The NF-κB Inhibitor, IMD-0354, Affects Immune Gene Expression, Bacterial Microbiota and Trypanosoma cruzi Infection in Rhodnius prolixus Midgut

Rhodnius prolixus is an insect vector of Trypanosoma cruzi, the causative agent of Chagas disease in Latin America. Nuclear factor-κB (NF-κB) transcription factors (TF) are conserved components of the innate immune system in several multicellular organisms including insects. The drug IMD-0354 [N-(3,5-bis-trifluoromethyl-phenyl)-5-chloro-2-hydroxy-benzamide] is a selective inhibitor of IκB kinases. It blocks IκBα phosphorylation thus preventing nuclear translocation of the NF-κb TF. In humans, NF-κB is involved in several biological processes such as inflammation, cell proliferation and immunity. In insects, the activation of the immune system upon microbial challenge can be controlled by signaling pathways such as the immune deficiency (IMD) and Toll, to combat infection. These activated pathways signal to downstream NF-κB TF to stimulate specific immune genes, triggering the synthesis of several molecules such as the antimicrobial peptides. In Drosophila melanogaster, the activation and regulation of NF-κB TF have been elucidated, while in triatomines these mechanisms are not fully understood Therefore, the present study investigated the effects of oral administration of the drug IMD-0354 on the R. prolixus immune response to challenge with bacteria and T. cruzi, as well as the impact on the gut bacterial microbiota. R. prolixus were fed with rabbit blood containing IMD-0354 and Escherichia coli, Staphylococcus aureus, or T. cruzi. The effects of IMD-0354 on insect mortality and antimicrobial activity in insect midgut samples, as well as the relative expression of R. prolixus immune genes were recorded. The bacterial microbiota was analyzed, and viable parasites were counted in insect midgut samples. The IMD-0354 treatment modulated antibacterial activity and the gene expression patterns of defensin A, defensin B, defensin C, and prolixicin, and the genes involved in the IMD and Toll pathways. Additionally, there was an increase of bacterial microbiota in treated insects. Insects treated with IMD-0354 and concomitantly infected with bacteria or T. cruzi through the blood meal had increased mortality, while the T. cruzi population in R. prolixus midgut was reduced. The inhibitory effect of IMD-0354 indicates the importance of NF-κB TF in the innate immune responses involved in the control of bacteria and parasite infections in the R. prolixus midgut.

