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

Abstract

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.

Keywords: Immunohistochemistry; Kinin; Muscle contraction; Salivary glands; qPCR.