Rhodnius prolixus Hemolymph Immuno-Physiology: Deciphering the Systemic Immune Response Triggered by Trypanosoma cruzi Establishment in the Vector Using Quantitative Proteomics

Understanding the development of Trypanosoma cruzi within the triatomine vector at the molecular level should provide novel targets for interrupting parasitic life cycle and affect vectorial competence. The aim of the current study is to provide new insights into triatomines immunology through the characterization of the hemolymph proteome of Rhodnius prolixus, a major Chagas disease vector, in order to gain an overview of its immune physiology. Surprisingly, proteomics investigation of the immunomodulation of T. cruzi-infected blood reveals that the parasite triggers an early systemic response in the hemolymph. The analysis of the expression profiles of hemolymph proteins from 6 h to 24 h allowed the identification of a broad range of immune proteins expressed already in the early hours post-blood-feeding regardless of the presence of the parasite, ready to mount a rapid response exemplified by the significant phenol oxidase activation. Nevertheless, we have also observed a remarkable induction of the immune response triggered by an rpPGRP-LC and the overexpression of defensins 6 h post-T. cruzi infection. Moreover, we have identified novel proteins with immune properties such as the putative c1q-like protein and the immunoglobulin I-set domain-containing protein, which have never been described in triatomines and could play a role in T. cruzi recognition. Twelve proteins with unknown function are modulated by the presence of T. cruzi in the hemolymph. Determining the function of these parasite-induced proteins represents an exciting challenge for increasing our knowledge about the diversity of the immune response from the universal one studied in holometabolous insects. This will provide us with clear answers for misunderstood mechanisms in host–parasite interaction, leading to the development of new generation strategies to control vector populations and pathogen transmission.


Introduction
Vector-borne diseases (VBDs) account for more than 17% of all infectious diseases, causing more than 700,000 deaths annually [1]. They can be caused by either parasites, bacteria, or viruses. These diseases are mainly transmitted to humans and other mammalians by hematophagous arthropods, such as flies, ticks, and bugs. These arthropods acquire pathogens when they ingest a blood meal from an infected host and eventually transmit them to the next host through vectorial competence [2]. Subsequently, transmission represents a vulnerable and attractive point of control. In addition, vector control remains the most effective method for preventing VBDs transmission in the absence of a safe and effective preventive alternative [2]. Insect vectors have a powerful immune system that has evolved to respond to these different pathogenic agents and the immune challenges  (Table S1); (B) Representation of the distribution of total hemolymph proteome between intracellular and extracellular proteins. Extracellular proteins are recognized using OutCyte prediction tool either by the presence of a predicted signal peptide using the SignalP algorithm, transmembrane or potential unconventional protein secretions (UPS) from intracellular proteins. Numbers in brackets indicate the percentage of proteins in each category.
The hemolymph ensures the exchange between the different tissues and consequently its proteome constitutes their secretome. Therefore, we investigated the localization of the identified proteins. Thus, among the identified proteins 121 are secreted through the classical endoplasmic reticulum-Golgi pathway with the guidance of a signal peptide (Table S1 and Figure 1B). However, proteins could also reach the hemolymph by following an unconventional secretory pathway (UPS). By using OutCyte [30], we have predicted 120 more proteins which are secreted by UPS. The remaining 124 proteins were predicted to be intracellular from which 11 were predicted as transmembrane proteins ( Figure 1B).

Functional Annotation of Hemolymph Proteins
Hemolymph proteins were functionally characterized through GO term analysis using Panther and g:Profiler classification tools. The proteins were classified according to their GO molecular function and biological process ( Figure 2). Hence, proteins with peptidase activity are the most represented functional category in the hemolymph with 40 proteins mainly involved in proteolysis and cellular protein catabolism (Figure 2 and Table S1). Among the identified peptidases, 29 are predicted to be secreted.  (Table S1); (B) Representation of the distribution of total hemolymph proteome between intracellular and extracellular proteins. Extracellular proteins are recognized using OutCyte prediction tool either by the presence of a predicted signal peptide using the SignalP algorithm, transmembrane or potential unconventional protein secretions (UPS) from intracellular proteins. Numbers in brackets indicate the percentage of proteins in each category.
The hemolymph ensures the exchange between the different tissues and consequently its proteome constitutes their secretome. Therefore, we investigated the localization of the identified proteins. Thus, among the identified proteins 121 are secreted through the classical endoplasmic reticulum-Golgi pathway with the guidance of a signal peptide (Table S1 and Figure 1B). However, proteins could also reach the hemolymph by following an unconventional secretory pathway (UPS). By using OutCyte [30], we have predicted 120 more proteins which are secreted by UPS. The remaining 124 proteins were predicted to be intracellular from which 11 were predicted as transmembrane proteins ( Figure 1B).

