by a variety of parasites and pathogens. Our understanding of how triatomines react to such agents can be used in a variety of contexts, including biological control. To our knowledge, critical information about immune mechanisms used by triatomines against attacking agents has not been gathered in a systematic fashion. Thus, in the present review, we summarize the still scarce information about how triatomines make use of their immune mechanisms against bacteria, virus, fungi and parasites (for a summary, see supplementary material, fig. 1). Our intention is twofold: (a) to provide a concise update of triatomine immune mechanisms during infection against these pathogens, and (b) to suggest potential areas of future research. Despite the fact that studies of triatomine immunity are scarce, we try to gain some insights from these studies to put forward some ideas of biological control. For a better reading of the present review, the immune response of triatomines is considered separately for each of their distinct attacking agents.
General factors involved in the immune response of insects
Insect immune machinery is composed by cells, molecules and reactions aimed to resist and/or ameliorate the cost of pathogens. A first defensive line is a tough one: the exoskeleton cuticle, epidermic physical and chemical properties, gut epithelium and reproductive accessory glands (Gillespie et al., Reference Gillespie, Kanost and Trenczek1997; Casteels, Reference Casteels, Brey and Hultmark1998). These components are capable of secreting lysozymes and cytotoxic compounds. A key feature in this immune machinery is the recognition of non-self-parts via pattern recognition receptors (PRRs) that identify pathogen-associated molecular patterns (PAMPs) (Gillespie et al., Reference Gillespie, Kanost and Trenczek1997). Recent research has shown that recognition mechanisms like this, allow the insect to recognize and respond to pathogens more effectively in secondary encounters in an analogous fashion to that of vertebrate memory (e.g. Dong et al., Reference Dong, Taylor and Dimopoulos2006; Cisarovsky et al., Reference Cisarovsky, Schmid-Hempel and Sadd2012; Nava-Sánchez et al., Reference Nava-Sánchez, Munguía-Steyer, González-Tokman and Córdoba-Aguilar2015). Once such recognition has taken place, then cellular and humoral immune responses are activated. Typical cellular responses are phagocytosis, nodulation and encapsulation, while humoral include antimicrobial systemic molecules, nitric oxide (NO) production, lysozymes (Gillespie et al., Reference Gillespie, Kanost and Trenczek1997). However, both cellular and humoral responses frequently act together. A key player here in immune response is the phenoloxidase (PO) cascade whose intermediate (e.g. reactive oxygen species) and final products (e.g. surrounding of pathogens using melanin) deal with the elimination (the case of reactive oxygen species) or isolation (the case of melanin) of pathogens (González-Santoyo & Córdoba-Aguilar, Reference González-Santoyo and Córdoba-Aguilar2012).
Bacteria
All instars of the triatomine life cycle ingest blood, which is sterile (i.e. free of bacteria). However, by inhaling air before ecdysis and through coprophagia, triatomines acquire bacteria that eventually reach the intestine (Beard et al., Reference Beard, Cordon-Rosales and Durvasula2002; Balczun et al., Reference Balczun, Knorr, Topal, Meiser, Kollien and Schaub2008; Sassera et al., Reference Sassera, Epis, Pajoro and Bandi2013). In fact, the intestinal microbiota in triatomines is composed principally of bacteria, which multiply rapidly (100–10,000-fold) after the ingestion of blood (Azambuja et al., Reference Azambuja, Feder and Garcia2004). However, to obtain some missing nutrients, symbiotic bacteria may also participate in triatomine metabolism either by providing particular dietetic elements (e.g. vitamin B) or by being digested (Beard et al., Reference Beard, Cordon-Rosales and Durvasula2002; Sassera et al., Reference Sassera, Epis, Pajoro and Bandi2013).
Included in this microbiota is Serratia marcescens, a bacteria whose development correlates with a decrease in T. cruzi populations, because it directly attacks the parasite membrane (Azambuja et al., Reference Azambuja, Feder and Garcia2004; Gourbière et al., Reference Gourbière, Dorn, Tripet and Dumonteil2012). The mechanism of this interaction is not entirely clear but, in response to the colonization of T. cruzi in the gut, S. marcescens seems to act as a haemolytic bacteria that lyses erythrocytes via the production of a pigment called prodigiosin, that ultimately impedes the establishment of T. cruzi, without killing the insect (Azambuja et al., Reference Azambuja, Feder and Garcia2004). We are unaware, however, whether this possible negative effect of S. marcescens has been implemented as a tool for biological control (e.g. using S. marcescens as a potential dietary component in triatomine blood sources). On the other hand, it has been observed that the insect immune system produces antimicrobial peptides (AMPs) such as defensins and lysozymes (see subsections ‘Defensins’ and ‘Lysozymes ’ below ) in response to a population increase of pathogens and symbiotic bacteria, an immune response that negatively affects trypanosomes (Gourbiére et al., Reference Gourbière, Dorn, Tripet and Dumonteil2012). Some immune components against bacteria are cited below.
Antimicrobial peptides
It is known that insects use a variety of AMPs, such as defensins, lysozymes, attacins, cecropins and prolixicins to combat Gram-positive or -negative bacteria. Production of AMPs takes place mainly in the fat body, haemocytes and digestive tract (Boulanger et al., Reference Boulanger, Bulet and Lowenberger2006; Vieira et al., Reference Vieira, Waniek, Mattos, Castro, Mello, Ratcliffe, Garcia and Azambuja2014). The primary role of AMPs in triatomine, as well as in other insects, is defending them against pathogens (including, but not limited to T. cruzi) (e.g. Ursic-Bedoya et al., Reference Ursic-Bedoya, Nazzari, Cooper, Triana, Wolff and Lowenberger2008). For example, Rhodococcus rhodnii (a genetically modified bacteria) produces Cecropin A, an AMP that eliminates T. cruzi in the insect (Beard et al., Reference Beard, Cordon-Rosales and Durvasula2002; Dotson et al., Reference Dotson, Plikaytis, Shinnick, Durvasula and Beard2003; Kollien et al., Reference Kollien, Fechner, Waniek and Schaub2003).
That pathogens have the ability to trigger AMPs in triatomines was elucidated by the presence of different types of bacteria such as Staphylococcus aureus (Gram-positive) or Escherichia coli (Gram-negative) (Vieira et al., Reference Vieira, Waniek, Mattos, Castro, Mello, Ratcliffe, Garcia and Azambuja2014). In the first case S. aureus was able to induce transcripts of mRNA for Defensin a and b. In relation to transcripts, a recent study in Rhodnius prolixus detected that the largest expression of transcripts for AMPs took place in the anterior midgut via the presence of Defensins or lysozymes. In contrast, a novel AMP, called Prolixicin, was detected in the posterior midgut (Vieira et al., Reference Vieira, Waniek, Mattos, Castro, Mello, Ratcliffe, Garcia and Azambuja2014).
