S. culicis epimastigotes were maintained at 28°C in liver infusion and tryptose (LIT) medium, supplemented with 10% heat-inactivated fetal bovine serum (FBS; Cultilab, Campinas, Brazil). Briefly, WT, aposymbiotic (Apo), and WTR strains were counted every 4 days and added to a fresh medium at a final concentration of 3 × 10⁶ parasites/ml. All A. aegypti infections were performed with 3-day-old culture epimastigotes, which correspond to parasite exponential growth phase. In addition, for the resistance induction, WT strain was successively cultivated with increasing concentrations of H₂O₂ (Merck, Darmstadt, Germany) up to 1 mM (Bombaça et al., 2017).

A. aegypti (Red Eye strain) was reared in insectaries at Universidade Federal do Rio de Janeiro or at Instituto Oswaldo Cruz, Brazil, under a 12-h light/dark cycle at 25°C ± 2°C and 40%–80% relative humidity. Briefly, 300 mosquito larvae were fed with fish food (TetraMin Tablets; Tetra, Melle, Germany) every 2 days until pupae development, while adult mosquitoes (males and females) were kept in cages and fed with a solution of 10% sucrose ad libitum (Farnesi et al., 2009). Here, 4–7-day-old females were starved for up to 12 h and then artificially fed for 1 h with S. culicis suspension (10⁸ parasites/ml in a solution of 10% sucrose + 1 mM ATP) using water-jacketed artificial feeders maintained at 37°C and sealed with parafilm membranes (Bombaça et al., 2017). Assays were performed in female mosquitoes 4 days post-infection (dpi) and under three different ad libitum diet conditions: (i) 10% sucrose, (ii) 10% sucrose + 5 mM ASC (Sigma-Aldrich, St. Louis, USA), or (iii) 10% sucrose + 10 µM diphenylene iodonium (DPI; Sigma-Aldrich) solutions.

For fecundity and fertility assays, inseminated females (5 days old) were collected and infected with the three S. culicis strains as described above. On the next day, blood meals required for egg production were allowed for 30–60 min in anesthetized Swiss mice (CEUA/FIOCRUZ LW-20/14), and engorged females were collected to continue the experiment. After 3 days, the synchronized oviposition was stimulated through the distribution of individual mosquitoes in Petri dishes (150 mm diameter, lined with filter paper) and incubation with 4 ml filtered water for 1 h in the dark. The females were then discarded, and the number of eggs per female was counted (Rezende et al., 2008; Vargas et al., 2014). For egg viability, 50 ml of 0.15% yeast solution (w/v) was added in each Petri dish and maintained for 24 h at 25°C ± 2°C and 40%–80% relative humidity. After that, the hatching of L1 larvae was calculated (Farnesi et al., 2009). The parameters analyzed were (a) the percentage of efficient females (egg production equal to or greater than the median production of control mosquitoes), (b) null females (egg production equal to zero), and (c) infertile females (egg hatching equal to zero) (Resck et al., 2020).