Functional Annotation of Hemolymph Proteins
Hemolymph proteins were functionally characterized through GO term analysis using Panther and g:Profiler classification tools. The proteins were classified according to their GO molecular function and biological process ( Figure 2). Hence, proteins with peptidase activity are the most represented functional category in the hemolymph with 40 proteins mainly involved in proteolysis and cellular protein catabolism ( Figure 2 and Table S1). Among the identified peptidases, 29 are predicted to be secreted.
Proteins of cytoskeleton, such as actin, myosin, and tubulin are also among the most represented category with 38 proteins involved in actin filament organization and cytoskeleton assembly ( Figure 2B and Table S1). On the other hand, 28 proteins amongst the most abundant proteins are involved in lipid transport and metabolism ( Figure 2 and Table S1). Twenty-two proteins of this category are predicted to be secreted.
Twenty-five proteins of the extracellular matrix involved in cell adhesion and communication were identified, among which ten are secreted ( Figure 2). Twenty-four proteins are involved in carbohydrate metabolism of which fifteen are secreted ( Figure 2). In addition, eighteen proteins involved in protein folding, mainly related to heat shock cellular response have been identified, from which eight are predicted to be secreted ( Figure 2).
Seventeen odorant binding proteins (OBPs) have been sequenced in this work and sixteen of them are secreted mainly through a signal peptide. Two OBP isoforms (T1H845 and R4G3B3) are amongst the most abundant proteins in the hemolymph proteome ( Figure 3 and Table S1).  Table S1.
Proteins of cytoskeleton, such as actin, myosin, and tubulin are also among the most represented category with 38 proteins involved in actin filament organization and cytoskeleton assembly ( Figure 2B and Table S1). On the other hand, 28 proteins amongst the most abundant proteins are involved in lipid transport and metabolism (Figure 2 and Table S1). Twenty-two proteins of this category are predicted to be secreted.
Twenty-five proteins of the extracellular matrix involved in cell adhesion and communication were identified, among which ten are secreted ( Figure 2). Twenty-four proteins are involved in carbohydrate metabolism of which fifteen are secreted ( Figure 2). In addition, eighteen proteins involved in protein folding, mainly related to heat shock cellular response have been identified, from which eight are predicted to be secreted ( Figure  2).
Seventeen odorant binding proteins (OBPs) have been sequenced in this work and sixteen of them are secreted mainly through a signal peptide. Two OBP isoforms (T1H845 and R4G3B3) are amongst the most abundant proteins in the hemolymph proteome (Figure 3 and Table S1).
Sixteen proteins are directly related to the insect immune response according to GO biological process (Table S1), among which we have identified 2 lysozymes. T1I5M5 has a temporal expression profile repressed at 24 h post-feeding, while A9LN32 is expressed constitutively (Table S1). Moreover, their expression level is low compared to the other identified immune proteins ( Figure 3). Seven proteins involved in pathogen recognition and pathogen-associated molecular patterns (PAMPs) have been identified and are represented by a protein containing an MD-2-related lipid-recognition (ML) domain (T1HU92) related to the recognition of pathogen-related molecules, a putative c1q domain protein (R4FJF3), GH16 domain-containing proteins (B8LJ39, T1HGN7, and T1I650), and peptidoglycan recognition receptors rpPGRP-LC/LAa and rpPGRP-LC/LAb (Table S1). Seven antimicrobial peptides (AMPs) comprising attacin-C domain-containing protein (T1I7V7), diptericins (D6BJP6 and E6Y430), prolixin (B8QEI8), and defensin domain-containing proteins (T1I7B0, R4G8B6, and R4FNJ9) were identified ( Figure 3 and Table S1).
Eight proteins involved in amino acids metabolism, five in oxygen transport, four in signal transduction, four in vasodilatation, two putative salivary lipocalins (T1HF25 and R4FN82), and two putative triabin-like lipocalins (R4G4J2 and T1H7Q9) have also been identified in this work. The other functional categories are underrepresented and belong to different insect physiological processes ( Figure 2 and Table S1). Interestingly, 63 identified proteins are of unknown function due to the absence of GO and known functional domains, among which 45 are secreted (Table S1). Their expression profiles is steady at 6 h et 24 h ( Figure 3 and Table S1). The height of each bar is proportional to the LFQ intensity of expression of the corresponding protein, and each bar is related to the protein's UniProt ID. Protein categories in the right panel are listed from the histogram clockwise.

Effect of T. cruzi on the Dynamic of Hemolymph Protein Expression
The expression of 71 proteins is significantly modulated in the hemolymph following the ingestion of T. cruzi, among which, 45 proteins are regulated during the first hours following the establishment of the parasite infection and 26 proteins 24 h post-infection. Amid the differentially expressed proteins, 30 are significantly induced by T. cruzi and their expression increases up to 13-fold ( Figure 4 and Table S1), and 41 are down-regulated, with a decrease in their expression level up to 54-fold ( Figure 4 and Table S1). Sixteen proteins are directly related to the insect immune response according to GO biological process (Table S1), among which we have identified 2 lysozymes. T1I5M5 has a temporal expression profile repressed at 24 h post-feeding, while A9LN32 is expressed constitutively (Table S1). Moreover, their expression level is low compared to the other identified immune proteins ( Figure 3). Seven proteins involved in pathogen recognition and pathogen-associated molecular patterns (PAMPs) have been identified and are represented by a protein containing an MD-2-related lipid-recognition (ML) domain (T1HU92) related to the recognition of pathogen-related molecules, a putative c1q domain protein (R4FJF3), GH16 domain-containing proteins (B8LJ39, T1HGN7, and T1I650), and peptidoglycan recognition receptors rpPGRP-LC/LAa and rpPGRP-LC/LAb (Table S1). Seven antimicrobial peptides (AMPs) comprising attacin-C domain-containing protein (T1I7V7), diptericins (D6BJP6 and E6Y430), prolixin (B8QEI8), and defensin domain-containing proteins (T1I7B0, R4G8B6, and R4FNJ9) were identified ( Figure 3 and Table S1).
Interestingly, seven transglutaminases (TGc domain-containing proteins) with aminoacyl transferase activity, involved in post-translational modification have been identified (Table S1). Iron ion homeostasis process is also overrepresented by ten unique proteins, among which seven ferritins with ferroxidase activity and three transferrins. The expression level of these proteins is relatively high, in particular B8LJ43 ( Figure 3). The next GO term is related to the melanization process of pathogens, which includes five POs (T1I7V8, A0A1B2G385, T1HW62, A0A1B2G381, and T1HW22), which together with lipid transporters and OBS constitute the most abundant proteins of the hemolymph.
Eight proteins involved in amino acids metabolism, five in oxygen transport, four in signal transduction, four in vasodilatation, two putative salivary lipocalins (T1HF25 and R4FN82), and two putative triabin-like lipocalins (R4G4J2 and T1H7Q9) have also been identified in this work. The other functional categories are underrepresented and belong to different insect physiological processes ( Figure 2 and Table S1). Interestingly, 63 identified proteins are of unknown function due to the absence of GO and known functional domains, among which 45 are secreted (Table S1). Their expression profiles is steady at 6 h et 24 h ( Figure 3 and Table S1).