Defensins
Defensins (with a molecular weight of 4 kDa) are cysteine-rich proteins that were first analysed in the vector R. prolixus (Lopez et al., Reference Lopez, Morales, Ursic, Wolff and Lowenberger2003; Araújo et al., Reference Araújo, Waniek, Stock, Mayer, Jansen and Schaub2006; Boulanger et al., Reference Boulanger, Bulet and Lowenberger2006). Defensins are highly widespread products in different insect orders that include Diptera, Hymenoptera, Hemiptera, Coleoptera, Lepidoptera, Odonata and Hemiptera. The primary role of defensins is to fight Gram-positive and -negative bacteria as well as fungi. Their mechanism of action is the formation of holes and canals that disrupt the cytoplasmic membrane of bacteria (Yi et al., Reference Yi, Chowdhury, Huang and Yu2014).
From the haemolymph of R. prolixus, three isoforms of genes encoding different defensins a, b and c have been isolated, with 46, 56 and 51 base pairs (bp), respectively. These peptides have sequences that are similar to those of the defensins found in other insects of the Hemipteran order, and are mainly used against Gram-positive bacteria (Lopez et al., Reference Lopez, Morales, Ursic, Wolff and Lowenberger2003). Interestingly, in R. prolixus, the peptide transcription did not take place immediately in the haemocoel, but it did in tissues that were not directly stimulated such as the intestine, suggesting a delayed or systemic transcription of defensins (Lopez et al., Reference Lopez, Morales, Ursic, Wolff and Lowenberger2003). Actually, this systemic effect could be seen in the expression of other AMPs (Ursic-Bedoya et al., Reference Ursic-Bedoya, Nazzari, Cooper, Triana, Wolff and Lowenberger2008). It can be inferred that the transcription of peptides was due to a signaling cascade, because such transcription did not occur immediately after inoculation (Lopez et al., Reference Lopez, Morales, Ursic, Wolff and Lowenberger2003). However, the molecules involved in this signalling cascade have not yet been described in detail (Lopez et al., Reference Lopez, Morales, Ursic, Wolff and Lowenberger2003).
Another vector in which defensins have been isolated is Triatoma brasiliensis – def1 and def2- (Araújo et al., Reference Araújo, Waniek, Stock, Mayer, Jansen and Schaub2006; Waniek et al., Reference Waniek, Jansen and Araújo2011). In the majority of insect taxa, the sequence of defensins begins with alanine–threonine aminoacids, which is similar to the sequence found in R. prolixus and T. brasiliensis (Lopez et al., Reference Lopez, Morales, Ursic, Wolff and Lowenberger2003; Araújo et al., Reference Araújo, Waniek, Stock, Mayer, Jansen and Schaub2006). This was supported by sequence alignment and identity of R. prolixus with other Hemiptera and Coleoptera (Lopez et al., Reference Lopez, Morales, Ursic, Wolff and Lowenberger2003). The fact that all these insect species, including triatomines, share such defensin sequences, imply that defensins have changed very little among insect lineages; thus, these defensins seem highly conserved enzymes. Despite the similarity in the structure of defensins, their expression varies in different host organs. The expression of def1 was very low in the small intestine (the site where ingested blood is digested), rectum and salivary glands, but high in the stomach (the site where the greatest quantity of symbionts exists) (Araújo et al., Reference Araújo, Waniek, Stock, Mayer, Jansen and Schaub2006). This suggests a role of this defensin in controlling symbionts in the intestinal tract (Araújo et al., Reference Araújo, Waniek, Stock, Mayer, Jansen and Schaub2006). The second defensin encoding gene, def2, showed a coding region of 282 bp, and its active form was similar to that of def1 (identity of 88.3%), suggesting similar functions.
Finally, other defensins, def3 and def4, have been studied in T. brasiliensis (Waniek et al., Reference Waniek, Castro, Sathler, Miceli, Jansen and Araújo2009). At days 3 and 5 post-feeding, def3 was principally expressed in the fat body, salivary glands and small intestine, and was activated mainly in the stomach and fat body (Araújo et al., Reference Araújo, Waniek, Stock, Mayer, Jansen and Schaub2006; Waniek et al., Reference Waniek, Castro, Sathler, Miceli, Jansen and Araújo2009).
Lysozymes
Lysozymes (with a molecular weight of 15 kDa) were the first AMPs isolated and purified from insects, initially in Galeria mellonella and Bombyx mori (Azambuja et al., Reference Azambuja, Mello, Feder, Carcavallo, Galíndez-Girón and Jurberg1997) and then corroborated in other insects orders (Fujita, Reference Fujita2004). These enzymes hydrolyse the β−1, 4-glycosidic bond between the N-acetylmuramic and N-acetylglucosamine acids of peptidoglycans that are present on the cellular wall of Gram-positive bacteria, including Bacillus megatarium and Micrococcus luteus (Azambuja et al., Reference Azambuja, Mello, Feder, Carcavallo, Galíndez-Girón and Jurberg1997; Kollien et al., Reference Kollien, Fechner, Waniek and Schaub2003). This hydrolysation causes cellular rupture (Azambuja et al., Reference Azambuja, Mello, Feder, Carcavallo, Galíndez-Girón and Jurberg1997; Araújo et al., Reference Araújo, Waniek, Stock, Mayer, Jansen and Schaub2006; Balczun et al., Reference Balczun, Knorr, Topal, Meiser, Kollien and Schaub2008; Ursic-Bedoya et al., Reference Ursic-Bedoya, Nazzari, Cooper, Triana, Wolff and Lowenberger2008).
Four groups of lysosymes have been described: chicken type (c), goose-type (g), invertebrate type (i) and viral type (v), with insects belonging to the c group (Araújo et al., Reference Araújo, Waniek, Stock, Mayer, Jansen and Schaub2006; Balczun et al., Reference Balczun, Knorr, Topal, Meiser, Kollien and Schaub2008). Although lysozymes are only active against Gram-positive bacteria, they can create a synergic effect when associated with other AMPs such as cecropins and attacins (Azambuja et al., Reference Azambuja, Mello, Feder, Carcavallo, Galíndez-Girón and Jurberg1997; Ursic-Bedoya et al., Reference Ursic-Bedoya, Nazzari, Cooper, Triana, Wolff and Lowenberger2008; Vieira et al., Reference Vieira, Waniek, Mattos, Castro, Mello, Ratcliffe, Garcia and Azambuja2014). This synergism implies an augmented antibacterial activity such as degradation of the bacterial cell-wall, lytic activity that affects the permeability of the outer membrane and extended action against Gram-negative bacteria such as E. coli (Engstrom et al., Reference Engstrom, Carlsson, Engstrom and Bennich1984; Azambuja et al., Reference Azambuja, Mello, Feder, Carcavallo, Galíndez-Girón and Jurberg1997; Kollien et al., Reference Kollien, Fechner, Waniek and Schaub2003). Another example of this synergism is the combination of different AMPs, extracted previously from other insect orders, which cause a potential toxic effect to T. cruzi (Fieck et al., Reference Fieck, Hurwitz, Kang and Durvasula2010).