Rhodnius prolixus is an insect vector of Trypanosoma cruzi, the causative agent of Chagas disease in Latin America. Nuclear factor-κB (NF-κB) transcription factors (TF) are conserved components of the innate immune system in several multicellular organisms including insects. The drug IMD-0354 [N-(3,5-bis-trifluoromethyl-phenyl)-5-chloro-2-hydroxy-benzamide] is a selective inhibitor of IκB kinases. It blocks IκBα phosphorylation thus preventing nuclear translocation of the NF-κb TF. In humans, NF-κB is involved in several biological processes such as inflammation, cell proliferation and immunity. In insects, the activation of the immune system upon microbial challenge can be controlled by signaling pathways such as the immune deficiency (IMD) and Toll, to combat infection. These activated pathways signal to downstream NF-κB TF to stimulate specific immune genes, triggering the synthesis of several molecules such as the antimicrobial peptides. In Drosophila melanogaster, the activation and regulation of NF-κB TF have been elucidated, while in triatomines these mechanisms are not fully understood Therefore, the present study investigated the effects of oral administration of the drug IMD-0354 on the R. prolixus immune response to challenge with bacteria and T. cruzi, as well as the impact on the gut bacterial microbiota. R. prolixus were fed with rabbit blood containing IMD-0354 and Escherichia coli, Staphylococcus aureus, or T. cruzi. The effects of IMD-0354 on insect mortality and antimicrobial activity in insect midgut samples, as well as the relative expression of R. prolixus immune genes were recorded. The bacterial microbiota was analyzed, and viable parasites were counted in insect midgut samples. The IMD-0354 treatment modulated antibacterial activity and the gene expression patterns of defensin A, defensin B, defensin C, and prolixicin, and the genes involved in the IMD and Toll pathways. Additionally, there was an increase of INTRODUCTION The invertebrate immune system relies on innate responses and lacks the classical adaptive immunity observed in vertebrates (Hoffmann and Reichhart, 2002). However, innate immunity is very effective as the first-line of host defense against infection in all eukaryotic organisms (Fearon and Locksley, 1996;Hoffmann and Reichhart, 2002). Nuclear Factor-Kappa B (NF-κB) transcription factors (TF) are important and conserved multi-components from pathways that regulate the expression of many genes including those responsible for the innate immune system (Silverman and Maniatis, 2001;Li and Verma, 2002;Kumar et al., 2004). NF-κB proteins have been identified in a range of multicellular organisms including cnidarians (Sullivan et al., 2007;Sinkovics, 2017), insects (Hetru and Hoffmann, 2009;Ganesan et al., 2011;Dong et al., 2012;Mesquita et al., 2015) and humans (Hayden and Ghosh, 2004;Senegas et al., 2015). Thus, these pathways emerged early in evolution, more than 500 million years ago, so that and the basic mechanisms of recognition and activation of response against pathogens are conserved in the animal kingdom (Hoffmann et al., 1999;Palmer and Jiggins, 2015).
The drug IMD-0354 [N-(3,5-bis-trifluoromethyl-phenyl)-5chloro-2-hydroxy-benzamide] is a selective molecular inhibitor of IκK-2 (Tanaka et al., 2005;Sugita et al., 2009). It blocks IκBα phosphorylation thus preventing nuclear translocation of the transcription factor NF-κB (Uota et al., 2012;Kong et al., 2015). Rhodnius prolixus is a triatomine species of medical importance vectoring Trypanosoma cruzi, the causative agent of Chagas disease in Latin America (Chagas, 1909;Vallejo et al., 2009;Coura, 2015;World Health Organization WHO, 2017). Therefore, studying aspects of R. prolixus immune system is relevant, for providing potential targets for insect control. In D. melanogaster, although the processes of activation and regulation of NF-κB TF are well-known (Ferrandon et al., 2007;Ganesan et al., 2011;Buchon et al., 2014;Imler, 2014), in triatomines these pathways are not fully understood. For example, the gene encoding the protein Relish, a conserved TF related to IMD signaling pathway (Ferrandon et al., 2007), was identified in R. prolixus as RpRelish, but its inhibitory protein Caspar was not detected (Mesquita et al., 2015). In addition, Dorsal, a NF-κB TF involved in the Toll pathway and its inhibitory protein, Cactus, a member of IκB-family protein (Geisler et al., 1992;Ferrandon et al., 2007) were detected in R. prolixus (Ursic-Bedoya et al., 2009;Ribeiro et al., 2014). Regarding effector molecules regulated by immune signaling pathways, AMPs have been isolated from R. prolixus midgut and fat body, including defensins (Lopez et al., 2003), lysozymes (Ursic-Bedoya et al., 2008) and prolixicin (Ursic-Bedoya et al., 2011). The induction of these effector molecules has been studied in R. prolixus challenged with different microorganisms. AMPs are differentially regulated, depending upon whether infection occurs with Gram-positive or Gram-negative bacteria or protozoans (Vieira et al., 2014(Vieira et al., , 2015(Vieira et al., , 2016. Therefore, the main objective of the present work was to determine the effect of IMD-0354 in the regulation of genes involved in NF-κB pathways (relish, dorsal and cactus, respectively) and effector AMPs (defensins and prolixicin) in R. prolixus challenged with T. cruzi or bacteria. The results indicate that R. prolixus NF-κB TF have a pivotal role in the modulation of the innate immune system and in the ability of triatomines to deal with different infections.

Bacterial Maintenance
Staphylococcus aureus 9518 and Escherichia coli K12 4401, both obtained from the National Collections of Industrial and Marine Bacteria (NCIMB), Aberdeen, United Kingdom, and Serratia marcescens RPH, previously isolated from R. prolixus midgut (Azambuja et al., 2004) were used in all experiments. Bacteria were maintained frozen at −70 • C in brain heart infusion (BHI) plus 10% glycerol until use. For all experimental procedures, bacteria were cultivated with shaking (90 revolutions per minute) in 20 mL of tryptone soy broth (TSB) for 17 h at 30 • C, and then 10 mL of fresh TSB were inoculated with 100 µL of the respective bacterial culture and incubated for a further 4 h under the same conditions. The bacteria were then washed in phosphate buffered saline (PBS, 0.01 M phosphate buffer, 0.0027 M potassium chloride and 0.137 M sodium chloride, pH 7.4) and diluted in TSB for use.

Insect Feeding, Infection, and Treatment in vivo
The R. prolixus colony was maintained at Laboratório de Bioquímica e Fisiologia de Insetos, IOC/FIOCRUZ, at a relative humidity of 50-60% and at 27 • C. For the experiments, R. prolixus 5th instar nymphs were randomly chosen and then fed with defibrinated rabbit blood through a membrane feeding device (Azambuja and Garcia, 1997). IMD-0354 (Sigma-Aldrich) was diluted in dimethyl sulfoxide (DMSO) (20 mg/mL) as a stock solution, according to the manufacturer's instructions. For assaying the effects of IMD-0354 on bacterial and parasite infections insects were fed with defibrinated rabbit blood containing 5 or 10 µg of IMD-0354/mL. Additionally, the insects treated with IMD-0354 were concomitantly orally infected with bacteria, E. coli or S. aureus at a final concentration of 10 4 bacteria/mL of blood or with T. cruzi epimastigotes at 1 × 10 6 parasites/mL of blood (Vieira et al., 2014). IMD-0354 untreated control groups, infected or not, were fed on blood containing the same final concentration of the solvent DMSO (0,004%) as the treated groups. Parasite quantification (detailed below) was assessed in a Neubauer chamber under an optical microscope. Insects not fully engorged were discarded.