Effect of T. cruzi on the Dynamic of Hemolymph Protein Expression
The expression of 71 proteins is significantly modulated in the hemolymph following the ingestion of T. cruzi, among which, 45 proteins are regulated during the first hours following the establishment of the parasite infection and 26 proteins 24 h post-infection. Amid the differentially expressed proteins, 30 are significantly induced by T. cruzi and their expression increases up to 13-fold ( Figure 4 and Table S1), and 41 are down-regulated, with a decrease in their expression level up to 54-fold ( Figure 4 and Table S1).
Four proteases are up-regulated at 6 h post-infection, two of which are predicted to be secreted. These proteases are cysteine proteinase cathepsin L (T1HS87, T1HS97, and R4G406) and serine-type endopeptidase (T1H816) which expression is induced by two-fold in response to T. cruzi infection.
Among the two superoxide dismutases (SODs) induced by the parasite 6 h postinfection, only R4FMI6 is predicted to be secreted (Table S1). Interestingly, we noticed that T. cruzi strongly induces (seven-fold) the expression of the three defensins (T1I7B0, R4G8B6, and R4FNJ9) only at 6 h post-infection. In contrast, the expression of the two diptericins (E6Y430 and D6BJP6) is simultaneously strongly down-regulated by the parasite (eighteenfold) solely at 6 h post-infection ( Figure 4). The putative c1q domain protein (R4FJF3) shows a seven-fold expression level increase following infection and its expression profile follows that of defensins ( Figure 4 and Table S1).
Among the highly induced proteins at 6 h post-infection were rpPGRP-LC/LAa and rpPGRP-LC/Lab showing expression increase by twelve-fold at 6 h and are completely repressed at 24 h ( Figure 4 and Table S1).

Discussion
We investigated the effect of T. cruzi presence in the digestive tract of R. prolixus on the hemolymph proteome using quantitative label-free proteomics. Because T. cruzi colonization of the anterior midgut was previously shown to achieve peaks at 3 h post-infection and declines after 24 h post-infection [31], we compared hemolymph proteome from insect female adults at 6 h and 24 h post-blood feeding of uninfected and T. cruzi-infected blood. These time scales should help to decipher the early regulation of hemolymph protein expression in response to T. cruzi journey in the insect's digestive tract.
These analyses allowed the identification of (i) the comprehensive hemolymph proteome (ii) the temporal modulation of hemolymph protein expression at 6 h and 24 h postblood feeding, and (iii) hemolymph protein differential expression in response to T. cruzi infection.

R. prolixus Hemolymph Proteome Homeostasis under Blood Feeding Condition
Our analysis led to the identification of 269 proteins (71,54%) in the hemolymph which are constitutively expressed at 6 h and 24 h post-blood feeding ( Figure 1 and Table  S1). Further bioinformatics analysis of the molecular functions of these proteins revealed that immune, cytoskeleton organization, metabolic pathways, and redox are the most represented processes in the hemolymph proteome independently of the post-feeding time ( Figure 2). In addition, this work revealed for the first time the expression at the protein level of numerous R. prolixus genes with altered protein expression post-blood ingestion from 6 h to 24 h. Among them, several are of unknown function, which represents 16% of the total hemolymph proteome (Table S1).
As the hemolymph extraction process was achieved by cutting the insect's legs, some tissue damage of the cuticle and the fat body can lead to wound repair processes [32]. Moreover, we have applied a separation process of hemocytes from plasma by centrifugation which could result in cell lysis leading to hemocytes release of cellular proteins into the plasma. We therefore sorted the identified proteins according to whether they are predicted to be extracellular (hemolymph plasma proteins) or resulting from the alteration of The other up-regulated proteins at 6 h post-infection were pacifastin (R4G3U6), a putative chitinase (R4G8S4), an alpha-galactosidase (T1HM73), a ferritin (R4G4L4), three putative gamma-interferon-inducible lysosomal thiol reductases (T1HUV3, R4G4A3, and T1I217), a putative fasciclin (T1HD74), an OBP (T1I0U4), and five proteins with unknown functions (T1HWK7, T1I3G7, A0A4P6D8Z0, T1HKD6, and A0A4P6DAQ4) ( Figure 4 and Table S1).
At 24 h post-infection, we identified that the putative hemolymph juvenile hormone binding protein (JHBP) (R4FK69) and the chemosensory protein (T1IAF9) are the only two significantly up-regulated proteins in the hemolymph in response to T. cruzi infection, while their expression was unmodulated by the infection at 6 h ( Figure 4 and Table S1).
The expression of seventeen proteins is down-regulated in the hemolymph at 6 h following the ingestion of the parasite, among which four heat shock proteins 70 (HSP70) (T1I0D9, T1HA76, R4FLS6, and A0A4P6DEQ0) all being predicted intracellular. Their expression level decreases by twenty-fold upon infection ( Figure 4 and Table S1). Furthermore, the presence of the parasite represses the expression of a transglutaminase (T1HFV3) by twenty-fold, which has been identified only in the hemolymph at 6 h ( Table S1). The expression level of the predicted secreted peptidase S1 (T1I1M7) and two serpin domaincontaining proteins (T1IF83 and R4FJD2) decreases by 1.6-fold by the presence of T. cruzi ( Figure 4). The expression profile of the peptidase and their putative regulator are similar (Table S1). Surprisingly, the putative vitellogenin R4G3Y1 is strongly repressed by the parasite (Figure 4). Indeed, its expression decreases by 54-fold post-infection. The analysis of its expression profile indicates a prolonged down-regulation up to 24 h with a less apparent difference (five-fold).
Regarding the proteins affected negatively at 24 h by the parasite, we have identified four proteins involved in the metabolism of carbohydrates (R4G4U2, T1HB69, T1I0J6, and R4G5J0), a ferritin (R4G4L4), and a putative glutathione S-transferase (T1HUM1). Additionally, six unknown proteins are negatively modulated by T. cruzi ingestion, among which three are exclusively expressed at 24 h, such as T1I0S5, which expression decreases by ten-fold (Table S1).