To analyze the ROS amounts produced during A. aegypti infection, midguts were dissected and the oxidation products of DHE (Invitrogen, Carlsbad, USA) were assessed (Fernandes et al., 2007). Briefly, midguts were opened and washed with 10 mM phosphate buffer saline (PBS) to remove the intestinal contents. After that, pools of 15 gut epithelia each were incubated with 100 µM DHE for 30 min at 28°C, washed twice with PBS, frozen in liquid N₂, and homogenized in 100% acetonitrile (500 µl). The samples were sonicated and centrifuged at 13,000 g for 10 min; the resulting supernatant was dried under vacuum, and the pellet obtained was stored at -70°C until use. The dried pellets were resuspended in PBS containing 100 µM diethylenetriaminepentaacetic acid (DTPA; Sigma-Aldrich) and injected into a HPLC LC-10AT device (Shimadzu, Kyoto, Japan) equipped with a diode array (SPD-M10A) and fluorescence (RF-20A) detectors. Chromatographic separation of DHE oxidation products was performed using NovaPak C18 column, with 4 µM particle size and 3.9 mm × 150 mm (Waters, Milford, USA), equilibrated in solution A (10% acetonitrile and 0.1% trifluoroacetic acid ) at a flow rate of 0.4 ml/min. After sample injection, a 0%–40% linear gradient of solution B (100% acetonitrile) was applied for 10 min, followed by 10 min of 40% solution B, 5 min of 100% solution B, and 10 min of 100% solution A. The amount of DHE was measured by light absorption at 245 nm, and the DHE oxidation products, hydroxyethidium (EOH) and ethidium (E), were monitored by fluorescence detection with excitation at 510 nm and emission at 595 nm. For fluorescence microscopy analysis, A. aegypti midguts were dissected and incubated with 50 µM DHE as described above. After the incubation period, midguts were washed twice, fixed in 4% paraformaldehyde (Sigma-Aldrich) for 30 min at room temperature, and transferred for a glass slide. Semiquantitative evaluation of fluorescence levels was performed by acquiring three images under identical conditions of each midgut using a ×40 objective and 100 ms exposure time. The images were acquired by a Zeiss AxioObserver M1 microscope (Carl Zeiss, Oberkochen, Germany), and the data were analyzed using the ImageJ software (Bethesda, MD, USA). Representative sections of midguts of each experimental condition were analyzed under an LSM 710 confocal microscope (Carl Zeiss, Germany) at Plataforma de Microscopia Confocal (Instituto Oswaldo Cruz, Brazil).

A. aegypti Aag2 cells (Grace, 1966) were maintained at 28°C in Schneider’s Insect Medium (Sigma-Aldrich) supplemented with L-glutamine and 10% FBS. Cultures with 3-days-old (80%–90% confluence) were enzymatically dissociated with 1% trypsin (Sigma-Aldrich) and plated for 72 h to establish a cellular monolayer. After that, cells were quantified and infected with WT and WTR strains [multiplicity of infection (MOI) 1:1] for 4 h, washed with PBS to remove the non-adhered parasite, and maintained until 24 h at 28°C. The culture was then stained with fast panoptic (Laborclin, Pinhais, Brazil), and the percentage of cells with adhered parasite and the number of parasites per 100 cells were calculated. Alternatively, cells were treated with different pro-oxidant or antioxidant agents for 2 h before S. culicis infection. Treatments with 0.25 µM DPI, 5 µM menadione (Sigma-Aldrich), 50 U/ml superoxide dismutase (SOD; Sigma-Aldrich) from bovine erythrocytes, 50 U/ml catalase (Sigma-Aldrich) from bovine liver, and 1 µM mitoTEMPO (Santa Cruz Biotechnology, Dallas, USA) were maintained up to 24 h.

To evaluate the H₂O₂ production, infected midguts were dissected, and the intestinal contents were washed out with PBS. Pools of 10–20 gut epithelia were incubated in 100 µl PBS under dim light and at room temperature with 100 µM Amplex Red reagent (Invitrogen) and 50 U/ml horseradish peroxidase (HRP; Sigma-Aldrich) for 30 min. After this period, the midguts were spun, and the supernatant was analyzed to H₂O₂ presence. Since DUOX activity depends on calcium presence to H₂O₂ generation, the evaluation was made before the pre-incubation of midguts (pools of 15 epithelia each) in 200 µM Schneider’s Insect Medium with 1 µM ionomycin (Iono; Sigma-Aldrich), 5 mM ethylene glycol bis(2-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA; Sigma-Aldrich), and 5 µM DPI for 10 min at room temperature. After that, samples were incubated with Amplex Red and HRP for 60 min, and the supernatant was assessed for H₂O₂ release (Dias et al., 2013). Resorufin fluorescence was monitored by a SpectraMax M3 fluorimeter (Molecular Devices, Sunnyvale, USA) with excitation at 530 nm and emission at 590 nm and compared to H₂O₂ standard curves. Infected Aag2 cells were also evaluated for H₂O₂ production and DUOX activity. Thus, the cells were submitted to the abovementioned protocol, and the supernatants (100 µl) were used for fluorescence assessment. After that, the cellular culture was washed twice with PBS, and the cells were mechanically dissociated. Homogenates were obtained by sonication, and the protein concentration was assessed by Pierce™ BCA protein assay kit (Thermo Fisher Scientific, Waltham, USA) according to the manufacturer’s instructions.

