Nitric oxide effects on Rhodnius prolixus’s immune responses, gut microbiota and Trypanosoma cruzi development

https://doi.org/10.1016/j.jinsphys.2020.104100Get rights and content

Highlights

  • L-arginine treatment decreased the number of Trypanosoma cruzi in Rhodnius prolixus.

  • Rhodnius prolixus treated with L-NAME enhances Trypanosoma cruzi infection.

  • NO levels caused by L-arginine and L-NAME treatments affect other immune responses.

  • L-arginine and L-NAME treatment reduces gut microbiota of Rhodnius prolixus.

Abstract

The immune system of Rhodnius prolixus comprehends the synthesis of different effectors that modulate the intestinal microbiota population and the life cycle of the parasite Trypanosoma cruzi inside the vector midgut. One of these immune responses is the production of reactive nitrogen species (RNS) derived by the action of nitric oxide synthase (NOS). Therefore, we investigated the effects of L-arginine, the substrate for nitric oxide (NO) production and Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME), an inhibitor of NOS, added in the insect blood meal. We analyzed the impact of these treatments on the immune responses and development of intestinal bacteria and parasites on R. prolixus nymphs. The L-arginine treatment in R. prolixus nymphs induced a higher NOS gene expression in the fat body and increased NO production, but reduced catalase and antimicrobial activities in the midgut. As expected, L-NAME treatment reduced NOS gene expression in the fat body. In addition, L-NAME treatment diminished catalase activity in the hemolymph and posterior midgut reduced phenoloxidase activity in the anterior midgut and increased the antimicrobial activity in the hemolymph. Both treatments caused a reduction in the cultivatable intestinal microbiota, especially in insects treated with L-NAME. However, T. cruzi development in the insect’s digestive tract was suppressed after L-arginine treatment and the opposite was observed with L-NAME, which resulted in higher parasite counts. Therefore, we conclude that induction and inhibition of NOS and NO production are associated with other R. prolixus humoral immune responses, such as catalase, phenoloxidase, and antibacterial activities in different insect organs. These alterations reflect on intestinal microbiota and T. cruzi development.

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

References (51)

  • A. Herrera-Ortiz et al.

    The effect of nitric oxide and hydrogen peroxide in the activation of the systemic immune response of Anopheles albimanus infected with Plasmodium berghei

    Dev Comp Immunol.

    (2011)
  • K.J. Livak et al.

    Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method

    Methods.

    (2001)
  • E.M. Machado et al.

    WEB 2086, a platelet-activating factor antagonist, inhibits prophenoloxidase-activating system and hemocyte microaggregation reactions induced by Trypanosoma rangeli infection in Rhodnius prolixus hemolymph

    J Insect Physiol.

    (2006)
  • C.C. Murdock et al.

    Ambient temperature and dietary supplementation interact to shape mosquito vector competence for malaria

    J Insect Physiol.

    (2014)
  • L.S. Pereira et al.

    Production of reactive oxygen species by hemocytes from the cattle tick Boophilus microplus

    Exp Parasitol

    (2001)
  • T.M. Peterson et al.

    Nitric oxide metabolites induced in Anopheles stephensi control malaria parasite infection

    Free Radic. Biol. Med.

    (2007)
  • J.M. Ribeiro et al.

    Nitric oxide synthase activity from a hematophagous insect salivary gland

    FEBS Lett.

    (1993)
  • A. Rivero

    Nitric oxide: an antiparasitic molecule of invertebrates

    Trends Parasitol

    (2006)
  • T.S. Soares et al.

    A Kazal-type inhibitor is modulated by Trypanosoma cruzi to control microbiota inside the anterior midgut of Rhodnius prolixus

    Biochimie.

    (2015)
  • M.M.A. Whitten et al.

    Role of superoxide and reactive nitrogen intermediates in Rhodnius prolixus (Reduviidae)/Trypanosoma rangeli interactions

    Exp Parasitol

    (2001)
  • Albajar-Viñas, P., and Jannin, J. (2011). The hidden Chagas disease burden in Europe. Euro Surveill. 16(38):pii=19975....
  • P. Ascenzi et al.

    Nitric oxide limits parasite development in vectors and in invertebrate intermediate hosts

    IUBMB Life

    (2002)
  • P. Azambuja et al.

    Care and maintenance of triatomine colonies

  • Azambuja, P., Garcia, E.S., Waniek, P.J., Vieira, C.S., Figueiredo, M.B., Gonzalez, M.S., Mello, C.B., Castro, D.P.,...
  • Y. Carton et al.

    Parasite-induced changes in nitric oxide levels in Drosophila paramelanica

    J Parasitol

    (2009)
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