Blood-Feeding Induces Reversible Functional Changes in Flight Muscle Mitochondria of Aedes aegypti Mosquito

Background: Hematophagy poses a challenge to blood-feeding organisms since products of blood digestion can exert cellular deleterious effects. Mitochondria perform multiple roles in cell biology acting as the site of aerobic energytransducing pathways, and also an important source of reactive oxygen species (ROS), modulating redox metabolism. Therefore, regulation of mitochondrial function should be relevant for hematophagous arthropods. Here, we investigated the effects of blood-feeding on flight muscle (FM) mitochondria from the mosquito Aedes aegypti, a vector of dengue and yellow fever. Methodology/Principal Findings: Blood-feeding caused a reversible reduction in mitochondrial oxygen consumption, an event that was parallel to blood digestion. These changes were most intense at 24 h after blood meal (ABM), the peak of blood digestion, when oxygen consumption was inhibited by 68%. Cytochromes c and a+a3 levels and cytochrome c oxidase activity of the electron transport chain were all reduced at 24 h ABM. Ultrastructural and molecular analyses of FM revealed that mitochondria fuse upon blood meal, a condition related to reduced ROS generation. Consistently, BF induced a reversible decrease in mitochondrial H2O2 formation during blood digestion, reaching their lowest values at 24 h ABM where a reduction of 51% was observed. Conclusion: Blood-feeding triggers functional and structural changes in hematophagous insect mitochondria, which may represent an important adaptation to blood feedin

Blood Meal-Derived Heme Decreases ROS Levels in the Midgut of Aedes aegypti and Allows Proliferation of Intestinal Microbiota

The presence of bacteria in the midgut of mosquitoes antagonizes infectious agents, such as Dengue and Plasmodium, acting as a negative factor in the vectorial competence of the mosquito. Therefore, knowledge of the molecular mechanisms involved in the control of midgut microbiota could help in the development of new tools to reduce transmission. We hypothesized that toxic reactive oxygen species (ROS) generated by epithelial cells control bacterial growth in the midgut of Aedes aegypti, the vector of Yellow fever and Dengue viruses. We show that ROS are continuously present in the midgut of sugar-fed (SF) mosquitoes and a blood-meal immediately decreased ROS through a mechanism involving heme-mediated activation of PKC. This event occurred in parallel with an expansion of gut bacteria. Treatment of sugar-fed mosquitoes with increased concentrations of heme led to a dose dependent decrease in ROS levels and a consequent increase in midgut endogenous bacteria. In addition, gene silencing of dual oxidase (Duox) reduced ROS levels and also increased gut flora. Using a model of bacterial oral infection in the gut, we show that the absence of ROS resulted in decreased mosquito resistance to infection, increased midgut epithelial damage, transcriptional modulation of immune-related genes and mortality. As heme is a pro-oxidant molecule released in large amounts upon hemoglobin degradation, oxidative killing of bacteria in the gut would represent a burden to the insect, thereby creating an extra oxidative challenge to the mosquito. We propose that a controlled decrease in ROS levels in the midgut of Aedes aegypti is an adaptation to compensate for the ingestion of heme

Catalase protects Aedes aegypti from oxidative stress and increases midgut infection prevalence of Dengue but not Zika.

BACKGROUND:Digestion of blood in the midgut of Aedes aegypti results in the release of pro-oxidant molecules that can be toxic to the mosquito. We hypothesized that after a blood meal, the antioxidant capacity of the midgut is increased to protect cells against oxidative stress. Concomitantly, pathogens present in the blood ingested by mosquitoes, such as the arboviruses Dengue and Zika, also have to overcome the same oxidative challenge, and the antioxidant program induced by the insect is likely to influence infection status of the mosquito and its vectorial competence. METHODOLOGY/PRINCIPAL FINDINGS:We found that blood-induced catalase mRNA and activity in the midgut peaked 24 h after feeding and returned to basal levels after the completion of digestion. RNAi-mediated silencing of catalase (AAEL013407-RB) reduced enzyme activity in the midgut epithelia, increased H2O2 leakage and decreased fecundity and lifespan when mosquitoes were fed H2O2. When infected with Dengue 4 and Zika virus, catalase-silenced mosquitoes showed no alteration in infection intensity (number of plaque forming units/midgut) 7 days after the infectious meal. However, catalase knockdown reduced Dengue 4, but not Zika, infection prevalence (percent of infected midguts). CONCLUSION/SIGNIFICANCE:Here, we showed that blood ingestion triggers an antioxidant response in the midgut through the induction of catalase. This protection facilitates the establishment of Dengue virus in the midgut. Importantly, this mechanism appears to be specific for Dengue because catalase silencing did not change Zika virus prevalence. In summary, our data suggest that redox balance in the midgut modulates mosquito vectorial competence to arboviral infections