Pools of 15 midguts were used for catalase and citrate synthase (CS) activity assessment. Briefly, the guts were dissected, washed out for content elimination, and disrupted by sonication for protein determination. In parallel, infected Aag2 cells were processed as described above. For catalase activity measurement, the homogenates were added to the reaction mixture containing 100 mM potassium phosphate buffer and 100 µM H₂O₂ (Weydert and Cullen, 2010). Peroxide disappearance was monitored for 2 min and recorded every 30 s at 240 nm, and positive control was made with 4 U/ml catalase. CS was measured through the reduction of coenzyme A using the thiol reagent 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) at 412 nm. The reaction mixture consisted of 75 mM Tris-HCl buffer (pH 8.0), with 30 µM acetyl-CoA (Sigma-Aldrich), 250 µM DTNB, and 500 µM oxaloacetate (Sigma-Aldrich) to start the reaction. Aag2 homogenates were also evaluated for mitochondrial complex I–III activity through ferricytochrome c reduction at 550 nm. Briefly, the samples were added to 50 mM potassium phosphate buffer (pH 7.5) with 1 mg/ml bovine serum albumin (BSA), 300 µM potassium cyanide (KCN; Merck, Darmstadt, Germany), 50 µM equine heart cytochrome c (Sigma-Aldrich), and 200 µM β-Nicotinamide adenine dinucleotide (NADH; Sigma-Aldrich). All assays were performed at room temperature in a SpectraMax M3 spectrometer and calculated by molar extinction coefficient of H₂O₂ (ε = 39.4 mmol⁻¹cm⁻¹), DTNB (ε = 13.6 mmol^-1cm^-1), and cytochrome c (ε = 18.5mmol⁻¹cm⁻¹) (Spinazzi et al., 2012).

Aag2 cells infected or not with S. culicis strains were fixed with 2.5% glutaraldehyde (Sigma-Aldrich) diluted in 0.1 M Na-cacodylate buffer (pH 7.2) for 40 min and postfixed with 1% osmium tetroxide (Sigma-Aldrich) with 2.5 mM calcium chloride and 0.8% potassium ferricyanide for 20 min, both at room temperature. After the washings, dehydration was performed in ethanol (50%, 70%, 90%, and 100%), dried by the critical point method with CO₂, mounted on aluminum stubs, coated with a 20-nm-thick gold layer, and analyzed in Jeol JSM6390LV (Jeol Ltd., Tokyo, Japan) scanning electron microscope, located in Plataforma de Microscopia Eletrônica Rudolf Barth (Instituto Oswaldo Cruz, Brazil).

For gene expression analysis, total RNA was isolated from 10–20 midguts using TRIzol reagent (Thermo Fisher Scientific) according to the manufacturer’s protocol. Complementary DNA was synthesized with SuperScript Vilo kit (Invitrogen) using oligo(dT) primers. All primers were tested by conventional PCR using a 40-cycle reaction (denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s), and PCR products were separated in 2% agarose gel. The qPCR was performed in an ABI Prism 7500 FAST (Applied Biosystems, Foster City, CA, USA) using ~40 ng/μl cDNA, specific primers, and Go-Taq PCR Master Mix (Promega, Madison, WI, USA). The comparative ΔΔCt method was used to compare changes in gene expression levels (Livak and Schmittgen, 2001). Genes of ribosomal protein S7 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as reference genes of A. aegypti and S. culicis, respectively. All oligonucleotide sequences used in qPCR assays are listed in Table S1.