Among haematophagous insects, the production of lysozymes has been detected in the salivary glands of Tsetse flies (Glossina spp.) and the digestive tract of R. prolixus (Azambuja et al., Reference Azambuja, Mello, Feder, Carcavallo, Galíndez-Girón and Jurberg1997; Fujita, Reference Fujita2004). Furthermore, an increase in the concentration of this enzyme was induced in the haemolymph of R. prolixus after a direct injection of Micrococcus lysodeicticus bacteria into the haemocoel (Azambuja & Garcia, Reference Azambuja and Garcia1987). Given all these sites where lysozymes have been detected, it is unclear where they are regulated. However, evidence in T. infestans suggests that lysozyme1 (lys1) is regulated in the digestive tract but only after ecdysis and feeding, and that its role is a digestive one (Kollien et al., Reference Kollien, Fechner, Waniek and Schaub2003). A second lysozyme was characterized in the intestine of T. infestans too, lysozyme2 (lys2), after a bloodmeal, and its catalytic active residues are valine and tyrosine, although it is unclear whether it is involved in immune functions (Balczun et al., Reference Balczun, Knorr, Topal, Meiser, Kollien and Schaub2008). Further research is needed to clarify whether there are other body regions where these lysozymes are regulated.
The function of lysozymes in triatomines is still unclear. Some evidence suggests that lysozymes seem involved in the digestion of symbiotic bacteria or with the movement of bacteria through the digestive tract. For example, in T. brasiliensis, lys1 shows a large expression in the stomach which, as aforementioned, is where bacteria multiply rapidly after a bloodmeal (Araújo et al., Reference Araújo, Waniek, Stock, Mayer, Jansen and Schaub2006). Another example comes from R. prolixus from which two lysozymes, RpLys-A and RpLys-B (with a molecular weight of 15.8 and 15.1 kDa, respectively), were found after inoculation with E. coli and M. luteus in the haemocoel (Ursic-Bedoya et al., Reference Ursic-Bedoya, Nazzari, Cooper, Triana, Wolff and Lowenberger2008). These lysozymes showed different expression in spite of their localization in the vector, where RpLys-A was among the enzymes in the digestive tract (anterior midgut), and their function correlated with digestion, whereas RpLys-B participates in the immune response and was located in the fat body (Ursic-Bedoya et al., Reference Ursic-Bedoya, Nazzari, Cooper, Triana, Wolff and Lowenberger2008). One final example is that of lysozyme RpLys-A which has a role in immune response after its activity was induced with blood that contained S. aureus or E. coli (Vieira et al., Reference Vieira, Waniek, Mattos, Castro, Mello, Ratcliffe, Garcia and Azambuja2014).
Cecropins
Cecropins are highly alkaline molecules with activity against Gram-positive and -negative bacteria, as well as fungi (Azambuja et al., Reference Azambuja, Mello, Feder, Carcavallo, Galíndez-Girón and Jurberg1997; Yi et al., Reference Yi, Chowdhury, Huang and Yu2014). Hultmark et al. (Reference Hultmark, Steiner, Rasmuson and Boman1980) inoculated the pupae of Hyalophora cecropia (Lepidoptera: Saturniidae) with genetically modified bacteria that induced the production of peptides with antibacterial activity. These peptides were called cecropins and have a molecular weight of 4 kDa (Azambuja et al., Reference Azambuja, Mello, Feder, Carcavallo, Galíndez-Girón and Jurberg1997). To date cecropins have not been identified in Hemiptera.
Regarding the use of cecropins in T. cruzi control, genetically modified bacteria, such as R. rhodnii, induced cecropin A production of H. cecropia in the lumen of the small intestine of R. prolixus, and these impeded the development of T. cruzi (Beard et al., Reference Beard, Dotson, Pennigton, Eichler, Cordon-Rosales and Durvasula2001). As expected, T. cruzi was able to develop in samples of R. prolixus that were not inoculated with genetically modified symbionts (Azambuja et al., Reference Azambuja, Mello, Feder, Carcavallo, Galíndez-Girón and Jurberg1997; Lopez et al., Reference Lopez, Morales, Ursic, Wolff and Lowenberger2003; Ursic-Bedoya et al., Reference Ursic-Bedoya, Nazzari, Cooper, Triana, Wolff and Lowenberger2008, Reference Ursic-Bedoya, Buchhop, Joy, Durvasula and Lowenberger2011). The inoculation of insects with transformed symbionts has been called paratransgenesis (Durvasula et al., Reference Durvasula, Gumbs, Panackal, Kruglov, Taneja, Kang, Cordon-Rosales, Richards, Whitham and Beard1999; Beard et al., Reference Beard, Dotson, Pennigton, Eichler, Cordon-Rosales and Durvasula2001, Reference Beard, Cordon-Rosales and Durvasula2002), and has been proposed as an alternative for control of Chagas disease through the expression of toxic cecropins in the insect vector to kill T. cruzi. Interestingly, also Cecropin A has been combined with other potential AMPs extracted from different insects: Apis mellifera (apidaecin and melittin), Xenopus laevis (magainin II), B. mori (moricin) and Penaeus monodon (penaidin) (Fieck et al., Reference Fieck, Hurwitz, Kang and Durvasula2010). The results showed a potential effect against T. cruzi through the inhibition of parasite growth (Fieck et al., Reference Fieck, Hurwitz, Kang and Durvasula2010).
Attacins and prolixicins
Attacins with a molecular weight of 20–23 kDa are a family of proteins rich in glycine whose activity is limited to a few Gram-negative bacteria such as E. coli (Engstrom et al., Reference Engstrom, Carlsson, Engstrom and Bennich1984; Azambuja et al., Reference Azambuja, Mello, Feder, Carcavallo, Galíndez-Girón and Jurberg1997). Of all the proteins in the insect humoral system, these possibly have the most specific bactericidal activity. This AMP was purified first from haemolymph of H. cecropia that was inoculated with bacteria, and two classes of attacins were described: basic (A–D) and acidic (E–F). The characterization of these attacins was achieved from insects such as lepidopteran and dipteran species (Yi et al., Reference Yi, Chowdhury, Huang and Yu2014). The fact that these attacins play a role in Glossina Tsetse flies against Trypanosoma brucei (Hu & Aksoy, Reference Hu and Aksoy2005), suggests that they can be used against T. cruzi in triatomines.
