Nitric oxide effects on Rhodnius prolixus’s immune responses, gut microbiota and Trypanosoma cruzi development
Graphical abstract
Introduction
Rhodnius prolixus is an insect vector that transmits the protist parasite, Trypanosoma cruzi, that causes Chagas disease. This disease is a public health problem, especially in Latin America, but it has been expanding to North America, Western Pacific Region and Europe (Albajar-Viñas and Jannin, 2011, World Health Organization (WHO)., 2018). The triatomines are associated to T. cruzi transmission to vertebrates through the classical via where the infected insect defecates near the bite site during the blood intake, or through oral transmission by food contamination with triturated infected insect or its feces (de Noya and González, 2015, Coura, 2015). R. prolixus is an important T. cruzi vector in Colombia, Guyana, French Guyana, Suriname and Venezuela (Coura, 2015), and is an important model for Triatominae physiological studies (Mesquita et al., 2015).
Trypanosoma cruzi develops inside the insect́s digestive tract, where lytic factors, lectins, enzymes, microbiota-derived factors and humoral immune responses have been described (Garcia et al., 2010, Soares et al., 2015, Azambuja et al., 2017). Therefore, a successful T. cruzi infection depends not only on parasite adhesion, multiplication, and differentiation inside the triatomine gut (Gonzalez et al., 1999 Jun, Gonzalez et al., 2006 Dec, Gonzalez et al., 2013, Azambuja et al., 2017) but also on the modulation of the insect immune responses and competition with the gut microbiota (Castro et al., 2012, Vieira et al., 2016).
The immune responses in the midgut of R. prolixus are composed of humoral defenses that may be secreted directly into the midgut lumen to eliminate potential pathogens acquired during feeding. In this category, we find antimicrobial peptides (AMPs), reactive nitrogen and oxygen species (RNS and ROS), and phenoloxidase activity (PO). The RNS, mainly nitric oxide (NO), is an immune response molecule related to the control of parasite infection in the invertebrate and vertebrate hosts (Dimopoulos et al., 1998, Luckhart et al., 1998, Hahn et al., 2001, Ascenzi and Gradoni, 2002, Radi, 2004). NO is generated by the activity of nitric oxide synthase (NOS) when it converts L-arginine to L-citrulline. Besides being a response to infections, NO is also associated to immune signaling, long-term memory, nervous system function, and blood-feeding (Foley and O'Farrell, 2003, Herrera-Ortiz et al., 2011, Matsumoto et al., 2013, Sadekuzzaman et al., 2018, Davies, 2000, Ribeiro and Nussenzveig, 1993).
In R. prolixus, the production of RNS seems to be modulated by T. cruzi infections depending on the parasite strain (Whitten et al., 2001, Castro et al., 2012). Whitten et al. (2001) observed that, in R. prolixus infected with T. cruzi, the NOS was upregulated in the hemocoel, and downregulated in the gut. Moreover, Castro et al. (2012) observed a reduction in the production of RNS in the digestive tract after T. cruzi infection. To comprehend the role of NO, different research groups have used L-arginine, as a precursor of NO, and L-NAME, as an inhibitor of NOS, in different insect vectors (Luckhart et al., 1998, Rivero, 2006, Peterson et al., 2007, Carton et al., 2009, Inamdar and Bennett, 2014 Feb, Murdock et al., 2014, Chavez et al., 2015 Jun). In the mosquito Anopheles, the treatment of different species with L-arginine or L-NAME impacted the survival of the malaria parasite. The increase of NOS and NO production negatively impact the survival of different Plasmodium species inside the insect vector (Luckhart et al., 1998, Vijay et al., 2011, Herrera-Ortiz et al., 2011, Murdock et al., 2014).
In this context, in this work, we treated R. prolixus with L-arginine and L-NAME. We followed the humoral immune responses, such as AMPs, PO, and catalase in different organs and tissues, and the effects on cultivatable intestinal microbiota and T. cruzi infections. The results obtained here indicate that the oral administration of L-NAME and L-arginine interfere in NO production, NOS expression level, and other immune responses such as PO, catalase and antibacterial activity of R. prolixus. Furthermore, these results suggest that NO homeostasis regulates the survival of T. cruzi in the insect vector, and could be used as a target to block the transmission of this parasite by inhibiting its development inside the triatomine.
Section snippets
Ethics statement
Defibrinated rabbit blood was provided by Instituto de Ciência e Tecnologia em Biomodelos at Fiocruz (ICTB-Fiocruz) that breed and maintain animals following the Ethical Principles in Animal Experimentation of Fiocruz. The license was obtained and approved by the Ethics Commission for Animal Use of Fiocruz (CEUA/Fiocruz) under the protocol number L-019/17.
Rhodnius prolixus maintenance and drug treatments
R. prolixus was maintained in a colony at Laboratório de Bioquímica e Fisiologia de Insetos, (LABFISI/IOC), under controlled temperature
Results
Using the fluorometric kit, we observed significant levels of NO in all organs tested (Fig. 1). In the hemolymph, L-arginine and L-NAME treatments do not result in changes in the NO levels when these groups are compared to controls (Fig. 1A). However, L-arginine treated insects had more NO in the hemolymph when compared to L-NAME treated insects (p < 0.05; Fig. 1A). L-arginine treated insects also showed higher NO levels in the anterior (p < 0.05; Fig. 1B) and posterior midgut (p < 0.05; Fig. 1
Discussion
In this work, we investigated the interference of L-arginine and L-NAME treatments in the innate immune responses, the cultivatable bacterial microbiota and the development of T. cruzi Dm28c in R. prolixus. L-arginine is the substrate for the production of NO. Oral treatment with L-arginine resulted in an increase in the nitric oxide (NO) levels and upregulation of NOS gene expression in the fat body of R. prolixus. This treatment also triggered a decrease in the catalase activity in the
CRediT authorship contribution statement
Kate Katherine da Silva Batista: Conceptualization, Methodology, Investigation. Cecília Stahl Vieira: Conceptualization, Investigation, Writing - review & editing. Emmanuelle Batista Florentino: Methodology, Investigation. Karina Francine Bravo Caruso: Methodology, Investigation. Paula Thais Pinheiro Teixeira: Investigation. Caroline da Silva Moraes: Methodology, Conceptualization, Writing - review & editing. Fernando Ariel Genta: Writing - review & editing. Patrícia de Azambuja: Funding
Acknowledgments
The authors would like to thank the fundings provided by Fundação Oswaldo Cruz (Fiocruz), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular (INCT-EM). KB (MSc) was a graduate student of the postgraduation Program in Biologia Parasitária of Instituto Oswaldo Cruz. EF was
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