Antibacterial Assays in vitro
Anterior midgut samples were collected from 5th instar nymphs of R. prolixus 7 days after feeding (DAF) on blood (control untreated group) or blood plus 5 µg of IMD-0354/mL (Sigma-Aldrich). From each group, 10 insect midguts were individually collected in 1.5 mL reaction tubes and diluted in 200 µL ultrapure water. The samples were homogenized, centrifuged at 10,000 × g for 10 min at 4 • C and the supernatants filtered using Millipore PVDF (0.22 µm) and maintained at −20 • C until use (Vieira et al., 2014(Vieira et al., , 2016. Midgut samples from control and treated insects were incubated for 19 h at 37 • C with E. coli, S. aureus or S. marcescens (Vieira et al., 2014). The midgut antibacterial activity was measured by the turbidimetric assays (TB) using a Spectra Max 190 Plate Reader (Molecular Devices, Sunnyvale, CA, United States), as described previously (Castro et al., 2012;Vieira et al., 2016).

RT-qPCR Assays
Temporal relative expression of R. prolixus antimicrobial peptides genes, defensins and prolixicin (defA, defB, defC, prol) and Toll and IMD pathway related genes (RpDorsal, RpCactus, and RpRelish) were analyzed through reverse transcription quantitative PCR. The specific primers for the genes of interest, as well as R. prolixus housekeeping genes (α-tubulin and GAPDH), were designed and used as previously published: α-tubulin and GAPDH (Paim et al., 2012), defA, defB and defC (Lopez et al., 2003;Vieira et al., 2016); prol (Ursic-Bedoya et al., 2011;Vieira et al., 2016), RpCactus (Ribeiro et al., 2014), RpRelish, RpDorsal (Mesquita et al., 2015) (Supplementary Material). The relative quantification of gene expression was estimated using untreated insects (controls) as calibrators. Untreated control and IMD-0354-treated R. prolixus 5th instar nymphs were dissected at different days after feeding to collect and separate midgut samples in three pools containing five anterior midguts each (Vieira et al., 2016). Total RNA was extracted and quantified using a NucleoSpin R RNA II Kit (Macherey-Nagel, Düren, Germany) and a NanoDrop 2000 Spectrophotometer (Thermo Scientific, Waltham, MA, United States), respectively. Synthesis of cDNA was with a First-Strand cDNA Synthesis Kit (GE Healthcare, Buckinghamshire, United Kingdom) using 2.5 µg of total RNA and the pd(N)6 primer. Quantification of cDNA was assessed by fluorescence in a Qubit Fluorimeter (Life Technologies) with the ssDNA assay kit. Real-time quantitative polymerase chain reactions (RT-qPCR) were conducted in an ABI PRISM 7500 Sequence Detection System (Applied Biosystems) at the FIOCRUZ facilities (Real-Time PCR Platform RPT-09A) using GoTaq R qPCR Master Mix (PROMEGA). Gene expression assays and analyses were performed as previously described by Vieira et al. (2016).

Microbiota Analysis
Total midguts from R. prolixus 5th instar nymphs were collected 7 days after feeding with blood with or without IMD-0354. It was performed three independent experiments with 10 insects from each group (n = 30). Cultivable bacteria microbiota population were quantified using the colony forming units (CFU) procedure. The samples were serially tenfold diluted with sterile PBS in 1.5 mL reaction tubes. Then 20 µL of each dilution was spread on BHI agar (Sigma-Aldrich) plates and immediately incubated at 30 • C for 24 h and the CFU counted. Autoclaved PBS was also plated and incubated to assess the sterility of all experiments. Additionally, RT-qPCR was performed to analyze relative expression of 16S-rRNA from S. marcescens, Rhodococcus rhodnii and Enterococcaceae using cDNA from 3 pools of 10 anterior midguts from insects: untreated; treated with IMD-0354; infected with T. cruzi epimastigotes; treated with IMD-0354 simultaneously infected with T. cruzie pimastigotes.

Analysis of IMD-0354 Effects on Rhodnius prolixus Mortality
To determine the toxicity of IMD-0354 in R. prolixus 5th instar nymphs, mortalities were evaluated 10 days after feeding.
The groups analyzed were: control (fed only on blood), insects fed on blood containing 5 µg IMD-0354/mL, insects fed on blood containing 5 µg IMD-0354/mL plus T. cruzi Dm28c or E. coli or S. aureus. Each blood meal type Was replicated twice, and 30 insects Were used for each replicate.