Recently, a glycine AMP called prolixicin was characterized in R. prolixus. Its sequence showed a region related to the diptericin/attacin family (Ursic-Bedoya et al., Reference Ursic-Bedoya, Buchhop, Joy, Durvasula and Lowenberger2011). This peptide showed a strong action against Gram-negative bacteria even when it did not show high activity (Ursic-Bedoya et al., Reference Ursic-Bedoya, Buchhop, Joy, Durvasula and Lowenberger2011). A possible site of expression for this AMP is the posterior midgut and so its function could be the avoidance of bacterial expansion (Vieira et al., Reference Vieira, Waniek, Mattos, Castro, Mello, Ratcliffe, Garcia and Azambuja2014).
Virus
The only virus known to attack triatomines is the Triatoma virus (TrV). The discovery of TrV was triggered by the death of fifth instar nymphs of T. infestans after feeding (Muscio et al., Reference Muscio, La Torre and Scodeller1987). Upon observing the intestinal content of the dead insects, spherical particles of 30 nm in diameter were found (Muscio et al., Reference Muscio, La Torre and Scodeller1987). The content was processed and used to inoculate fifth instar nymphs, which died within 36 h. The immediate symptom was hind leg paralysis (Muscio et al., Reference Muscio, La Torre and Scodeller1987). These spherical particles were characterized as uncoated viral particles whose viral genome is composed of a positive-sense single-stranded RNA (+ssRNA) (Muscio et al., Reference Muscio, La Torre and Scodeller1987). The capsid is composed of three main polypeptides of 33, 37 and 39 kDa and a minor one of 45 kDa (Muscio et al., Reference Muscio, La Torre and Scodeller1987). TrV replicates within the gut cells of triatomines (Rozas-Dennis et al., Reference Rozas-Dennis, La Torre, Muscio and Guérin2000).
In relation to the specificity of TrV, Rozas-Dennis et al. (Reference Rozas-Dennis, La Torre, Muscio and Guérin2000) reported that it not only infects T. infestans, but also T. platensis, T. delpontei, T. pallidipennis, T. rubrovaria and R. prolixus. However, TrV is not pathogenic for vertebrates (Querido et al., Reference Querido, Agirre, Marti, Guérin and Silva2013). The ultimate effects of TrV, belonging to the Dicistroviridae family (previously called the Picorna virus), are a high mortality rate, delayed development and reduced fecundity (Muscio et al., Reference Muscio, La Torre, Bonder and Scodeller1997; Rozas-Dennis et al., Reference Rozas-Dennis, La Torre, Muscio and Guérin2000).
TrV can be transmitted among triatomines transovarially and through the faecal–oral route (Muscio et al., Reference Muscio, Bonder, La Torre and Scodeller2000). The triatomine immune response to TrV is as yet unknown. It would be expected that triatomines rely on PO, NO and/or endosymbiotic bacteria in response to a TrV infection, as has been reported for other insects (Ourth & Renis, Reference Ourth and Renis1993; Johnson, Reference Johnson2015).
Fungi
There is a shortage of information about the fungi–triatomine relationship. R. prolixus was the first triatomines species from which fungi were isolated, first finding Nocardia rhodnii (Moraes et al., Reference Moraes, Junqueira, Costa, Celano and Coura2000) and later Aspergillus versicolor (Moraes et al., Reference Moraes, Junqueira, Costa, Celano and Coura2000, Reference Moraes, Junqueira, Celano, Lara da Costa and Rodrigues Coura2004). Moreover, the principal fungi from the digestive tract of triatomine adults and nymphs (including T. brasiliensis, T. infestans, T. pseudomaculata and T. sordida under laboratory conditions and T. vitticeps in wild specimens) were Aspergillus flavus, A. ochraceus, A. parasiticus, Fusarium sp., Trichoderma harzianum and Verticillium sp. (Moraes et al., Reference Moraes, Junqueira, Costa, Celano and Coura2000). In spite of the presence of these fungi, the affected triatomines showed no signs of infection (Moraes et al., Reference Moraes, Junqueira, Costa, Celano and Coura2000). In the digestive tract of nymphs and adults of Panstrongylus megistus, R. prolixus, R. neglectus and Dipetalogaster maxima, the following fungal species were also observed: Aspergillus niger, Penicillium corylophilum and Acremonium sp. (Moraes et al., Reference Moraes, Junqueira, Costa, Celano and Coura2000).
Interestingly, T. cruzi was not present when these fungi were found in nymphs, suggesting that natural fungal flora present in the digestive tract may influence T. cruzi colonization (Moraes et al., Reference Moraes, Junqueira, Celano, Lara da Costa and Rodrigues Coura2004). Actually, it is not clear if fungus could deal with T. cruzi directly, as has been shown in other vectors, such as Anopheles spp. infected with Plasmodium species (Thomas & Read, Reference Thomas and Read2007). Moraes et al. (Reference Moraes, Junqueira, Celano, Lara da Costa and Rodrigues Coura2004) just reported high susceptibility to both fungi and T. cruzi in P. megistus and R. prolixus, and low susceptibility of these pathogens in D. maxima and R. neglectus. Perhaps the immune response used by triatomines has the same effect on both the fungus and parasite. Further experimental research is needed to test whether the main function of these fungi is to prevent T. cruzi colonization.
In general, the fungus–insect interaction has been well documented. If a fungus can overcome the insect immune response, it proceeds to degrade the cuticle, leading to insect death (Thomas & Read, Reference Thomas and Read2007). Indeed, fungi have two principal strategies when facing the host immune response: cryptic forms of growth (blastospores) that evade host defence mechanisms, and the production of immunomodulatory substances (mycotoxins such as dextruxins) that inhibit the host immune response (Thomas & Read, Reference Thomas and Read2007). Furthermore, the two fungal strategies work complementarily: while the component present in the cell wall of the conidia (β−1, 3-glucans) acts as an immunosuppressor, blastospores avoid recognition by the insect immune system (Boucias & Pendland, Reference Boucias, Pendland, Boucias and Latgé1991). How these functions work in triatomines is as yet unknown.
As aforementioned, extremely little is known about the triatomine immune response to fungi. However, some details can be extrapolated from the study of other insects. Different types of immunomodulatory mycotoxins in insects are specific to each type of entomopathogenic fungus. For example, Metarhizium anisopliae produces dextruxins and Beauveria bassiana beauvericins (Rohlfs & Churchill, Reference Rohlfs and Churchill2011), both being important in pathogenesis (Boucias & Pendland, Reference Boucias, Pendland, Boucias and Latgé1991). The action of dextruxin A was evaluated in R. prolixus in the Malpighian tubule system, where it inhibits fluid secretion and leads to a decrease in electrical potential (Ruiz-Sanchez et al., Reference Ruiz-Sanchez, Orchard and Lange2010). This inhibitory capacity against fungi has been found in the insects of agricultural importance (Gillespie et al., Reference Gillespie, Burnett and Charnley2000), and likely also occurs in triatomines.