Analyses were performed with GraphPad Prism version 5.0 for Windows (GraphPad Software, San Diego, USA) or IBM SPSS Statistics 22.0 software (IBM Corporation, New York, NY, USA). Asterisks indicate significant differences with the threshold for significance set at p ≤ 0.05. The pairwise comparisons and each used posttest analysis are described in respective figure legends.

As previous data pointed out that an antioxidant-enriched diet increases S. culicis infection in A. aegypti females (Bombaça et al., 2017), we decided to evaluate the contribution of ROS in response to infection. DHE oxidation analysis and H₂O₂ quantification were performed to assess the redox state of infected midguts from sucrose- or ASC-fed females. Primarily, as DHE oxidation is known to yield two distinct products according to oxidative species available, the concentration of EOH (selectively formed when DHE is oxidized by superoxide) and E (generated through reactions with different oxidant species) (Zhao et al., 2003) in tissues 4 dpi was differentially assessed through HPLC. WT infection increases the EOH levels 2.1-fold in sugar-fed mosquitoes, while no significant difference was detected in E amount. In contrast, WTR infection exacerbated only the production of E, increasing its detection 9.7-fold in infected midguts. The antioxidant-enriched diet prevented ROS production in infected tissues and conserved the EOH and E levels as in the diet-matched control group (Figures 1A, B). To further confirm that non-superoxide oxygen species are increased in WTR-infected mosquitoes, H₂O₂ release in midguts was measured. Our data pointed out that H₂O₂ was produced during S. culicis infection, increasing detection 1.8- and 7.0-fold after WT and WTR infections, respectively. Once again, the antioxidant-enriched diet limited ROS generation upon infection, decreasing up to 65.0% the H₂O₂ release compared to sugar-fed infected mosquitoes (Figure 1C).

Midguts of non-infected (NI) and infected mosquitoes were also incubated with DHE to determine the oxidative state by fluorescence microscopy. Infected midguts showed a clear increase in DHE staining comparing to NI controls, especially after WTR infection. These data reflect the increase of 1.6- and 2.3-fold in fluorescence intensity observed after the infection with WT and WTR, respectively. Microscopy analysis also allowed the detection of the effects of diet supplementation. ASC-fed mosquitoes presented that ROS production in response to S. culicis infection was reduced up to 70% (Figures 1D, E). Our microscopy findings also reflected the general observations about oxidative state of infected midguts, being corroborated by the analysis of DHE oxidation products (Figures 1A, B, E). To confirm that ROS production is a direct immunological response against S. culicis, DHE fluorescence analysis was performed in midguts infected with Apo strain, given that ASC feeding is able to modulate protist infection in the midguts (Bombaça et al., 2017). Fluorescence intensity was greater in infected midguts (up to 5.4-fold) 1 dpi regardless of antioxidant feeding. This profile changes 4 dpi in sugar-fed insects, when similar fluorescence detection was observed between NI and infected mosquitoes. However, at 4 dpi, fluorescence intensity was 2.9-fold higher in Apo-infected ASC-fed group compared to diet-matched NI mosquitoes, demonstrating a time-dependent increase of ROS production upon infection (Figure S1).