Discussion
We investigated the effect of T. cruzi presence in the digestive tract of R. prolixus on the hemolymph proteome using quantitative label-free proteomics. Because T. cruzi colonization of the anterior midgut was previously shown to achieve peaks at 3 h post-infection and declines after 24 h post-infection [31], we compared hemolymph proteome from insect female adults at 6 h and 24 h post-blood feeding of uninfected and T. cruzi-infected blood. These time scales should help to decipher the early regulation of hemolymph protein expression in response to T. cruzi journey in the insect's digestive tract.
These analyses allowed the identification of (i) the comprehensive hemolymph proteome (ii) the temporal modulation of hemolymph protein expression at 6 h and 24 h post-blood feeding, and (iii) hemolymph protein differential expression in response to T. cruzi infection.

R. prolixus Hemolymph Proteome Homeostasis under Blood Feeding Condition
Our analysis led to the identification of 269 proteins (71,54%) in the hemolymph which are constitutively expressed at 6 h and 24 h post-blood feeding ( Figure 1 and Table S1). Further bioinformatics analysis of the molecular functions of these proteins revealed that immune, cytoskeleton organization, metabolic pathways, and redox are the most represented processes in the hemolymph proteome independently of the post-feeding time ( Figure 2). In addition, this work revealed for the first time the expression at the protein level of numerous R. prolixus genes with altered protein expression post-blood ingestion from 6 h to 24 h. Among them, several are of unknown function, which represents 16% of the total hemolymph proteome (Table S1).
As the hemolymph extraction process was achieved by cutting the insect's legs, some tissue damage of the cuticle and the fat body can lead to wound repair processes [32]. Moreover, we have applied a separation process of hemocytes from plasma by centrifugation which could result in cell lysis leading to hemocytes release of cellular proteins into the plasma. We therefore sorted the identified proteins according to whether they are predicted to be extracellular (hemolymph plasma proteins) or resulting from the alteration of hemocytes and surrounding tissues. Proteins were defined as extracellular if they are secreted through the classic endoplasmic reticulum-Golgi pathway with the guidance of a signal peptide or following UPS pathway. Based on these criteria, the dataset was composed of 241 extracellular proteins representing 64.09% of the total hemolymph proteome and 124 intracellular proteins representing 32.97% of the total proteome ( Figure 1B). The remaining eleven proteins (2.92%) were predicted transmembrane proteins ( Figure 1B). Previous comparative proteomic analyses of hemocytes and plasma of Dreissena polymorpha [33] and Mytilus edilus [34] revealed that up to 60% of the plasma proteins could result from hemocytes.

Exploring the Hemolymph Immunoproteins
Hemolymph is the site of important defense mechanisms in insects, relying on the humoral and cellular innate immune response. Insects have developed an array of common strategies to defend themselves against intruders as exemplified by the model Drosophila (for review, see [35]); however, recent studies have revealed unique immune adaptations across arthropods taxonomic groups [36]. In this section, a focus will be given to R. prolixus hemolymph immune proteins identified in this work, their temporal expression profiles post-blood feeding as well as their modulation by T. cruzi infection. 30%, and T1HGN7 with 29%. However, GNBP-1 was shown to be a GPI-anchored m brane protein [41] while T1I650 sequence, although predicted UPS, it is lacking a s peptide, a transmembrane domain, and GPI-anchor unless T1I650 N-terminal sequen incompletely annotated. In addition to their immune role, these proteins seem to have a digestive implic when they are expressed in the insect's midgut [42]. Interestingly, we have shown i comparative proteome of R. prolixus midgut [43,44] that T1HU92 (ML domain pro expression level increases in response to blood feeding. This protein is related to the re nition of pathogen-related molecules [45] and a homologous protein, which expre was also constitutive, was identified in the hemolymph proteome of A. gambiae [25]. I hemolymph, T1HU92 is expressed constitutively from 6 h to 24 h post-feeding.
These published data and our results suggest that different PRRs with different tern recognition specificities are constitutively present primed to identify different p gens that may arrive with the blood meal to induce rapidly both humoral and cel responses. We suggest the adoption of a "watchdog" strategy comparable to the com ment alternative pathway [46]. We have also observed a remarkable mounting of a sp pathogen recognition against T. cruzi demonstrated by the overexpression of rpPG LC/LAa and rpPGRP-LC/Lab. 30%, and T1HGN7 with 29%. However, GNBP-1 was shown to be a GPI-anchored membrane protein [41] while T1I650 sequence, although predicted UPS, it is lacking a signal peptide, a transmembrane domain, and GPI-anchor unless T1I650 N-terminal sequence is incompletely annotated.
In addition to their immune role, these proteins seem to have a digestive implication when they are expressed in the insect's midgut [42]. Interestingly, we have shown in the comparative proteome of R. prolixus midgut [43,44] that T1HU92 (ML domain protein) expression level increases in response to blood feeding. This protein is related to the recognition of pathogen-related molecules [45] and a homologous protein, which expression was also constitutive, was identified in the hemolymph proteome of A. gambiae [25]. In the hemolymph, T1HU92 is expressed constitutively from 6 h to 24 h post-feeding.
These published data and our results suggest that different PRRs with different pattern recognition specificities are constitutively present primed to identify different pathogens that may arrive with the blood meal to induce rapidly both humoral and cellular responses. We suggest the adoption of a "watchdog" strategy comparable to the complement alternative pathway [46]. We have also observed a remarkable mounting of a specific pathogen recognition against T. cruzi demonstrated by the overexpression of rpPGRP-LC/LAa and rpPGRP-LC/Lab. 30%, and T1HGN7 with 29%. However, GNBP-1 was shown to be a GPI-anchored membrane protein [41] while T1I650 sequence, although predicted UPS, it is lacking a signal peptide, a transmembrane domain, and GPI-anchor unless T1I650 N-terminal sequence is incompletely annotated.
In addition to their immune role, these proteins seem to have a digestive implication when they are expressed in the insect's midgut [42]. Interestingly, we have shown in the comparative proteome of R. prolixus midgut [43,44] that T1HU92 (ML domain protein) expression level increases in response to blood feeding. This protein is related to the recognition of pathogen-related molecules [45] and a homologous protein, which expression was also constitutive, was identified in the hemolymph proteome of A. gambiae [25]. In the hemolymph, T1HU92 is expressed constitutively from 6 h to 24 h post-feeding.
These published data and our results suggest that different PRRs with different pattern recognition specificities are constitutively present primed to identify different pathogens that may arrive with the blood meal to induce rapidly both humoral and cellular responses. We suggest the adoption of a "watchdog" strategy comparable to the complement alternative pathway [46]. We have also observed a remarkable mounting of a specific pathogen recognition against T. cruzi demonstrated by the overexpression of rpPGRP-LC/LAa and rpPGRP-LC/Lab. 30%, and T1HGN7 with 29%. However, GNBP-1 was shown to be a GPI-anchored membrane protein [41] while T1I650 sequence, although predicted UPS, it is lacking a signal peptide, a transmembrane domain, and GPI-anchor unless T1I650 N-terminal sequence is incompletely annotated.
In addition to their immune role, these proteins seem to have a digestive implication when they are expressed in the insect's midgut [42]. Interestingly, we have shown in the comparative proteome of R. prolixus midgut [43,44] that T1HU92 (ML domain protein) expression level increases in response to blood feeding. This protein is related to the recognition of pathogen-related molecules [45] and a homologous protein, which expression was also constitutive, was identified in the hemolymph proteome of A. gambiae [25]. In the hemolymph, T1HU92 is expressed constitutively from 6 h to 24 h post-feeding.
These published data and our results suggest that different PRRs with different pattern recognition specificities are constitutively present primed to identify different pathogens that may arrive with the blood meal to induce rapidly both humoral and cellular responses. We suggest the adoption of a "watchdog" strategy comparable to the complement alternative pathway [46]. We have also observed a remarkable mounting of a specific pathogen recognition against T. cruzi demonstrated by the overexpression of rpPGRP-LC/LAa and rpPGRP-LC/Lab. Table 1. Expression pattern of immunity related proteins identified in R. prolixus hemolymph.