Fungi produce a number of enzymes (e.g. proteases, lipases, esterases and chitinases) that facilitate invasion of the haemocoel in a number of insects (Boucias & Pendland, Reference Boucias, Pendland, Boucias and Latgé1991; Thomas & Read, Reference Thomas and Read2007). The entire invasion process occurs within 24 h after the adhesion of the conidia to the cuticle and the secretion of dextruxin by the fungus. This mycotoxin acts as an inhibitor and alters haemocyte morphology (Avulova & Rosengaus, Reference Avulova and Rosengaus2011). Negative effects of dextruxins include a reduction of insect PO activity (see ‘Parasites’ subsection ‘The PO system’ below), and a decreased rate of phagocytosis and encapsulation (Rohlfs & Churchill, Reference Rohlfs and Churchill2011). The hyphal bodies, another form of reproduction in the host, initiate the production of a protective covering of collagen that encapsulates β−1, 3-glucans and in this way avoids the encapsulating immune response of the host (Avulova & Rosengaus, Reference Avulova and Rosengaus2011). The interactions at immune level between symbiotic or entomopathogenic fungi need to be assessed in triatomines.
Parasites
To date, the only parasites of triatomines whose biology has been studied are T. cruzi and Trypanosoma rangeli. It is known that T. cruzi is able to cause important physiological changes in triatomines such as a delay in nymphal development in T. infestans (Schaub, Reference Schaub1989), and a reduction in adult longevity in R. prolixus (Schaub, Reference Schaub1989). However, T. cruzi did not have such effects in Triatoma dimidiata (Schaub, Reference Schaub1989), although in this species it reduces bloodmeal ingestion in infected nymphs, which leads to an immunosuppression as the triatomine is not obtaining enough metabolites (e.g. iron; Schaub, Reference Schaub1989; Schaub et al., Reference Schaub, Meiser, Balczun and Mehlhorn2011). These pathological effects have not been considered by other authors and require further study to determine whether or not T. cruzi is capable of stressing its vector.
Antimicrobial peptides
Even when AMPs have been studied in the context of antibacterial function, some research has been done with T. cruzi (Ursic-Bedoya et al., Reference Ursic-Bedoya, Buchhop, Joy, Durvasula and Lowenberger2011; Waniek et al., Reference Waniek, Jansen and Araújo2011; Moreira et al., Reference Moreira, Waniek, Valente, Carvalho, Perales, Feder, Geraldo, Castro, Azambuja, Ratcliffe and Mello2014). These AMPs include defensins, lysozymes and prolixicins, which could participate in clearing of ingested blood meal and as signalling molecules (Boulanger et al., Reference Boulanger, Bulet and Lowenberger2006). For instance, it has been shown that the defensin def1 of T. brasiliensis fifth instar nymphs attenuates T. cruzi proliferation but does not eliminate the parasite (Araújo et al., Reference Araújo, Waniek, Stock, Mayer, Jansen and Schaub2006). Moreover, the role of def1 depends on the intestinal region: in the stomach it regulates the quantity of symbiotic organisms while in the small intestine it may control T. cruzi populations (Waniek et al., Reference Waniek, Jansen and Araújo2011).
Another AMP, prolixicin (with a molecular weight of 11 kDa), was recently isolated from R. prolixus. Paradoxically, it showed no activity against T. cruzi (Ursic-Bedoya et al., Reference Ursic-Bedoya, Buchhop, Joy, Durvasula and Lowenberger2011). This enzyme is produced in the fat body and the small intestine of adult insects as a response to infection by Gram-negative bacteria such as E. coli (Ursic-Bedoya et al., Reference Ursic-Bedoya, Buchhop, Joy, Durvasula and Lowenberger2011; Vieira et al., Reference Vieira, Waniek, Mattos, Castro, Mello, Ratcliffe, Garcia and Azambuja2014). A recent study demonstrated that the quantity of prolixicin in the small intestine was insufficient for eliminating T. cruzi. One reason for this action is that proteases present in the digestive tract of the host could affect the production of prolixicin peptide (Ursic-Bedoya et al., Reference Ursic-Bedoya, Buchhop, Joy, Durvasula and Lowenberger2011). The question of why R. prolixus still produces prolixicin despite its apparent inefficacy awaits further investigation. One possibility is that this response was useful in the evolutionary past but is no longer effective. The fact, however, that immune responses are energetically costly (Schmid-Hempel, Reference Schmid-Hempel2005) implies that if a response is not needed, it should be selected against. This does not seem to be the case for R. prolixus.
The PO system
PO is an enzyme involved in the oxidation of phenols and quinones. This oxidation consists of a cascade of reactions that hydrolyses monophenols and O-diphenols and oxidizes the latter to form O-quinones (Gregorio & Ratcliffe, Reference Gregorio and Ratcliffe1991; Cerenius et al., Reference Cerenius, Lee and Soderhall2008; González-Santoyo & Córdoba-Aguilar, Reference González-Santoyo and Córdoba-Aguilar2012). PO is highly reactive and covalently binds to proteins to form polymers like melanin that encapsulate pathogens. PO normally exists as an inactive precursor called proPO, found in the plasmatic fraction of the haemolymph or in haemocytes of invertebrates (Gomes et al., Reference Gomes, Feder, Garcia and Azambuja2003; Genta et al., Reference Genta, Souza, García and Azambuja2010; González-Santoyo & Córdoba-Aguilar, Reference González-Santoyo and Córdoba-Aguilar2012). PO can be activated by enzymes such as trypsin, chymotrypsin and components on the cellular wall of bacteria and fungi (Laughton & Siva-Jothy, Reference Laughton and Siva-Jothy2011).
PO acts against many invasive agents in insects (González-Santoyo & Córdoba-Aguilar, Reference González-Santoyo and Córdoba-Aguilar2012) but has only been assessed in a few species of triatomines. For example, as a response to the presence of T. rangeli in R. prolixus, higher levels of PO led to an increase in the number of lysozymes and haemocytes as well as to the formation of melanin nodules (Azambuja et al., Reference Azambuja, Feder, Mello, Gomes and Garcia1999). Interestingly, T. rangeli seems to have mechanisms for evading the negative effects of PO, according to the following evidence: (a) although T. rangeli is recognized and encapsulated by host defence cells, it is capable of surviving and utilizing haemocytes for proliferation (Azambuja et al., Reference Azambuja, Feder, Mello, Gomes and Garcia1999); (b) T. rangeli has been shown to inhibit PO activity in R. prolixus; (c) in R. prolixus adults that were infected orally with T. rangeli epimastigotes, the proPO system was inhibited at the haemolymph level, the site where the parasite develops (Gregorio & Ratcliffe, Reference Gregorio and Ratcliffe1991; Gomes et al., Reference Gomes, Feder, Garcia and Azambuja2003). This inhibition may actually be the explanation for the negative effect of invasive agents in spite of PO production that has been found a number of insect studies (reviewed by González-Santoyo & Córdoba-Aguilar, Reference González-Santoyo and Córdoba-Aguilar2012). Interestingly, the inhibitory action by T. rangeli may not be direct, but instead via the disruption of juvenile hormone regulation, which in turn would affect PO production (Nakamura et al., Reference Nakamura, Stiebler, Fantappié, Fialho, Masuda and Oliveira2007).