To investigate the mechanisms involved in strain-specific response produced by A. aegypti, the DUOX activity in infected midguts was analyzed. DUOX enzymes are calcium-dependent NADPH oxidases, and the incubation of NI midguts with ionomycin markedly stimulated H₂O₂ production. Additionally, the incubation of tissues with DPI, an inhibitor of flavoenzymes, or EGTA, a calcium-chelating agent, prevented the ionomycin-induced increase in H₂O₂ production up to 80% (Figure S2A). The contribution of DUOX to the oxidative burst during S. culicis infection was confirmed by analyses of enzyme activity and ROS production and the addition of 10 µM DPI into mosquitoes’ diet. First, we observed an increase of 3.2-fold in DUOX activity in WTR-infected mosquitoes, while DPI feeding decreased it significantly, in response to S. culicis infection (Figure 2A). DHE and Amplex Red assays evidenced that ROS generation in infected midguts was prevented by DPI, decreasing the fluorescence intensity 51.0% and the H₂O₂ release 78.3% after WTR infection (Figures 2B, C and Figure S2B). As our results demonstrated that WT infection did not change DUOX activity, and oxidative stress remained high even in the DPI-fed group (Figure 2), the mitochondrial activity of epithelial midgut cells was analyzed. The evaluation of CS activity, a mitochondrial content marker enzyme (Spinazzi et al., 2012), revealed an impairment of 52.5% and 37.3% after WT and WTR infections, respectively (Figure 3A). Since the balance between ROS-generating systems and ROS-detoxifying reactions is critical for oxidative homeostasis (Sterkel et al., 2017), the antioxidant state of A. aegypti midguts and S. culicis strains during infection was assessed. WTR-infected midguts showed a decrease of 63.5% catalase activity, while no significant differences were detected after WT infection (Figure 3B). As a counterpart, qPCR analysis presented a modulation of the parasite’s antioxidant gene expression. In relation to WT strain, WTR showed an increment of up to 1.3-fold in trypanothione reductase (TR) and mitochondrial tryparedoxin peroxidase (mTXNPx) expression and a decrease of 50.0% in cytoplasmic tryparedoxin peroxidase (cTXNPx) in infected midguts (Figure 3C).

Using Aag2 cell line, an interesting model for insect immune studies (Barletta et al., 2012), we evaluated the early steps (4 and 24 h post-infection) of S. culicis infection in midgut cells. Scanning electron microscopy analysis revealed that parasite–host interaction occurs in a similar manner independently of the strain used mainly by flagellum insertion in epithelial cells and in some cases with protist body participation. On the other hand, there is a remarkable difference in the level of infection between the strains, especially at 24 h (Figure 4 and Figure S3). Quantitative analysis revealed that both strains interact similarly in an early time, with up to 25% infected cells and an average of 120.7 parasites per 100 cells. Comparing 4 and 24 h, an increase in WT and WTR infections was detected. The percentage of WT-infected cells increased 1.2-fold in the later time point, while in the WTR group, this increase was more expressive, reaching up to 2.0-fold. The number of parasites per 100 cells was also higher, detecting an increase of 1.3- and 1.7-fold in WT and WTR infections, respectively (Figure 5).

After that, non-lethal doses (data not shown) of pro-oxidant and antioxidant molecules were tested in Aag2 cell cultures, aiming to evaluate their participation to S. culicis establishment. The treatment of Aag2 with 0.25 µM DPI or 50 U/ml catalase did not impact cell infection by both strains at 4 h (Figures S4A, B). At 24 h, both compounds increased the number of parasites per 100 cells up to 1.4-fold in the WTR-infected group, while no effect was detected in the WT-infected one (Figure 6A and Figure S4C). The evaluation of H₂O₂ release by Aag2 revealed that ROS production was time-dependent, increasing 1.3- and 2.5-fold in WT and WTR-infected cells, respectively (Figure 6B). Our data showed that both infections induced DUOX activity up to 1.6-fold at 4 h. DUOX activity was upregulated in all groups comparing both time points, being the most prominent increase found in infected groups (1.6- and 1.4-fold in WT and WTR, respectively). Despite the similar catalase activity of NI and infected groups at 4 h, an increase of 1.7-fold in NI and WT groups was observed after 24 h. In contrast, Aag2 cells had a significant decrease of 53.1% in this enzymatic activity comparing both time points of WTR infection (Figures 6C, D).

To evaluate the contributions of Aag2 mitochondrial metabolism and superoxide radical production during parasite interaction, the treatment of epithelial cells with 5 µM menadione, 1 µM mitoTEMPO, and 50 U/ml SOD was performed. Menadione strongly impacts WT infection, decreasing 44.5% infected cells and 27.5% the number of adhered parasites at 4 h (Figures S4D, E). This phenotype was exacerbated after 24 h, and the decrease of infected cells and parasites per 100 cells reaches 63.1% and 36.0%, respectively. On the other hand, pretreatment with mitoTEMPO and SOD increased the WT infection parameters up to 1.4-fold at 24 h (Figures 7A, B). We also observed a significant impairment in Aag2 mitochondrial complex I–III activity caused by WT infection at 24 h. No differences were observed in infection quantification or in complex I–III activity of Aag2 cultures infected with WTR (Figure 7C).