Immune category
IDs Protein names Fold increase 30%, and T1HGN7 with 29%. However, GNBP-1 was shown to be a GPI-anchored membrane protein [41] while T1I650 sequence, although predicted UPS, it is lacking a signal peptide, a transmembrane domain, and GPI-anchor unless T1I650 N-terminal sequence is incompletely annotated.
In addition to their immune role, these proteins seem to have a digestive implication when they are expressed in the insect's midgut [42]. Interestingly, we have shown in the comparative proteome of R. prolixus midgut [43,44] that T1HU92 (ML domain protein) expression level increases in response to blood feeding. This protein is related to the recognition of pathogen-related molecules [45] and a homologous protein, which expression was also constitutive, was identified in the hemolymph proteome of A. gambiae [25]. In the hemolymph, T1HU92 is expressed constitutively from 6 h to 24 h post-feeding.
These published data and our results suggest that different PRRs with different pattern recognition specificities are constitutively present primed to identify different pathogens that may arrive with the blood meal to induce rapidly both humoral and cellular responses. We suggest the adoption of a "watchdog" strategy comparable to the complement alternative pathway [46]. We have also observed a remarkable mounting of a specific pathogen recognition against T. cruzi demonstrated by the overexpression of rpPGRP-LC/LAa and rpPGRP-LC/Lab. Table 1. Expression pattern of immunity related proteins identified in R. prolixus hemolymph.

Immune category
IDs Protein names Fold increase 30%, and T1HGN7 with 29%. However, GNBP-1 was shown to be a GPI-anchored membrane protein [41] while T1I650 sequence, although predicted UPS, it is lacking a signal peptide, a transmembrane domain, and GPI-anchor unless T1I650 N-terminal sequence is incompletely annotated.
In addition to their immune role, these proteins seem to have a digestive implication when they are expressed in the insect's midgut [42]. Interestingly, we have shown in the comparative proteome of R. prolixus midgut [43,44] that T1HU92 (ML domain protein) expression level increases in response to blood feeding. This protein is related to the recognition of pathogen-related molecules [45] and a homologous protein, which expression was also constitutive, was identified in the hemolymph proteome of A. gambiae [25]. In the hemolymph, T1HU92 is expressed constitutively from 6 h to 24 h post-feeding.
These published data and our results suggest that different PRRs with different pattern recognition specificities are constitutively present primed to identify different pathogens that may arrive with the blood meal to induce rapidly both humoral and cellular responses. We suggest the adoption of a "watchdog" strategy comparable to the complement alternative pathway [46]. We have also observed a remarkable mounting of a specific pathogen recognition against T. cruzi demonstrated by the overexpression of rpPGRP-LC/LAa and rpPGRP-LC/Lab. Table 1. Expression pattern of immunity related proteins identified in R. prolixus hemolymph.

Immune category
IDs Protein names Fold increase 30%, and T1HGN7 with 29%. However, GNBP-1 was shown to be a GPI-anchored membrane protein [41] while T1I650 sequence, although predicted UPS, it is lacking a signal peptide, a transmembrane domain, and GPI-anchor unless T1I650 N-terminal sequence is incompletely annotated.
In addition to their immune role, these proteins seem to have a digestive implication when they are expressed in the insect's midgut [42]. Interestingly, we have shown in the comparative proteome of R. prolixus midgut [43,44] that T1HU92 (ML domain protein) expression level increases in response to blood feeding. This protein is related to the recognition of pathogen-related molecules [45] and a homologous protein, which expression was also constitutive, was identified in the hemolymph proteome of A. gambiae [25]. In the hemolymph, T1HU92 is expressed constitutively from 6 h to 24 h post-feeding.
These published data and our results suggest that different PRRs with different pattern recognition specificities are constitutively present primed to identify different pathogens that may arrive with the blood meal to induce rapidly both humoral and cellular responses. We suggest the adoption of a "watchdog" strategy comparable to the complement alternative pathway [46]. We have also observed a remarkable mounting of a specific pathogen recognition against T. cruzi demonstrated by the overexpression of rpPGRP-LC/LAa and rpPGRP-LC/Lab. Table 1. Expression pattern of immunity related proteins identified in R. prolixus hemolymph.