Microbiota activates IMD pathway and limits Sindbis infection in Aedes aegypti

Submitted by Sandra Infurna ([email protected]) on 2017-03-07T12:09:56Z No. of bitstreams: 1 luiza_pereira_etal_IOC_2017.pdf: 946502 bytes, checksum: 9fbc3fe71c26955f1174456ceca1c987 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2017-03-07T12:21:23Z (GMT) No. of bitstreams: 1 luiza_pereira_etal_IOC_2017.pdf: 946502 bytes, checksum: 9fbc3fe71c26955f1174456ceca1c987 (MD5)Made available in DSpace on 2017-03-07T12:21:23Z (GMT). No. of bitstreams: 1 luiza_pereira_etal_IOC_2017.pdf: 946502 bytes, checksum: 9fbc3fe71c26955f1174456ceca1c987 (MD5) Previous issue date: 2017Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica Leopoldo de Meis. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica Leopoldo de Meis. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica Leopoldo de Meis. Rio de Janeiro, RJ, Brasil.Universidade Federal de Santa Catarina. Departamento de Microbiologia, Imunologia e Parasitologia. Florianópolis, SC, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisas em Leishmaniose. Rio de Janeiro, RJ. Brasil.Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica Leopoldo de Meis. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica Leopoldo de Meis. Rio de Janeiro, RJ, Brasil.Background: Aedes aegypti is the main vector of important arboviruses such as dengue, Zika and chikungunya. During infections mosquitoes can activate the immune pathways Toll, IMD and JAK/STAT to limit pathogen replication. Results: Here, we evaluate the immune response profile of Ae. aegypti against Sindbis virus (SINV). We analyzed gene expression of components of Toll, IMD and JAK/STAT pathways and showed that a blood meal and virus infection upregulated aaREL2 in a microbiota-dependent fashion, since this induction was prevented by antibiotic. The presence of the microbiota activates IMD and impaired the replication of SINV in the midgut. Constitutive activation of the IMD pathway, by Caspar depletion, leads to a decrease in microbiota levels and an increase in SINV loads. Conclusion: Together, these results suggest that a blood meal is able to activate innate immune pathways, through a nutrient induced growth of microbiota, leading to upregulation of aaREL2 and IMD activation. Microbiota levels seemed to have a reciprocal interaction, where the proliferation of the microbiota activates IMD pathway that in turn controls bacterial levels, allowing SINV replication in Ae. aegypti mosquitoes. The activation of the IMD pathway seems to have an indirect effect in SINV levels that is induced by the microbiota

Catalase knockdown in the midgut.

<p>(A) Two-day-old females were injected with dsRNA against catalase (dsCat) or an unrelated control gene (dsLac). Two days after the dsRNA injection, a group of mosquitoes were fed blood, while others were fed exclusively with sugar. Twenty-four hours later, RNA was extracted for qPCR analysis. (B) Catalase activity in the epithelia was measured 24 h after a blood meal in dsRNA-treated mosquitoes. (C) Hydrogen peroxide leakage was measured in the midgut epithelia of SF mosquitoes injected with dsCat or dsLacZ. (A) *** <i>p <</i> 0.001 (t-test). (B) **** <i>p <</i> 0.0001 (t-test). (C) * <i>p</i> = 0.0404 (t-test).</p

BYC, an atypical aspartic endopeptidase from Rhipicephalus (Boophilus) microplus eggs

