Marina S. Defferrari

Stage-specific gut proteinases of the cotton stainer bug Dysdercus peruvianus: Role in the release of entomotoxic peptides from Canavalia ensiformis urease

Canavalia ensiformis ureases are toxic to insects of different orders. The entomotoxicity of urease is due to a 10 kDa internal peptide released by proteinases in the insect digestive tract. We previously observed that, given orally, urease is toxic to nymphs of Dysdercus peruvianus, but does not affect adults. Here we characterized the major proteolytic activities of D. peruvianus midgut homogenates and investigated their in vitro-catalyzed release of the 10 kDa entomotoxic peptide from urease. Cysteine, aspartic and metalloproteinases are present in both homogenates. Variations in optimal pH and susceptibility to inhibitors indicated differences in the enzyme profiles in the two developmental stages. Only nymph homogenates released approximately 10 kDa fragment(s) from urease, recognized by antibodies against the entomotoxic peptide. Fluorogenic substrates containing urease partial sequences flanking the N-terminal or the C-terminal portion of the entomotoxic peptide were efficiently cleaved by homogenates from nymphs, but much more slowly by the adult homogenate. Different classes of enzymes in the homogenates cleaved both substrates suggesting that in vivo the release of the entomotoxic peptide results from the concerted action of at least two different proteinases. Our findings support the view that a differential processing of ingested urease by the insects explains at least in part the lack of toxicity in adults.

Insecticidal effect of Canavalia ensiformis major urease on nymphs of the milkweed bug Oncopeltus fasciatus and characterization of digestive peptidases.

Jackbean (Canavalia ensiformis) ureases are entomotoxic upon the release of internal peptides by insects digestive enzymes. Here we studied the digestive peptidases of Oncopeltus fasciatus (milkweed bug) and its susceptibility to jackbean urease (JBU). O. fasciatus nymphs fed urease showed a mortality rate higher than 80% after two weeks. Homogenates of midguts dissected from fourth instars were used to perform proteolytic activity assays. The homogenates hydrolyzed JBU in vitro, yielding a fragment similar in size to known entomotoxic peptides. The major proteolytic activity at pH 4.0 upon protein substrates was blocked by specific inhibitors of aspartic and cysteine peptidases, but not significantly affected by inhibitors of metallopeptidases or serine peptidases. The optimal activity upon N-Cbz-Phe-Arg-MCA was at pH 5.0, with complete blockage by E-64 in all pH tested. Optimal activity upon Abz-AIAFFSRQ-EDDnp (a substrate for aspartic peptidases) was detected at pH 5.0, with partial inhibition by Pepstatin A in the pH range 2-8. Fluorogenic substrates corresponding to the N- and C-terminal regions flanking a known entomotoxic peptide within urease sequence were also tested. While the midgut homogenate did not hydrolyze the N-terminal peptide, it cleaved the C-terminal peptide maximally at pH 4.0-5.0, and this activity was inhibited by E-64 (10 μM). The midgut homogenate was submitted to ion-exchange chromatography followed by gel filtration. A 22 kDa active fraction was obtained, resolved in SDS-PAGE (12%), the corresponding band was in-gel digested by trypsin, the peptides were analyzed by mass spectrometry, retrieving a cathepsin L protein. The purified cathepsin L was shown to have at least two possible cleavage sites within the urease sequence, and might be able to release a known insecticidal peptide in a single or cascade event. The results suggest that susceptibility of O. fasciatus nymphs to jackbean urease is, like in other insect models, due mostly to limited proteolysis of ingested protein and subsequent release of entomotoxic peptide(s) by cathepsin-like digestive enzymes.

Structure-function studies on jaburetox, a recombinant insecticidal peptide derived from jack bean (Canavalia ensiformis) urease.