Quantification of Parasites in the Digestive Tract
Fifth instar R. prolixus nymphs fed With T. cruzi epimastigotes Were dissected and the total digestive tract Was collected 20 DAF in 1.5 mL microtubes and homogenized in 1.0 mL of sterile PBS. Parasite number Was determined by counting in a Neubauer haemocytometer and expressed as parasites/mL. Parasites quantification Was performed in three independent experiments With five individually insects each (n = 15).

Statistical Analysis
The statistical analysis of data was performed using one-way ANOVA, two-way ANOVA, Student's T, Tukey's or Mann-Whitney tests on GraphPad Prism 5 software, depending on data distribution. Significant differences between groups are displayed in each figure and legends in the present study and were considered statistically different when p < 0.05.

Ethics Declaration
The colony of R. prolixus was maintained in controlled environmental conditions at Laboratório de Bioquímica e Fisiologia de Insetos of IOC-FIOCRUZ and fed with defibrinated rabbit blood obtained at the Instituto de Ciência e Tecnologia em Biomodelos (ICTB), according to the Ethical Principles in Animal Experimentation approved by the Comitê de Ética de Experimentação Animal (CEUA/FIOCRUZ, under the protocol number LW019/17). The protocol was developed by CONCEA/MCT 2 , which is associated with the American Association for Animal Science (AAAS), the Federation of European Laboratory Animal Science Associations (FELASA), the International Council for Animal Science (ICLAS) and the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). 2 http://www.sbcal.org.br/

Preliminary Observations
To evaluate IMD-0454 toxicity, initial experiments, tested feeding 5 or 10 µg IMD-0354/mL blood to insects. At the 10 µg level, R. prolixus mortality rate was greater than 60%, a fact that impaired the infection experiments (data not shown). So, at 5 µg IMD-0354/mL a similar mortality rate (15%) occurred as in the control insects (12%) (data not shown) then, that dosage was used in all subsequent experiments. Additionally, at 5 µg IMD-0354/mL had no direct effect on parasite and bacteria cultures after 24 h of incubation.

IMD-0354 Affects the Expression of Genes Related to R. prolixus Immunity in the Midgut
To test if oral administration of the drug IMD-0354 (5ug/mL) alters the regulation of the NF-κB TF genes in the R. prolixus midgut, quantification of the temporal gene expression of TF RpRelish, RpDorsal and the inhibitory TF gene RpCactus, as well as some AMPs, was performed by RT-qPCR. The IMD-0354 treatment induced a significant downregulation of TF RpRelish gene expression and an upregulation of RpCactus and RpDorsal 1 DAF in comparison to control insects ( Figure 1A, p < 0.05; Figure 1B, p < 0.05; and Figure 1C, p < 0.01). At 5 DAF, however, both RpRelish and RpCactus expressions are similar to untreated control insects while Dorsal expression is higher in treated insects (p < 0.05). In contrast, at 7 DAF, RpRelish levels were significantly higher, RpCactus mRNA levels were significantly lower than control insects (Figures 1A,B, p < 0.05). RpDorsal levels in treated or untreated insects are the same at 7 DAF ( Figure 1C). The IMD-0354 treatment induced a significantly downregulation of the defA, defB and defC genes in the anterior midgut compared with control insects at 1 and 7 DAF (Figure 2A, p < 0.001, p < 0.01; Figure 2B, p < 0.001, p < 0.001; and Figure 2C, p < 0.01, p < 0.01). Prol expression was also lower at 1DAF in treated insects in comparison to control insects 1 DAF ( Figure 2D, p < 0.01) but, in contrast, at 7 DAF, prol was higher than the control group ( Figure 2D, p < 0.001).
FIGURE 1 | IMD-0354 effects on the relative gene expression of Relish, Cactus and Dorsal in the anterior midgut of Rhodnius prolixus. Relish, Cactus and Dorsal gene expressions were analyzed using the anterior midgut of fifth instar R. prolixus nymphs at different days after feeding on blood containing IMD-0354 (5 µg/ml). Data were quantified using the gene expression of untreated control insects as the calibrator (black columns) and shown as the relative expression of (A) RpRelish, (B) RpCactus, (C) RpDorsal on the 1st, 5th, and 7th days after feeding (DAF). Bars represent the mean ± SEM of 3 independent experiments with 3 pools of insects (n = 3). Means were compared using Student's T-test; * p < 0.05, * * p < 0.01, ns, non-significant.
Frontiers in Physiology | www.frontiersin.org FIGURE 2 | IMD-0354 effects on the relative gene expression of antimicrobial peptides in the anterior midgut of Rhodnius prolixus. Fifth instar nymphs of R. prolixus were fed on blood containing IMD-0354 (5 µg/ml). Data were quantified using the gene expression of untreated control insects as the calibrator (black columns). The grid columns show the relative expression of the antimicrobial peptide genes at the 1st and 7th days after feeding with IMD-0354. Relative expression of: (A) defA; (B) defB (C) defC (D) Prol at the 1st and 7th days after feeding. Treatments: black columns -insects fed with blood (control); grid columns -insects fed with blood containing the drug IMD-0354. Each bar represents 3 independent experiments performed in duplicate (n = 6). Means were compared using one-way ANOVA and Student T-test; * * * p < 0.001, * * p < 0.01, * p < 0.1.