The assumed inhibition of PO by T. rangeli, however, needs to be considered in a broader context. PO production in triatomines is condition-dependent, and one key factor driving condition is diet (González-Santoyo & Córdoba-Aguilar, Reference González-Santoyo and Córdoba-Aguilar2012). Thus, whether a reduced PO production is prompted by T. rangeli or diet needs to be clarified. On the other hand, inhibitory action cannot be considered a generalized property in triatomines. For example, infection by T. cruzi was followed by a high PO activity in the host haemocoel during the first 24 h, but decreased after this time (Mello et al., Reference Mello, Garcia, Ratcliffe and Azambuja1995).
Nitric oxide
NO is a powerful free radical and a highly toxic gas produced by the oxidation of L-arginine to citrulline mediated by the NO synthase (NOS) enzyme (Rivero, Reference Rivero2006). Although NO has been well described as a powerful defence mechanism against parasites, the information is limited about the role played by NO (and free radicals in general) against bacteria, fungi or virus. In a first exploration of this topic, Whitten et al. (Reference Whitten, Mello, Gomes, Nigam, Azambuja, Garcia and Ratcliffe2001) studied two strains of T. rangeli (H14 and Choachi) in R. prolixus. These authors concluded that high levels of free radicals (including superoxide and NO) reduced parasite survival and completion of the life cycle. At least for the defence of R. prolixus against T. rangeli, this suggests a key role of free radicals.
More recently, and also in relation to R. prolixus, it was found that the expression of NO takes place in the fat body, haemocytes, haemolymph, stomach epithelium, small intestine, rectum and salivary glands (Whitten et al., Reference Whitten, Sun, Tew, Schaub, Soukou, Nappi and Ratcliffe2007), areas where T. cruzi and T. rangeli are present. However, both parasites seem to interact differently with respect to NO. Whereas the presence of T. cruzi activates NOS expression in haemocytes and the stomach, T. rangeli suppresses the expression of this molecule (Whitten et al., Reference Whitten, Mello, Gomes, Nigam, Azambuja, Garcia and Ratcliffe2001). One explanation is that the early stimulation of NO production in haemocytes in response to T. cruzi impedes its colonization in the haemocoel (Whitten et al., Reference Whitten, Mello, Gomes, Nigam, Azambuja, Garcia and Ratcliffe2001). On the other hand, the inhibition of NO in haemocytes and the fat body by T. rangeli likely represents a defence mechanism of the parasite that allows for its development in the haemocoel and therefore its later passage to the salivary glands (Whitten et al., Reference Whitten, Mello, Gomes, Nigam, Azambuja, Garcia and Ratcliffe2001). Furthermore, a high concentration of NO is induced in the rectum during a T. rangeli infection, which could force the parasite to leave the middle intestine and then complete its life cycle in the salivary glands (Whitten et al., Reference Whitten, Sun, Tew, Schaub, Soukou, Nappi and Ratcliffe2007). However, whether the migration of T. rangeli to these glands is a consequence of escaping the triatomine's immune response or a use of NO by the parasite as a signal to complete its life cycle in the glands is unknown.
There are other much less studied effects of nitrogen-based compounds involved in triatomine immune defence. The high concentration of nitrites in the small intestine could possibly avoid penetration of the intestinal epithelium by T. cruzi (Whitten et al., Reference Whitten, Sun, Tew, Schaub, Soukou, Nappi and Ratcliffe2007; Castro et al., Reference Castro, Moraes, Gonzalez, Ratcliffe, Azambuja and Garcia2012), which would block its dissemination to the haemocoel. Though it is unclear why nitrite concentrations decrease in the small intestine following infection by T. rangeli (Whitten et al., Reference Whitten, Sun, Tew, Schaub, Soukou, Nappi and Ratcliffe2007), the reason may lie in an inhibitory effect similar to that that occurs with PO. Further studies are needed on the role of NO and its oxygen intermediates in the capacity of triatomines to combat parasitic infections.
Lectins (agglutinins)
Lectins or agglutinins are glycoproteins that have the capacity to agglutinate cells and precipitate glycoconjugates (Azambuja et al., Reference Azambuja, Mello, Feder, Carcavallo, Galíndez-Girón and Jurberg1997). Lectins are found in the haemolymph and digestive tract of a number of invertebrates, and their synthesis is carried out principally in haemocytes and the fat body (Azambuja et al., Reference Azambuja, Feder and Garcia2004). It has been reported that they can act as opsonins, facilitating phagocytosis by haemocytes (Azambuja et al., Reference Azambuja, Mello, Feder, Carcavallo, Galíndez-Girón and Jurberg1997).
Very little is known about the action of lectins in triatomines. A study that described the activity of agglutinins in the intestine and haemolymph of R. prolixus suggested a role for these glycoproteins in the regulation of parasite-vector interactions (Mello et al., Reference Mello, Garcia, Ratcliffe and Azambuja1995; Ratcliffe et al., Reference Ratcliffe, Nigam, Mello, Garcia and Azambuja1996). Mello et al. (Reference Mello, Garcia, Ratcliffe and Azambuja1995) showed that there are differences in the agglutination of three different strains of T. cruzi – Dm28c, CI and Y – in the stomach as well as the haemolymph of R. prolixus, and that these differences correlate with a variation in the carbohydrates on the surface of the parasite. The ability to agglutinate trypanosomes may depend on the amount of lectins produced by the host. T. infestans has more lectins than R. prolixus, and the former has greater resistance to infection by T. rangeli (Azambuja et al., Reference Azambuja, Feder, Mello, Gomes and Garcia1999).
Cellular defence mediated by haemocytes
The mechanisms of cellular defence are mediated mainly by haemocytes in insects. Once again, very little information is available on this subject in relation to triatomines. The types of haemocytes in insects vary among taxa, with four main classes that are based on their morphology, histochemistry and functional characteristics (Lavine & Strand, Reference Lavine and Strand2002; Borges et al., Reference Borges, Santos, Furtado and Figueiredo2008; Strand, Reference Strand and Beckage2008): (a) granulocytes, which have a function of phagocytosis; (b) plasmatocytes, which adhere to parasites to form cellular capsules; (c) oenocitoids, which contain precursors of PO that are involved in the production of melanin; and (d) prohaemocytes, which are stem cells. It is now known that there are six types of haemocytes in R. prolixus, R. neglectus, Triatoma infestans, P. megistus and D. maxima: prohaemocytes, plasmocytes, cistocysts, oenocytoids, adipohaemocytes and giant granular cells (Azambuja et al., Reference Azambuja, Mello, Feder, Carcavallo, Galíndez-Girón and Jurberg1997).