An aspartic endopeptidase was purified in our laboratory from Rhipicephalus (Boophilus) microplus eggs [Logullo, C., Vaz, I.S., Sorgine, M.H., Paiva-Silva, G.O., Faria, F.S., Zingali, R.B., De Lima, M.F., Abreu, L., Oliveira, E.F., Alves, E.W, Masuda, H., Gonzales, J.C., Masuda, A., and Oliveira, P.L., 1998. Isolation of an aspartic proteinase precursor from the egg of a hard tick, Rhipicephalus (Boophilus) microplus. Parasitology 116, 525-532]. Boophilus yolk cathepsin (BYC) was tested as component of a protective vaccine against the tick, inducing a significant immune response in cattle [da Silva, VI., Jr., Logullo, C., Sorgine, M., Velloso, F.F., Rosa de Lima, M.F., Gonzales, J.C., Masuda, H., Oliveira, P.L., and Masuda, A., 1998. Immunization of bovines with an aspartic proteinase precursor isolated from Rhipicephalus (Boophilus) microplus eggs. Vet. Immunol. Immunopathol. 66,331-341]. In this work, BYC was cloned and its primary sequence showed high similarity with other aspartic endopeptidases. In spite of this similarity, BYC sequence shows many important differences in relation to other aspartic peptidases, the most important being the lack of the second catalytic Asp residue, considered to be essential for the catalysis of this class of endopeptidases. When we determined BYC cleavage specificity by LC-MS, we found out that it presents a preference for hydrophobic residues in P1 and P1` in accordance to most aspartic endopeptidases. Also, when analyzed by circular dicroism, BYC presented high beta sheet content, also a characteristic of aspartic endopeptidases. On the other hand, although both native and recombinant BYC are catalytically active, they present a very low specific activity, what seems to indicate that this peptidase will digest its natural substrate, vitellin, very slowly. We speculate that such a slow Vn degradative process might constitute an important strategy to preserve egg protein content to the hatching larvae. (c) 2007 Elsevier Inc. All rights reserved

Catalase mRNA and activity increased in the midgut epithelia of blood-fed mosquitoes.

<p><i>Aedes aegypti</i> females were fed sugar (SF) or blood (BF) and dissected at the indicated time points after a blood meal. (A) Catalase mRNA expression in the epithelia was evaluated using qPCR analysis of SF and BF mosquitoes. (B) Enzymatic activity was measured as described in the Materials and Methods section. (C) Catalase inhibition by AT <i>in vitro</i>. Midgut epithelia was collected from blood-fed mosquitoes 24 h after feeding and was incubated with AT and H₂O₂ for 30 min at 4°C; then, catalase activity was assayed. (D) Mosquitoes were fed blood supplemented with 15 mM AT and assayed for catalase activity in the epithelia *** <i>p <</i> 0.001. Figure 1A-B–ANOVA followed by Dunnett's multiple comparison test.</p

Catalase knockdown in the midgut.

<p>(A) Two-day-old females were injected with dsRNA against catalase (dsCat) or an unrelated control gene (dsLac). Two days after the dsRNA injection, a group of mosquitoes were fed blood, while others were fed exclusively with sugar. Twenty-four hours later, RNA was extracted for qPCR analysis. (B) Catalase activity in the epithelia was measured 24 h after a blood meal in dsRNA-treated mosquitoes. (C) Hydrogen peroxide leakage was measured in the midgut epithelia of SF mosquitoes injected with dsCat or dsLacZ. (A) *** <i>p <</i> 0.001 (t-test). (B) **** <i>p <</i> 0.0001 (t-test). (C) * <i>p</i> = 0.0404 (t-test).</p

Catalase silencing impacted Dengue but not Zika midgut infection prevalence.

<p>(A) Females were fed blood contaminated with 10⁷ PFU/mL of Zika virus, and 7 days after feeding the number of PFU was determined in the midgut. (B) The percentage of infected midguts (infection prevalence) was scored from the same set of data as in A. (C) Mosquitoes were fed a lower dose of Zika-infected blood (10⁵/mL), and PFU/midgut was determined 7 days post-infection. (D) Infection prevalence of mosquitoes from C. (E) Mosquitoes were fed blood contaminated with 10⁶ PFU/mL of Dengue 4 virus, and PFU/midgut was counted 7 DPI. (F) Infection prevalence was determined from the same group of mosquitoes. Mann-Whitney <i>U</i>-tests were used for infection intensity (A, C, E), and chi-square tests were performed to determine the significance of infection prevalence analysis (B, D, F). Statistical values and number of replicates are depicted in the corresponding figures.</p

Catalase knockdown affected both resistance to H₂O₂ and oviposition.

<p>Catalase was silenced as described, and two days after dsRNA injection, SF (A) or BF (immediately after feeding) (B) mosquitoes were transferred to cages containing 5% sucrose supplemented with 1 M H₂O₂ <i>ad libitum</i> (day 0). A fresh H₂O₂ solution was provided daily. Survival was scored every 24 h. *** <i>p</i> < 0.0001 for the comparison between dsLacZ–H₂O₂ vs dsCat–H₂O₂ (log-rank test). (C) Catalase-silenced mosquitoes were blood-fed and allowed to lay eggs. Each dot represents an individual mosquito. LacZ–n = 21. Catalase–n = 25. * <i>p =</i> 0.37 (t-test).</p