IMD-0354 Affects Antimicrobial Peptides Activity in R. prolixus Midgut
The antibacterial assays performed with midgut samples from insects fed on blood with or without IMD-0354 was an attempt to corroborate AMP activity and gene expression. In fact, the IMD-0354 treatment modulated both AMP gene expression and antibacterial activity in the R. prolixus midgut (Figure 3). Anterior midgut samples from treated insects showed significantly lower activity against S. aureus and S. marcescens in comparison to the untreated control ( Figure 3B, p < 0.001, Figure 3C, p < 0.001). In contrast, a higher antibacterial activity against E. coli in vitro was detected in treated insects compared to the controls (Figure 3A, p < 0.01).

R. prolixus Midgut Bacterial Population Was Altered After IMD-0354 Treatment
To investigate whether the treatment with a NF-κB TF inhibitor could impact natural midgut homeostasis, the cultivable bacterial population was analyzed in the R. prolixus midgut at different times after drug ingestion. Insects fed with blood containing IMD-0354 had higher CFU counts than control insects at 1 and 7 DAF (p < 0.01) ( Figure 4A). Furthermore, analysis of 16S RNA confirmed a significant increase of S. marcescens and R. rhodnii population in insects treated with IMD-0354 comparing to control insects (p < 0.001, p < 0.05) ( Figure 4B). Additionally, in insects treated with IMD-0354 and concomitantly infected with T. cruzi, the population of S. marcescens and Enterococcaceae were significantly higher than untreated T. cruzi infected insects (p < 0.001, p < 0.001) ( Figure 4B).

R. prolixus Survival After Microbial Challenge
The importance of NF-κB TF in R. prolixus survival after a microbial challenge was analyzed by recording mortalities 72 h after a blood meal. Adding T. cruzi or bacteria to the blood of IMD-0354 treated insects increased the mortality to 31, 42, and 59% after T. cruzi, E. coli, and S. aureus oral infection, respectively (Figure 5). Insects infected with E. coli, S. aureus or T. cruzi without IMD-0354 treatment, had a similar mortality rate to control insects, lower than 18% (data not shown).

Quantification of T. cruzi in the R. prolixus Treated With IMD-0354
To investigate the role of NF-κB TF in the establishment of T. cruzi in R. prolixus gut, insects were concomitantly treated with IMD-0354 and infected with T. cruzi epimastigotes through the blood meal. The parasite population was quantified in the digestive tract collected from 15 insects 20 DAF. The T. cruzi population in the gut of insects treated with IMD-0354 was significantly lower in comparison to untreated infected insects (Figure 6, p < 0.001). Moreover, there was a decrease of 50% in the percentage of insects infected after IMD-0354 treatment.
FIGURE 3 | IMD-0354 effects on antibacterial activity in the anterior midgut of Rhodnius prolixus. Fifth instar nymphs of R. prolixus were fed on blood containing IMD-0354 (5 µg/ml). The antibacterial activities were measured in vitro using the anterior midgut samples 7 days after feeding and tested against (A) E. coli, (B) S. aureus, and (C) S. marcescens through the turbidimetric assay (OD 550 nm) after 19 h incubation. Black columnuntreated control insects fed only on blood; grid column -insects fed on blood containing IMD-0354. Bars represent the mean ± SEM of three independent experiments with nine pools of insects (n = 9). Means were compared using one-way ANOVA and Student's T-test; * * * p < 0.001, * * p < 0.01, NS, not significant.