BACKGROUND Ureases are metalloenzymes involved in defense mechanisms in plants. The insecticidal activity of Canavalia ensiformis (jack bean) ureases relies partially on an internal 10kDa peptide generated by enzymatic hydrolysis of the protein within susceptible insects. A recombinant version of this peptide, jaburetox, exhibits insecticidal, antifungal and membrane-disruptive properties. Molecular modeling of jaburetox revealed a prominent β-hairpin motif consistent with either neurotoxicity or pore formation. METHODS Aiming to identify structural motifs involved in its effects, mutated versions of jaburetox were built: 1) a peptide lacking the β-hairpin motif (residues 61-74), JbtxΔ-β; 2) a peptide corresponding the N-terminal half (residues 1-44), Jbtx N-ter, and 3) a peptide corresponding the C-terminal half (residues 45-93), Jbtx C-ter. RESULTS 1) JbtxΔ-β disrupts liposomes, and exhibited entomotoxic effects similar to the whole peptide, suggesting that the β-hairpin motif is not a determinant of these biological activities; 2) both Jbtx C-ter and Jbtx N-ter disrupted liposomes, the C-terminal peptide being the most active; and 3) while Jbtx N-ter persisted to be biologically active, Jbtx C-ter was less active when tested on different insect preparations. Molecular modeling and dynamics were applied to the urease-derived peptides to complement the structure-function analysis. MAJOR CONCLUSIONS The N-terminal portion of the Jbtx carries the most important entomotoxic domain which is fully active in the absence of the β-hairpin motif. Although the β-hairpin contributes to some extent, probably by interaction with insect membranes, it is not essential for the entomotoxic properties of Jbtx. GENERAL SIGNIFICANCE Jbtx represents a new type of insecticidal and membrane-active peptide.

Identification of the first insulin-like peptide in the disease vector Rhodnius prolixus: Involvement in metabolic homeostasis of lipids and carbohydrates.

Insulin-like peptides (ILPs) are functional analogs of insulin and have been identified in many insect species. The insulin cell signaling pathway is a conserved regulator of metabolism, and in insects, as well as in other animals, can modulate physiological functions associated with the metabolism of lipids and carbohydrates. In the present study, we have identified the first ILP from the Rhodnius prolixus genome (termed Rhopr-ILP) and investigated its involvement in energy metabolism of unfed and recently fed fifth instars. We have cloned the cDNA sequence and analyzed the expression profile of the transcript, which is predominantly present in neurosecretory cells in the brain, similar to other insect ILPs. Using RNAi, we have reduced the expression of this peptide transcript by 90% and subsequently measured the carbohydrate and lipid levels in the hemolymph, fat body and leg muscles. Reduced levels of Rhopr-ILP transcript induced increased carbohydrate and lipid levels in the hemolymph and increased lipid content in the fat body, in unfed insects and recently fed insects. Also their fat bodies displayed enlarged lipid droplets within the cells. On the other hand, the carbohydrate content in the fat body and in the leg muscles of unfed insects were decreased when compared to control insects. Our results indicate that Rhopr-ILP is a modulator of lipid and carbohydrate metabolism, probably through signaling the presence of available energy and nutrients in the hemolymph.

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

HighlightsThe tachykinin transcript encodes 8 Rhopr‐TKs, Rhopr‐TK 5 having 2 copies.TK‐like and kinin‐like immunoreactivity is co‐localized in processes on the hindgut.Rhopr‐TK 2 stimulates hindgut contractions.Rhopr‐TK 2 and Rhopr‐Kinin 2 are additive on hindgut contractions. 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.

An Insulin-Like Growth Factor in Rhodnius prolixus Is Involved in Post-feeding Nutrient Balance and Growth

Growth of organisms is modulated by the availability of nutrients and energy, and is mostly regulated by insulin-like growth factors (IGFs) through the insulin signaling system. In insects, IGFs produced by the fat body induce cell division during the molt cycle, regulate adult body size, and have metabolic effects. Here, we describe an IGF from the hematophagous hemipteran Rhodnius prolixus and show its activity in regulating growth and metabolism in the post-feeding period during the fifth, and last, nymphal instar. Rhopr-IGF transcript is present in a variety of tissues, with greatest expression in the fat body, the dorsal vessel, and the CNS. We silenced the expression of the transcript using RNA interference, and at 2 weeks after feeding, insects with reduced Rhopr-IGF expression showed increased hemolymph lipid and carbohydrate levels when compared to controls, but no differences were observed in fat body lipid or carbohydrate content. In order to assess the role of Rhopr-IGF in post-feeding growth, double stranded IGF-injected insects were followed through ecdysis, and this treatment resulted in shorter adults, with shorter and narrower wings, when compared to controls. The results suggest that Rhopr-IGF modulates growth in R. prolixus most likely through altering the usage of nutrients that are available in the hemolymph.