Immune category
IDs Protein names Fold increase 30%, and T1HGN7 with 29%. However, GNBP-1 was shown to be a GPI-anchored membrane protein [41] while T1I650 sequence, although predicted UPS, it is lacking a signal peptide, a transmembrane domain, and GPI-anchor unless T1I650 N-terminal sequence is incompletely annotated.
In addition to their immune role, these proteins seem to have a digestive implication when they are expressed in the insect's midgut [42]. Interestingly, we have shown in the comparative proteome of R. prolixus midgut [43,44] that T1HU92 (ML domain protein) expression level increases in response to blood feeding. This protein is related to the recognition of pathogen-related molecules [45] and a homologous protein, which expression was also constitutive, was identified in the hemolymph proteome of A. gambiae [25]. In the hemolymph, T1HU92 is expressed constitutively from 6 h to 24 h post-feeding.
These published data and our results suggest that different PRRs with different pattern recognition specificities are constitutively present primed to identify different pathogens that may arrive with the blood meal to induce rapidly both humoral and cellular responses. We suggest the adoption of a "watchdog" strategy comparable to the complement alternative pathway [46]. We have also observed a remarkable mounting of a specific pathogen recognition against T. cruzi demonstrated by the overexpression of rpPGRP-LC/LAa and rpPGRP-LC/Lab. 30%, and T1HGN7 with 29%. However, GNBP-1 was shown to be a GPI-anchored membrane protein [41] while T1I650 sequence, although predicted UPS, it is lacking a signal peptide, a transmembrane domain, and GPI-anchor unless T1I650 N-terminal sequence is incompletely annotated.
In addition to their immune role, these proteins seem to have a digestive implication when they are expressed in the insect's midgut [42]. Interestingly, we have shown in the comparative proteome of R. prolixus midgut [43,44] that T1HU92 (ML domain protein) expression level increases in response to blood feeding. This protein is related to the recognition of pathogen-related molecules [45] and a homologous protein, which expression was also constitutive, was identified in the hemolymph proteome of A. gambiae [25]. In the hemolymph, T1HU92 is expressed constitutively from 6 h to 24 h post-feeding.
These published data and our results suggest that different PRRs with different pattern recognition specificities are constitutively present primed to identify different pathogens that may arrive with the blood meal to induce rapidly both humoral and cellular responses. We suggest the adoption of a "watchdog" strategy comparable to the complement alternative pathway [46]. We have also observed a remarkable mounting of a specific pathogen recognition against T. cruzi demonstrated by the overexpression of rpPGRP-LC/LAa and rpPGRP-LC/Lab. 30%, and T1HGN7 with 29%. However, GNBP-1 was shown to be a GPI-anchored membrane protein [41] while T1I650 sequence, although predicted UPS, it is lacking a signal peptide, a transmembrane domain, and GPI-anchor unless T1I650 N-terminal sequence is incompletely annotated.
In addition to their immune role, these proteins seem to have a digestive implication when they are expressed in the insect's midgut [42]. Interestingly, we have shown in the comparative proteome of R. prolixus midgut [43,44] that T1HU92 (ML domain protein) expression level increases in response to blood feeding. This protein is related to the recognition of pathogen-related molecules [45] and a homologous protein, which expression was also constitutive, was identified in the hemolymph proteome of A. gambiae [25]. In the hemolymph, T1HU92 is expressed constitutively from 6 h to 24 h post-feeding.
These published data and our results suggest that different PRRs with different pattern recognition specificities are constitutively present primed to identify different pathogens that may arrive with the blood meal to induce rapidly both humoral and cellular responses. We suggest the adoption of a "watchdog" strategy comparable to the complement alternative pathway [46]. We have also observed a remarkable mounting of a specific pathogen recognition against T. cruzi demonstrated by the overexpression of rpPGRP-LC/LAa and rpPGRP-LC/Lab. 30%, and T1HGN7 with 29%. However, GNBP-1 was shown to be a GPI-anchored membrane protein [41] while T1I650 sequence, although predicted UPS, it is lacking a signal peptide, a transmembrane domain, and GPI-anchor unless T1I650 N-terminal sequence is incompletely annotated.
In addition to their immune role, these proteins seem to have a digestive implication when they are expressed in the insect's midgut [42]. Interestingly, we have shown in the comparative proteome of R. prolixus midgut [43,44] that T1HU92 (ML domain protein) expression level increases in response to blood feeding. This protein is related to the recognition of pathogen-related molecules [45] and a homologous protein, which expression was also constitutive, was identified in the hemolymph proteome of A. gambiae [25]. In the hemolymph, T1HU92 is expressed constitutively from 6 h to 24 h post-feeding.
These published data and our results suggest that different PRRs with different pattern recognition specificities are constitutively present primed to identify different pathogens that may arrive with the blood meal to induce rapidly both humoral and cellular responses. We suggest the adoption of a "watchdog" strategy comparable to the complement alternative pathway [46]. We have also observed a remarkable mounting of a specific pathogen recognition against T. cruzi demonstrated by the overexpression of rpPGRP-LC/LAa and rpPGRP-LC/Lab. 30%, and T1HGN7 with 29%. However, GNBP-1 was shown to be a GPI-anchored membrane protein [41] while T1I650 sequence, although predicted UPS, it is lacking a signal peptide, a transmembrane domain, and GPI-anchor unless T1I650 N-terminal sequence is incompletely annotated.
In addition to their immune role, these proteins seem to have a digestive implication when they are expressed in the insect's midgut [42]. Interestingly, we have shown in the comparative proteome of R. prolixus midgut [43,44] that T1HU92 (ML domain protein) expression level increases in response to blood feeding. This protein is related to the recognition of pathogen-related molecules [45] and a homologous protein, which expression was also constitutive, was identified in the hemolymph proteome of A. gambiae [25]. In the hemolymph, T1HU92 is expressed constitutively from 6 h to 24 h post-feeding.
These published data and our results suggest that different PRRs with different pattern recognition specificities are constitutively present primed to identify different pathogens that may arrive with the blood meal to induce rapidly both humoral and cellular responses. We suggest the adoption of a "watchdog" strategy comparable to the complement alternative pathway [46]. We have also observed a remarkable mounting of a specific pathogen recognition against T. cruzi demonstrated by the overexpression of rpPGRP-LC/LAa and rpPGRP-LC/Lab. 30%, and T1HGN7 with 29%. However, GNBP-1 was shown to be a GPI-anchored membrane protein [41] while T1I650 sequence, although predicted UPS, it is lacking a signal peptide, a transmembrane domain, and GPI-anchor unless T1I650 N-terminal sequence is incompletely annotated.
In addition to their immune role, these proteins seem to have a digestive implication when they are expressed in the insect's midgut [42]. Interestingly, we have shown in the comparative proteome of R. prolixus midgut [43,44] that T1HU92 (ML domain protein) expression level increases in response to blood feeding. This protein is related to the recognition of pathogen-related molecules [45] and a homologous protein, which expression was also constitutive, was identified in the hemolymph proteome of A. gambiae [25]. In the hemolymph, T1HU92 is expressed constitutively from 6 h to 24 h post-feeding.
These published data and our results suggest that different PRRs with different pattern recognition specificities are constitutively present primed to identify different pathogens that may arrive with the blood meal to induce rapidly both humoral and cellular responses. We suggest the adoption of a "watchdog" strategy comparable to the complement alternative pathway [46]. We have also observed a remarkable mounting of a specific pathogen recognition against T. cruzi demonstrated by the overexpression of rpPGRP-LC/LAa and rpPGRP-LC/Lab. responses. We suggest the adoption of a "watchdog" strategy comparable to the com ment alternative pathway [46]. We have also observed a remarkable mounting of a sp pathogen recognition against T. cruzi demonstrated by the overexpression of rpPG LC/LAa and rpPGRP-LC/Lab.