IMD-0354 Affects the Induction of defC and prol Genes After T. cruzi Infection in R. prolixus Midgut
After observing that IMD-0354 impedes T. cruzi infection in R. prolixus midgut whether in these infected insects the drug altered the expression of defC and prol was tested. Defensin C and prolixicin genes were chosen for these experiments since previous results demonstrated the upregulation of these genes after T. cruzi infection (Vieira et al., 2016). Quantification of gene expression was examined at 1 and 7 DAF. At 1 DAF, T. cruzi infected insects concomitantly treated with IMD-0354 had a higher prol expression in comparison to controls and to T. cruzi infected untreated insects (Figure 7A, p < 0.01, p < 0.001, respectively). In contrast, on 7DAF, prol levels in the midgut of infected insects concomitantly treated with IMD-0354 were 8-fold lower in comparison to T. cruzi infected untreated insects (Figure 7A, p < 0.05). Unlike the prol gene expression, that of the defC gene at 1DAF showed no differences between infected insects and insects infected with T. cruzi and concomitantly treated with IMD-0354. However, on the 7 DAF, defC levels were lower in T. cruzi infected/IMD-0354 treated insects in comparison to infected but untreated insects ( Figure 7B, p < 0.05). Both prol and defC genes were also significantly upregulated after T. cruzi infection in comparison to control insects 7DAF ( Figure 7A, p < 0.01; Figure 7B, p < 0.001, respectively).