To assess whether S. culicis strains trigger an innate immunological response during midgut infection, we determined gene expression of key molecules in the immune pathways Toll, Immune deficiency (IMD), and Janus kinase/signal transducer and activator of transcription (JAK/STAT). Our results demonstrate that both strains modulate the expression of all immune pathways analyzed. The gene expression of the negative regulator of Toll pathway cact was upregulated, but the expression of the negative regulator of IMD pathway casp was not altered, suggesting an inactivation of Toll pathway after S. culicis infection. In contrast, the transcription factor Rel2 was similarly increased by WT and WTR strains, reaching 2.2-fold in relation to NI; in addition, the receptor of JAK–STAT pathway domeless (dome) was also upregulated by both strains, suggesting activation of both immune pathways. Except for DefC, the transcriptional increase of antimicrobial peptide (AMP) genes was greater in WTR-infected midguts. WT-infected midguts showed an increase of 3.0- and 7.5-fold in the expression of DefA and DefC, compared to uninfected control, while WTR-infected tissues presented an increase of 6.8- and 1.8-fold in each of those AMPs, respectively. Additionally, the gene expression of Att increased 3.9- and 10.4-fold in WT- and WTR-infected midguts, respectively (Figure 8).

To determine whether S. culicis infection and the exacerbated ROS production affect A. aegypti reproductive fitness, the first gonotrophic cycle was analyzed. The overall results showed that both strains cause similar losses in egg production and viability after sucrose and ASC feeding (Figure 9). WT and WTR led to a reduction in egg production up to 55.7% in sugar-fed and 61.8% in ASC-fed groups compared to diet-matched NI mosquitoes. The addition of antioxidant potentiated the harmful effects caused by WTR infection, decreasing egg production 37.9% in relation to infected females (Figure 9A). NI females presented more than 60.0% egg viability in both diet groups, while midgut infection decreased this value until 30% (Figure 9B). To confirm that reproductive disruption was derived from S. culicis infection, we evaluated this process in Apo-infected females. The presence of Apo strain did not impact egg production in both diet strategies; however, a reduction of 20.2% in egg viability was observed after ASC feeding (Figure S5).

S. culicis infection, especially by WTR, had a strong impact on oviposition efficiency, decreasing the median of eggs laid per female more than 10-fold according to the diet used. Efficiency was determined according to the median of eggs laid by the diet-matched uninfected group. The proportion of efficient females was lowered during midgut infection, especially by WTR, which reduced the percentage from 50.0% in NI to 19.3% and 15.9% in sugar- and ASC-fed, respectively. Regarding null females, we observed a similar increase comparing NI and infected groups fed with sugar; however, when the ASC-enriched diet was provided, there is a greater increment in this percentage, reaching 63.5% in WTR infection. Curiously, the percentage of infertile females was affected only in WTR groups, increasing from 9.3% in NI to 21.7% in ASC-fed females (Table 1).

Although S. culicis-infected females maintained the positive correlation between fecundity (numbers of eggs laid) and fertility (number of eggs hatched), the strength of this correlation, as found in NI mosquitoes, was lost after the infection. As observed in Figure 10A, the control group had a homogeneous distribution and linear relationship (R = 0.8360, p < 0.0001). In contrast, infected mosquitoes had a dispersed distribution and linearity impairment after WT (R = 0.7861, p < 0.0001) and WTR (R = 0.6416, p < 0.0001) infections (Figures 10C, E). Moreover, antioxidant feeding did not impact fecundity and fertility correlation when compared to sugar-fed females (Figures 10B, D, F).