T1HEN7
Putative mucin Attacin_C domain-containing protein responses. We suggest the adoption of a "watchdog" strategy comparable to the com ment alternative pathway [46]. We have also observed a remarkable mounting of a sp pathogen recognition against T. cruzi demonstrated by the overexpression of rpPG LC/LAa and rpPGRP-LC/Lab.

Protease inhibitors
Serpins T1IF83 SERPIN domain-containing protein responses. We suggest the adoption of a "watchdog" strategy comparable to the com ment alternative pathway [46]. We have also observed a remarkable mounting of a sp pathogen recognition against T. cruzi demonstrated by the overexpression of rpPG LC/LAa and rpPGRP-LC/Lab. responses. We suggest the adoption of a "watchdog" strategy comparable to the com ment alternative pathway [46]. We have also observed a remarkable mounting of a sp pathogen recognition against T. cruzi demonstrated by the overexpression of rpPG LC/LAa and rpPGRP-LC/Lab. responses. We suggest the adoption of a "watchdog" strategy comparable to the com ment alternative pathway [46]. We have also observed a remarkable mounting of a sp pathogen recognition against T. cruzi demonstrated by the overexpression of rpPG LC/LAa and rpPGRP-LC/Lab. therefore the three isoforms are indistinguishable, and we cannot affirm which isoforms are unambiguously expressed. Interestingly, this peptide is in the pro-peptide region indicating that we have identified the unprocessed protein which can be activated by a pro-defensin processing enzyme [53]. RPRC012182 clusters separately from hemimetabolous arthropods defensins [39]. Interestingly, these defensins are expressed in the hemolymph 6 h postblood feeding (Table 1) as it has been observed for defensins A, B, and C transcripts in R. prolixus fat body post-hemocoel bacterial injection [54]. However, while defensins A, B, and C transcripts level stays high in R. prolixus fat body at 24 h post-hemocoel bacterial injection [54], T1I7B0, R4G8B6, and R4FNJ9 are absent at 24 h (Table 1). However, manual inspection of WEPAGEITEEHLAR peptide revealed its presence also at 24 h, but given its intensity being below the threshold, it was not quantified by MaxQuant algorithm. To confirm the expression of defensins at both 6 h and 24 h and compare their expression level between the different studied conditions, Western blotting experiment using a polyclonal anti-defensin antibody showed a significant increase in the defensins expression level at 6 h post-infection compared to blood-fed condition (p-value = 0.0014) ( Figure 5). Their expression level in the hemolymph decreases significantly at 24 h post-blood feeding and post-infection (p-value = 0.0035 and 0.0118, respectively).
Three defensin isoforms (T1I7B0, R4G8B6, and R4FNJ9) ( Table 1) were identified amongst the nine putative defensins encoded by R. prolixus genome [39]. They show a seven-fold increase in their expression level 6 h post-T. cruzi infection (Table S1). Of note, all three defensins are encoded by the same gene (RPRC012182) and were identified and quantified with a unique and common peptide (WEPAGEITEEHLAR) (Table S1), therefore the three isoforms are indistinguishable, and we cannot affirm which isoforms are unambiguously expressed. Interestingly, this peptide is in the pro-peptide region indicating that we have identified the unprocessed protein which can be activated by a pro-defensin processing enzyme [53]. RPRC012182 clusters separately from hemimetabolous arthropods defensins [39]. Interestingly, these defensins are expressed in the hemolymph 6 h post-blood feeding (Table 1) as it has been observed for defensins A, B, and C transcripts in R. prolixus fat body post-hemocoel bacterial injection [54]. However, while defensins A, B, and C transcripts level stays high in R. prolixus fat body at 24 h post-hemocoel bacterial injection [54], T1I7B0, R4G8B6, and R4FNJ9 are absent at 24 h (Table 1). However, manual inspection of WEPAGEITEEHLAR peptide revealed its presence also at 24 h, but given its intensity being below the threshold, it was not quantified by MaxQuant algorithm. To confirm the expression of defensins at both 6 h and 24 h and compare their expression level between the different studied conditions, Western blotting experiment using a polyclonal anti-defensin antibody showed a significant increase in the defensins expression level at 6 h post-infection compared to blood-fed condition (p-value = 0.0014) ( Figure 5). Their expression level in the hemolymph decreases significantly at 24 h post-blood feeding and post-infection (p-value = 0.0035 and 0.0118, respectively). Figure 5. Western blot validation of defensins' temporal expression profile in the hemolymph at 6 h and 24 h post-blood feeding and T. cruzi infection. The relative expression of defensins was calculated by normalizing the band intensity of defensins to the intensity of the total proteins signal. The results are expressed as the mean ± SEM (n = 3). Statistical significance is shown by * (* p ≤ 0.05 and ** p ≤ 0.01), calculated by unpaired t-test.
Interestingly, Vieira et al. [55] showed a differential and opposite response of defensins A and C transcripts levels in the fat body 24 h post-oral ingestion of T. cruzi. Hence, despite the vectorial life cycle of T. cruzi being restricted to the digestive tract, these defensins expression level increases seven-fold at 6 h post-T. cruzi infection (Figure 4 and Table 1). Stimulation of the systemic secretion of AMPs in the hemolymph in response to the presence of bacteria or protozoan parasites in the digestive tract has been reported in several insects, including species of Phlebotomus [56], Glossina [57] and Drosophila [58], even without the invasion of the infective pathogens of the hemocoel. Interestingly, a systemic response expression of AMPs in R. prolixus hemolymph and fat body following the colonization of its midgut by T. cruzi has been reported simultaneously with the local response in the midgut [55]. Moreover, it has been demonstrated that infection of R. prolixus with epimastigotes forms of T. cruzi Dm28c clone reduces bacteria density, and increases Figure 5. Western blot validation of defensins' temporal expression profile in the hemolymph at 6 h and 24 h post-blood feeding and T. cruzi infection. The relative expression of defensins was calculated by normalizing the band intensity of defensins to the intensity of the total proteins signal. The results are expressed as the mean ± SEM (n = 3). Statistical significance is shown by * (* p ≤ 0.05 and ** p ≤ 0.01), calculated by unpaired t-test.