DISCUSSION
The innate immune system represents the first line of defense against infection by pathogens. For decades, insect innate immunity has commonly been studied using the genetic malleable model, D. melanogaster. This model elucidated details of the genes involved in the Toll and IMD pathways, as well as their specific roles in combating infection, by challenging with Gram-positive and negative bacteria and fungi (Lemaitre and Hoffmann, 2007;Ferrandon et al., 2007;Hetru and Hoffmann, 2009). Nowadays, attention has also turned to the immune systems of insect vectors of disease in order to identify potential targets to develop new control strategies. Regarding the triatomine vector, R. prolixus, only recently its whole genome sequence was published (Mesquita et al., 2015). With these available data now, it is possible to study the pattern recognition mechanisms and genes involved in immune signaling pathways as well as the effector genes regulated by these pathways. In this context, in the present work, the compound IMD-0354 was used as an immunosuppressive drug, since it is selective NF-κB TF inhibitor, preventing the transcription of several antimicrobial peptides in mammals (Kamon et al., 2004;Tanaka et al., 2005;Sugita et al., 2009). The use of this drug in arthropods, as far as we are aware, has not previously been documented to study components of insect vector immunity. Through drug incorporation in the insect blood meal, the importance of genes regulated by NF-κB pathways in the activation and modulation of the R. prolixus immune genes against infection by different microorganisms can be evaluated, as well as the influence of Toll and IMD TF in the establishment of T. cruzi infections.
The lack of some canonical elements from the IMD pathway was observed in the R. prolixus genome, with the absence of some genes such as IMD, FADD (Fas-Associated protein with dead domain) and Caspar, the inhibitor of the IMD pathway FIGURE 4 | IMD-0354 effects on microbiota population in Rhodnius prolixus anterior midgut. Determination of bacterial load in the midgut of 5th instar nymphs of R. prolixus fed with blood containing IMD-0354 (5 µg/ml). (A) Colony forming units (CFU) were counted in the anterior midgut samples 1 and 7 days after feeding. (B) Relative expression of 16S-rRNA of Serratia marcescens, Enterococcaceae and Rhodococcus rhodnii analyzed by RT-qPCR. Data was normalized to the R. prolixus 18S RNA gene and quantified using the gene expression of untreated control insects as the calibrator represented by dotted lines on each graph. Black column -untreated control insects fed on blood; grid column -insects fed on blood containing IMD-0354; white column -insects fed on blood containing T. cruzi epimastigotes (10 6 parasites/mL of blood); striped columns -insects fed on blood containing IMD-0354 plus T. cruzi epimastigotes (10 6 parasites/mL of blood). Bars represent the mean ± SEM of three independent experiments with 10 insects (n = 30). Means were compared using Student's T-test or Mann-Whitney test; * * * p < 0.001, * * p < 0.01, * p < 0.05.
FIGURE 5 | IMD-0354 effects on the mortality of Rhodnius prolixus infected with bacteria or Trypanosoma cruzi. R. prolixus 5th instar nymphs were fed on blood containing IMD-0354 (5 µg/ml) and E. coli or S. aureus at a final concentration of 1x 10 4 bacteria/ml or T. cruzi at 1 × 10 6 epimastigotes/ml. Numbers of dead insects were counted 7 days after the blood meal. Treatments: black column -untreated control insects fed on blood; green column -insect feed on blood with -IMD-0354; red column -IMD-0354 plus T. cruzi; blue column -IMD-0354 plus Escherichia coli.; orange column -IMD-0354 plus Staphylococcus aureus. Bars represent the percentage of dead insects (among 30 insects in each treatment) in 3 independent experiments. Means were compared using one-way ANOVA and Tukey's post-test; ns * * p < 0.01, * * * p < 0.001, ns, not significant. (Mesquita et al., 2015). The gene encoding the TF Relish, however, was detected and its knockdown induced a decrease of some AMPs and allowed the proliferation of the intestinal microbiota in R. prolixus (Mesquita et al., 2015), indicating the presence of a functional IMD pathway. Some disparity in the IMD signaling pathway elements is also observed in other insects such as in Pediculus humanus (Kim et al., 2011), Acyrthosiphon pisum (Gerardo et al., 2010) and in the ticks, Ixodes scapularis (Shaw et al., 2017), Rhipicephalus microplus (Rosa et al., 2016) suggesting some plasticity in the IMD pathway among arthropods (Palmer and Jiggins, 2015;Shaw et al., 2017). When R. prolixus 5th instar nymphs were fed with blood containing IMD-0354, defA, defB, and defC expression and FIGURE 6 | IMD-0354 effect on Trypanosoma cruzi population in the digestive tract of Rhodnius prolixus. R. prolixus 5th instar nymphs were fed on blood containing IMD-0354 (5 µg/ml of blood) and T. cruzi epimastigotes at a final concentration of 10 6 parasites/mL of blood. Number of T. cruzi were estimated in R. prolixus 5 th instar nymphs in whole digestive tracts at 20 days after feeding (DAF). Each point represents the number of parasites in an individual insect, and bars indicate the median. Means were compared using Student T-test; * * * p < 0.001. activity were downregulated, suggesting that NF-κB TF activation and its translocation to the nucleus is essential to trigger the transcription of, at least, the defensins genes in R. prolixus. In contrast, the NF-κB inhibitor induced an increase in prol levels at 7 DAF. This discrepancy may be due to the fact that the R. prolixus defensins, and those of other triatomines such as Triatoma brasiliensis and Panstrongylus megistus, are regulated by NF-κB TF (Lopez et al., 2003;Waniek et al., 2009;de Araújo et al., 2015), whereas NF-κB binding sites are absent in the prolixicin gene sequence, which appears to be regulated by GATA transcription factors (Ursic-Bedoya et al., 2011). Further experiments are necessary to fully understand the signaling pathways that regulate prolixicin in R. prolixus and in other FIGURE 7 | IMD-0354 effects on relative gene expression of antimicrobial peptides in the midgut of Rhodnius prolixus infected with Trypanosoma cruzi. R. prolixus 5th instar nymphs were fed on blood containing IMD-0354 and T. cruzi epimastigotes at a final concentration of 10 6 parasites/mL. Data were quantified using the gene expression of untreated control insects as the calibrator represented by horizontal dotted lines on each graph to show the relative expression of antimicrobial peptide. (A) Prolixicin relative expression; (B) Defensin C relative expression on the 1st and 7th days after feeding. Treatments: Black column-insect feed on blood containing IMD-0354 alone; white columns -fed on T. cruzi alone; grid columns -insects fed on IMD-0354 and T. cruzi. Each bar represents 3 independent experiments performed in duplicate (n = 6). Means were compared using two-way ANOVA and Student T-test; * * * p < 0.001, * * p < 0.01, * p < 0.05.
hemipterans. The increase of prolixicin mRNA levels in the R. prolixus midgut after NF-κB inhibition by IMD-0354 could represent an indirect compensatory effect that implicates other pathways, such as those controlled by GATA TF, for regulating the genes.