Concerning the morphological and functional characterization of cell types involved in the immune response, five different haemocytes were identified in R. prolixus: prohaemocytes, plasmatocytes, oenocytoids, adipocytes and granulocytes (Borges et al., Reference Borges, Santos, Furtado and Figueiredo2008). Plasmatocytes and prohaemocytes are the most abundant cells, perhaps due to their direct action against bacteria via phagocytosis. On the other hand, granulocytes and oenocytoids also changed in response to S. aureus bacteria. In response to this bacterium no events concerning melanization were observed (Borges et al., Reference Borges, Santos, Furtado and Figueiredo2008). Despite the lack of evidence, it is likely that triatomine haemocytes are involved in other typical insect haemocyte functions such as nodule formation and encapsulation. Clearly, studying functions of triatomine haemocytes is an open research line.
Signalling mechanisms in triatomines/insect immune response
Our knowledge of signalling mechanism in triatomines is highly limited but one can gain some insights from other taxa (Hoffman & Reichhart, Reference Hoffman and Reichhart2002). Similar to other insects, triatomines are likely to rely on three principal pathways to attack pathogens such as bacteria and fungi: IMD, Toll and Jak-STAT (Kingsolver & Hardy, Reference Kingsolver and Hardy2012). To trigger any of these pathways, it is necessary to recognize foreign agents and one mechanism is via the PAMPs present on the pathogen's surface. For this, insects use host PRRs which have been identified such as peptidoglycan recognition proteins (PGRPs), Gram-negative binding proteins, thioester containing proteins, scavenger receptors, C-type lectins, galactosidose-binding lectins and fibrinogen-like domain molecules (Michel & Kafatos, Reference Michel and Kafatos2005; Kingsolver & Hardy, Reference Kingsolver and Hardy2012). The anterior PRRs could be circulating in the haemolymph and bounding to cells (Michel & Kafatos, Reference Michel and Kafatos2005). In R. prolixus (Vieira et al., Reference Vieira, Waniek, Mattos, Castro, Mello, Ratcliffe, Garcia and Azambuja2014) and other insects (Hoffman & Reichhart, Reference Hoffman and Reichhart2002; Kingsolver & Hardy, Reference Kingsolver and Hardy2012), the IMD pathway could be activated by Lipopolysaccharides present in Gram-negative bacteria, and then the induction of different AMPs like defensins could be achieved. The Toll pathway seems to be induced by the presence of PAMPs like β−1, 3-glucans in fungi, and peptidoglycans present in Gram-positive bacteria, that bind with their respective recognition protein: βGRP or PGRP (Boulanger et al., Reference Boulanger, Bulet and Lowenberger2006; Ursic-Bedoya & Lowenberger, Reference Ursic-Bedoya and Lowenberger2007). These two pathways induce a nuclear factor κB (NF-κB)-like transcription factor (Boulanger et al., Reference Boulanger, Bulet and Lowenberger2006), that could be involved in proteolytic cascades such as melanisation, coagulation, phagocytosis and encapsulation by haemocytes (Ursic-Bedoya & Lowenberger, Reference Ursic-Bedoya and Lowenberger2007).
With respect to T. cruzi or T. rangeli, there is scarce evidence of pathways activated. Worth mentioning is the study of Ursic-Bedoya & Lowenberger (Reference Ursic-Bedoya and Lowenberger2007) in R. prolixus. These authors found that the expression of genes in response to bacteria (E. coli and M. luteus) or T. cruzi, led to the identification of six molecules involved in the above pathways – Transferrin, Nitrophorins, β−1, 3-glucan recognition protein (GRP), haemolymph protein, Rel/Dorsal and Mucin/Peritrophin like.
Recently, the transcriptome of the digestive tract of R. prolixus has been sequenced (Ribeiro et al., Reference Ribeiro, Genta, Sorgine, Logullo, Mesquita, Paiva-Silva, Majerowicz, Medeiros, Koerich, Terra, Ferreira, Pimentel, Bisch, Leite, Diniz, Lídio da, Junior, Da Silva, Araujo, Caroline, Gandara, Brosson, Salmon, Bousbata, González-Caballero, Silber, Alves-Bezerra, Gondim, Silva-Neto, Atella, Araujo, Dias, Polycarpo, Vionette-Amaral, Fampa, Melo, Tanaka, Balczun, Oliveira, Gonçalves, Lazoski, Rivera-Pomar, Diambra, Schaub, Garcia, Azambuja, Braz and Oliveira2014). This study found a high number of genes expressed belonging to the Toll pathway in contrast with other groups of insects such as Dipterans. On the other hand, a low abundance of transcripts related to IMD and STAT pathways were present in the digestive tract (Ribeiro et al., Reference Ribeiro, Genta, Sorgine, Logullo, Mesquita, Paiva-Silva, Majerowicz, Medeiros, Koerich, Terra, Ferreira, Pimentel, Bisch, Leite, Diniz, Lídio da, Junior, Da Silva, Araujo, Caroline, Gandara, Brosson, Salmon, Bousbata, González-Caballero, Silber, Alves-Bezerra, Gondim, Silva-Neto, Atella, Araujo, Dias, Polycarpo, Vionette-Amaral, Fampa, Melo, Tanaka, Balczun, Oliveira, Gonçalves, Lazoski, Rivera-Pomar, Diambra, Schaub, Garcia, Azambuja, Braz and Oliveira2014). An explanation for these results is that lysozymes and lectins (Toll-regulated) are used more due to their defensive action in comparison with IMD and STAT pathways whose products are for digestive functions (Ribeiro et al., Reference Ribeiro, Genta, Sorgine, Logullo, Mesquita, Paiva-Silva, Majerowicz, Medeiros, Koerich, Terra, Ferreira, Pimentel, Bisch, Leite, Diniz, Lídio da, Junior, Da Silva, Araujo, Caroline, Gandara, Brosson, Salmon, Bousbata, González-Caballero, Silber, Alves-Bezerra, Gondim, Silva-Neto, Atella, Araujo, Dias, Polycarpo, Vionette-Amaral, Fampa, Melo, Tanaka, Balczun, Oliveira, Gonçalves, Lazoski, Rivera-Pomar, Diambra, Schaub, Garcia, Azambuja, Braz and Oliveira2014).