Interestingly, Vieira et al. [55] showed a differential and opposite response of defensins A and C transcripts levels in the fat body 24 h post-oral ingestion of T. cruzi. Hence, despite the vectorial life cycle of T. cruzi being restricted to the digestive tract, these defensins expression level increases seven-fold at 6 h post-T. cruzi infection (Figure 4 and Table 1). Stimulation of the systemic secretion of AMPs in the hemolymph in response to the presence of bacteria or protozoan parasites in the digestive tract has been reported in several insects, including species of Phlebotomus [56], Glossina [57] and Drosophila [58], even without the invasion of the infective pathogens of the hemocoel. Interestingly, a systemic response expression of AMPs in R. prolixus hemolymph and fat body following the colonization of its midgut by T. cruzi has been reported simultaneously with the local response in the midgut [55]. Moreover, it has been demonstrated that infection of R. prolixus with epimastigotes forms of T. cruzi Dm28c clone reduces bacteria density, and increases PO and antibacterial activities in the midgut [59]. We can speculate that T. cruzi ingestion induces defensin's expression in the first hours post-infection, which may participate in the control of microbiota population. However, the intensive lysis of T. cruzi during the first The active PO is generated by a proteolytic cleavage of PPO zymogen by clip domain serine proteinases (CLIPs), which are specific to invertebrates and act in cascades to modulate several immune responses including coagulation, melanization, and AMPs synthesis through Toll pathway activation [74,75]. The PPO cascade is also tightly controlled by serine protease inhibitors (serpins) to prevent their spontaneous and excessive activation [76].
CLIP proteases are non-digestive serine proteases, apparently unique to invertebrates, and present in the hemolymph. CLIP proteases with a serine protease-like domain with mutated residues of the catalytic triad needed for proteolysis are named serine protease homologs (SPHs). Such CLIP-SPHs with a proteolytic activity deficiency can function as cofactors for PPO activation [77], and contrastingly they can also induce its inhibition [78]. We have identified fifteen SP isoforms of the peptidase family S1 (Table 1). Three isoforms are CLIP-SPs (Table 1) with a conserved serine residue of the protease catalytic triad, a single amino-terminal clip domain, and a secretion signal peptide. They are isoforms of the same gene RPRC003090 which is one of the two annotated CLIP genes from R. prolixus genome [38,39]. The three isoforms share more than 97% sequence identity and their identification by MS/MS was based on the same tryptic peptides. Interestingly, the sequenced peptides cover solely the S1 domain probably due to the short and limited number of tryptic peptides of the clip domain. Consequently, we are tempted to suggest that the identified CLIP-SPs are active proteases. The analysis of their temporal expression shows that all isoforms are expressed at 6 h and 24 h post-feeding ( Figure 4 and Table 1). Worthy of note, are the three S1 proteins with a catalytically functional triad Once CLIPs are activated, they are tightly regulated by serine proteinase inhibitors (serpins) present in hemolymph plasma [79] to control the toxic by-products of melanization. Proteinases and proteinase inhibitors often exist in pairs. Thus, serine proteinase inhibitors from the serpin family constitute a group of proteins that are likely to be regulators of serine proteinases with a clip domain. Accordingly, four serpins have been identified and their expression profiles are steady at 6 h and 24h post-feeding. However, T1IF83 and R4FJD2 are 1,6-fold down-regulated (Figure 4) by the presence of T. cruzi at 6 h post-infection while their expression is unaffected by the parasite at 24 h.
Transglutaminases catalyze the deamidation and transamidation of glutamine, crosslinking of proteins by formation of ε-(γ-glutamyl) lysine isopeptide bonds, which play vital roles in blood clotting, regulation of cellular responses to stress, and formation of the epithelium [3]. It has been shown that transglutaminase activity from both Drosophila hemolymph and human blood accumulates on microbial surfaces, leading to their sequestration into the clot. Moreover, Drosophila larvae with reduced TG levels show increased mortality after septic injury and susceptibility to a natural infection involving entomopathogenic nematodes and symbiotic bacteria [80]. RNA interference directed against TG reduced the life span of flies reared under conventional conditions and enhanced the expression of AMPs in the IMD pathway [81]. This is probably a consequence of the fact that TG is involved in negative regulation of the IMD pathway. Indeed, TG catalyzes Relish cross-linking suppressing the IMD signaling pathway to enable immune tolerance against resident commensal microbes [81]. We have identified seven TGs among which T1HFV3 and T1HFV2 are only expressed at 6 h post-blood feeding ( Table 1). The other isoforms are stably expressed from