Expression and functional characterization of tachykinin-related peptides in the blood-feeding bug, Rhodnius prolixus.

Tachykinins (tachykinin-related peptides, TRPs) are multifunctional neuropeptides that have widespread distribution in the central nervous system (CNS) and in the gastrointestinal tract of many insects, and most have been shown to stimulate contractions of visceral muscles. Invertebrate TRPs carry a characteristic conserved C-terminal pentapeptide (FXGXR-amide) and most of them share some amino acid sequence similarities (approx. 45%) with the vertebrate and mammalian tachykinin family. We have functionally characterized the tachykinins in R. prolixus (Rhopr-TKs) and partially cloned the transcript that encodes for the peptide precursor. The transcript encodes 8 Rhopr-TKs, 7 of which are unique with Rhopr-TK 5 having 2 copies. The spatial distribution analysis of the Rhopr-TK transcript indicates that the highest expression levels are in the CNS, but transcript expression is also associated with salivary glands, fat body, dorsal vessel, and the various gut compartments. Rhopr-TK 1, 2 and 5 significantly increase the frequency and amplitude of peristaltic contractions of the salivary glands. Hindgut muscle also displayed a dose-dependent increase in basal tonus in response to Rhopr-TK1, 2 and 5. TK-like immunoreactivity was seen in a small group of processes that are situated on the lateral margins of the hindgut. Interestingly, kinin-like immunoreactivity is seen in immunoreactive processes on the lateral margin of the hindgut as well as fine processes covering the entire hindgut. Co-localization studies show that TK-like staining is always co-localized with kinin-like immunoreactivity, whereas kinin-like staining is seen in the fine processes that are devoid of TK-like immunoreactivity indicating that TKs are most likely released together with kinins to act on the hindgut. Rhopr-Kinin 2 is a potent stimulator of hindgut muscle contraction in R. prolixus. Addition of Rhopr-Kinin 2 and Rhopr-TK 2 to the hindgut leads to a contraction that was additive of the effects of Rhopr-Kinin 2 and Rhopr-TK 2 alone.

Jack bean (Canavalia ensiformis) urease induces eicosanoid-modulated hemocyte aggregation in the Chagas' disease vector Rhodnius prolixus.

Ureases are multifunctional proteins that display biological activities independently of their enzymatic function, such as induction of exocytosis and insecticidal effects. Rhodnius prolixus, a major vector of Chagas' disease, is a model for studies on the entomotoxicity of jack bean urease (JBU). We have previously shown that JBU induces the production of eicosanoids in isolated tissues of R. prolixus. In insects, the immune response comprises cellular and humoral reactions, and is centrally modulated by eicosanoids. Cyclooxygenase products signal immunity in insects, mainly cellular reactions, such as hemocyte aggregation. In searching for a link between JBU's toxic effects and immune reactions in insects, we have studied the effects of this toxin on R. prolixus hemocytes. JBU triggers aggregation of hemocytes after injection into the hemocoel and when applied to isolated cells. On in vitro assays, the eicosanoid synthesis inhibitors dexamethasone (phospholipase A2 indirect inhibitor) and indomethacin (cyclooxygenase inhibitor) counteracted JBU's effect, indicating that eicosanoids, more specifically cyclooxygenase products, are likely to mediate the aggregation response. Contrarily, the inhibitors esculetin and baicalein were inactive, suggesting that lipoxygenase products are not involved in JBU's effect. Extracellular calcium was also necessary for JBU's effect, in agreement to other cell models responsive to ureases. A progressive darkening of the medium of JBU-treated hemocytes was observed, suggestive of a humoral response. JBU was immunolocalized in the cultured cells upon treatment along with cytoskeleton damage. The highest concentration of JBU tested on cultured cells also led to nuclei aggregation of adherent hemocytes. This is the first time urease has been shown to affect insect hemocytes, contributing to our understanding of the entomotoxic mechanisms of action of this protein.

A phospholipase A2 gene is linked to Jack bean urease toxicity in the Chagas' disease vector Rhodnius prolixus.

BACKGROUND: Ureases are multifunctional enzymes that display biological activities independent of their enzymatic function, including exocytosis induction and insecticidal effects. The hemipteran Rhodnius prolixus is one of the known susceptible models for this toxicity. It has been shown that Jack bean urease (JBU) has deleterious effects on R. prolixus, and these effects are modulated by eicosanoids, which are synthesized in a cascade involving phospholipase A2 (PLA2) enzymes. METHODS: R. prolixus genome was screened for putative PLA2s and matching transcripts were cloned. Predicted amino acid sequences were analyzed and transcript distribution among tissues was determined by qPCR. RNAi techniques were used and subsequent JBU toxicity assays were performed. RESULTS: Two PLA2 genes were identified, Rhopr-PLA2III and Rhopr-PLA2XII. The transcripts are widely distributed in the tissues but at different levels. The analyses fit the putative proteins into groups III and XII of secretory PLA2s. After 70% of Rhopr-PLA2XII expression was knocked down, JBU's toxicity was decreased by more than 50% on 5th instars R. prolixus. CONCLUSIONS: Rhopr-PLA2XII gene is linked to JBU's toxic effect in R. prolixus and our findings support previous studies demonstrating that eicosanoids modulate this toxicity. GENERAL SIGNIFICANCE: Besides identifying and characterizing two PLA2 genes in the major Chagas' disease vector R. prolixus, we have shown that the potent toxicity of JBU is linked to one of these genes. Our results contribute to the general comprehension of urease's mechanisms of action in insects, and, potentially, to studies on the control of the Chagas' disease parasite transmission.