Conclusions
Scarce information is available on the immune response of triatomines to pathogens, whether in terms of quality (i.e. the description of the immune machinery or pathogenic agents in triatomines) or quantity (i.e. of the ca. 130 triatomine species, no more than 10 have been studied). This can be contrasted to the abundant research on mosquitoes (Cator et al., Reference Cator, Lynch, Read and Thomas2012; Clayton et al., Reference Clayton, Dong and Dimopoulos2014), whose interactions with other infective agents are well known. For example, by combining the information available on the antiviral responses of mosquitoes and genomics techniques, it may be possible to disrupt RNA interference and the JAK/STAT pathway, thus leaving mosquitoes immune depressed against several arbovirus types (reviewed by Rückert et al., Reference Rückert, Bell-Sakyi, Fazakerley and Fragkoudis2014). On the other hand, studies on the interaction between triatomines and invading agents have centred on describing the latter, providing very little information on the effects of invasion or the immune response of triatomines. In the case of fungi, for example, a number of species have been described, but their effects and, consequently, the immune response by triatomines are completely absent.
One does not need to start from scratch to understand triatomine defence mechanisms, as there is already information on other insects (e.g. agricultural insect pests; Shah & Pell, Reference Shah and Pell2003). One case is the study of Ribeiro et al. (Reference Ribeiro, Genta, Sorgine, Logullo, Mesquita, Paiva-Silva, Majerowicz, Medeiros, Koerich, Terra, Ferreira, Pimentel, Bisch, Leite, Diniz, Lídio da, Junior, Da Silva, Araujo, Caroline, Gandara, Brosson, Salmon, Bousbata, González-Caballero, Silber, Alves-Bezerra, Gondim, Silva-Neto, Atella, Araujo, Dias, Polycarpo, Vionette-Amaral, Fampa, Melo, Tanaka, Balczun, Oliveira, Gonçalves, Lazoski, Rivera-Pomar, Diambra, Schaub, Garcia, Azambuja, Braz and Oliveira2014) who identified the transcripts of the intestinal tract of R. prolixus. As indicated before, these authors found that Toll pathway-related genes that code for lysozymes and lectins were more highly expressed in comparison with other insect orders. The immune responses dictated by these genes were presumed from studies in other insects (Ribeiro et al., Reference Ribeiro, Genta, Sorgine, Logullo, Mesquita, Paiva-Silva, Majerowicz, Medeiros, Koerich, Terra, Ferreira, Pimentel, Bisch, Leite, Diniz, Lídio da, Junior, Da Silva, Araujo, Caroline, Gandara, Brosson, Salmon, Bousbata, González-Caballero, Silber, Alves-Bezerra, Gondim, Silva-Neto, Atella, Araujo, Dias, Polycarpo, Vionette-Amaral, Fampa, Melo, Tanaka, Balczun, Oliveira, Gonçalves, Lazoski, Rivera-Pomar, Diambra, Schaub, Garcia, Azambuja, Braz and Oliveira2014). Studies like this can serve to design studies on potential triatomine responses. Some strategies that can be explored are the following: (a) the use of already described powerful infective organisms, such as the bacteria R. rhodnii (Dotson et al., Reference Dotson, Plikaytis, Shinnick, Durvasula and Beard2003; Kollien et al., Reference Kollien, Fechner, Waniek and Schaub2003) and S. marcescens (Azambuja et al., Reference Azambuja, Feder and Garcia2004; Gourbiére et al., Reference Gourbière, Dorn, Tripet and Dumonteil2012), as well as TrV (Muscio et al., Reference Muscio, La Torre and Scodeller1987) and fungi (Moraes et al., Reference Moraes, Junqueira, Costa, Celano and Coura2000), to kill triatomines; and (b) enhancement of the triatomine immune response to trypanosomes, which consists of cecropins (Beard et al., Reference Beard, Dotson, Pennigton, Eichler, Cordon-Rosales and Durvasula2001), defensins, lysozymes and prolixicins (Mello et al., Reference Mello, Garcia, Ratcliffe and Azambuja1995; Gomes et al., Reference Gomes, Feder, Thomas, Garcia and Azambuja1999; Ursic-Bedoya et al., Reference Ursic-Bedoya, Buchhop, Joy, Durvasula and Lowenberger2011; Waniek et al., Reference Waniek, Jansen and Araújo2011; Moreira et al., Reference Moreira, Waniek, Valente, Carvalho, Perales, Feder, Geraldo, Castro, Azambuja, Ratcliffe and Mello2014), free radicals (Whitten et al., Reference Whitten, Mello, Gomes, Nigam, Azambuja, Garcia and Ratcliffe2001) and lectins (Azambuja et al., Reference Azambuja, Feder, Mello, Gomes and Garcia1999).
Finally and for biological control purposes, several other strategies can be explored. Now that the genome of T. cruzi has been elucidated (Grisard et al., Reference Grisard, Teixeira, De Almeida, Stoco, Gerber, Talavera-López, Lima, Andersson and De Vasconcelos2014), researchers should be able to establish new types of adaptive associations between triatomines and trypanosomes, as have been described between humans and trypanosomes (Sistrom et al., Reference Sistrom, Evans, Bjornson, Gibson, Balmer, Máser, Aksoy and Caccone2014; in this case using T. brucei). One necessary step here is to elucidate the genome of triatomines, which among other possibilities would allow for the identification of triatomine genes that respond to trypanosome infection, and the consequent use of this information to target the immune response for manipulation and biological control. Another possible strategy is to understand the biochemical intimacy of the T. cruzi–triatomine interaction. In this sense, Gutiérrez-Cabrera et al. (Reference Gutiérrez-Cabrera, Alejandre-Aguilar, Hernández-Martínez and Espinoza2014) identified the changes in glycoproteins that take place in the triatomine intestinal membrane, allowing for T. cruzi adhesion and development. With this information, such glycoprotein composition could perhaps be altered, leading to new strategies for the control of trypanosome establishment and colonization. By last, a multi-pathogen approach could be conceived. One example is the combined use of fungi and nematodes, as has been described for agricultural pests (e.g. Bedding & Molyneux, Reference Bedding and Molyneux1982). In this extent, results in those pests have shown that fungi can weaken the insect cuticle, which allows nematodes to penetrate (Bedding & Molyneux, Reference Bedding and Molyneux1982). Actually, one variable that can be added to this multipathogen approach is toxic elements such as what Fieck et al. (Reference Fieck, Hurwitz, Kang and Durvasula2010) when including different Cecropins.
Supplementary material
The supplementary material for this article can be found at http://www.journals.cambridge.org/S0007485315000504
Acknowledgements
A. Laura Flores-Villegas acknowledges the scholarship and financial support provided by the Consejo Nacional de Ciencia y Tecnología (CONACyT-326511). This paper constitutes a partial fulfillment of the doctoral work of A. Laura Flores-Villegas in the Doctorado en Ciencias Biológicas of the Universidad Nacional Autónoma de México (UNAM). M. Cabrera-Bravo, P. M. Salazar-Schettino and M. I. Bucio-Torres had a financial grant from PAPIIT-UNAM (IT 200913 and IN 211613), and so did A. Córdoba-Aguilar (IN 222312).