The low antibacterial activity detected in the midgut samples of insects treated with IMD-0354, against the Gram-positive S. aureus and the Gram-negative S. marcescens might be related to the negative regulation of defA, defB, and defC. In contrast, the increased antibacterial activity against E. coli which could be related to the enhancement of the prol expression. Previously, in in vitro studies, of purified prolixicin from R. prolixus fat body have been shown to have strong activity against E. coli (Ursic-Bedoya et al., 2011). In addition, the increase of cultivable bacterial numbers, as well S. marcescens, R. rhodnii and Enterococcaceae in the midgut in IMD-0354-treated insects suggests microbiota regulation by the defensins, highlighting the importance of NF-κB TF in R. prolixus intestinal homeostasis.
Moreover, knowledge of the role of immune signaling pathways and effector proteins (AMPs) in the regulation of insect intestinal bacterial microbiota is still fragmentary. Since AMP synthesis can inhibit the development of pathogens in their insect vectors (Vizioli et al., 2001a,b;Boulanger et al., 2002Boulanger et al., , 2004Telleria et al., 2013;Das De et al., 2018), innumerable microorganisms present in the intestinal tract are able to induce the insect immune system (Cirimotich et al., 2011;Eappen et al., 2013;Vieira et al., 2014), consequently interfering in the life cycle of the parasites. S. marcescens, one of the most abundant bacteria found in R. prolixus gut (da Mota et al., 2012), possesses lytic mechanisms that drastically reduce the success of T. cruzi infection in the R. prolixus digestive tract (Azambuja et al., 2004;Castro et al., 2007Castro et al., , 2012Vieira et al., 2016). It was suggested that the normal S. marcescens population growth is inhibited by R. prolixus AMPs, especially by defensins (Vieira et al., 2014(Vieira et al., , 2016. T. cruzi Dm28c infection in R. prolixus seems to overcome the trypanolytic effect of S. marcescens, inducing a strong upregulation of defC and prol (Vieira et al., 2015(Vieira et al., , 2016 which in turn decrease the S. marcescens population. In the present study, IMD-0354 affected the normal signaling requirements needed for AMP transcription and protein synthesis since the drug prevents the translocation of the TF to the nucleus (Tanaka et al., 2005). A transitory modulation in transcriptional levels of NF-kB TFs (RpRelish, RpDorsal)and of IkB RpCactus was observed in IMD-0354 treated insects. Since insects treated withIMD-0354recordedan increased population of some bacterial species in the midgut, this effect might be associated with the activation of the Toll pathway, marked by an enhancementinmRNA levels of Cactus (IkB) and Dorsal(NF-kBTF). Theopposite occurs in IMD pathways, in which the TF, Relish, is downregulated. Several studies using different insect species have demonstrated the role of the gut microbiota in stimulating immune signaling pathways (Eappen et al., 2013;Eleftherianos et al., 2013;Jiménez-Cortés et al., 2018). After the recognition of bacterial components by specific receptors, the Toll pathway is activated, requiring the degradation of the Cactus protein to release Dorsal protein, allowing its nuclear translocation to initiate the transcription of effector genes (Ferrandon et al., 2007;Ganesan et al., 2011). These processes are followed by the transcription of both Cactus and Dorsal mRNAs, in order to rescue and retain these regulatory proteins in the cytoplasm (Geisler et al., 1992;Nicolas et al., 1998;Rutschmann et al., 2002). It is known that the drug IMD-0354 acts downstream in microbial recognition and upstream of NF-kB TF (Relish and Dorsal) nuclear translocation, only impairing the phosphorylation of IkB proteins (Cactus) but not the transcription of the genes (NF-kB TF and IkB) (Tanaka et al., 2005). Thus, the alterations in Relish, Cactus and Dorsal gene expressions detected here could represent secondary effects of IMD-0354 treatment in R. prolixus.
The main effect of IMD-0354 treatment is to inhibit AMPs transcription. In the present work, the three defensins studied were suppressed in IMD-0354 treated insects. Downregulation of the AMP genes confirmed by the lower antibacterial activity detected in R. prolixus midgut samples was one of the possible reasons that allowed an intense proliferation of S. marcescens, R. rhodnii and Enterococcaceae. Thereby, defensins and its related NF-κB signaling pathways seem to play an essential role in regulating microbiota homeostasis in R. prolixus midgut. In this sense, the negative impact on the T. cruzi population was observed in insects fed with the NF-κB inhibitor could be related to the increased bacterial population of S. marcescens and Enterococcaceae. As discussed above, S. marcescens presents trypanolytic activity (Azambuja et al., 2004;Castro et al., 2007) and large amounts of this bacteria in the midgut probably interfered in T. cruzi development in IMD-0453 treated insects. Additionally, a decrease in the defC and prol levels in insects fed with IMD-0354 concurrently infected with T. cruzi, reinforcing the importance of the activation of these genes for the establishment of T. cruzi infection in R. prolixus. Together these results highlight the relevance of genes regulated by NF-κB TF in the R. prolixus immune system and gut homeostasis, as well as in the development of the parasite in the insect vector. Studying the genes involved in the signaling pathways regulated by NF-κB TF is essential for a broad knowledge of the mechanisms of activation and modulation of the immune response. More experiments are necessary to understand how different microorganisms selectively activate immune signaling pathways and different NF-κB TF molecules. Investigation of NF-κB TF genes and genomic analysis of AMPs promoter regions could provide relevant insights into the function of each pathway in controlling effector molecules against specific invading microorganisms.

AUTHOR CONTRIBUTIONS
CSV conceived and designed the experiments, performed all the experiments, participated in the analysis and interpretation of the data, wrote the manuscript, and corrected and approved the final manuscript. OCM conceived and designed the PCR experiments, participated in the analysis and interpretation of the data, and read, corrected, and approved the final manuscript. KSB performed the experiments of feeding and infection in insects, parasite quantification in insect midgut, participated in the analysis and interpretation of the data, and read, corrected, and approved the final manuscript. NAR conceived and designed the experiments, wrote the manuscript, and corrected and approved the final manuscript. DPC participated in the physiological and molecular experiments, analysis and interpretation of the data, and read, corrected, and approved the final manuscript. PA conceived and designed the experiments, contributed with reagents and materials, participated in the analysis and interpretation of the data, and read, corrected, and approved the final manuscript.

FUNDING
This work was sponsored by Brazilian Research Agencies FIOCRUZ, FAPERJ, and CNPq.