Brasiliensin: A novel intestinal thrombin inhibitor from Triatoma brasiliensis (Hemiptera: Reduviidae) with an important role in blood intake

Abstract

Every hematophagous invertebrate studied to date produces at least one inhibitor of coagulation. Among these, thrombin inhibitors have most frequently been isolated. In order to study the thrombin inhibitor from Triatoma brasiliensis and its biological significance for the bug, we sequenced the corresponding gene and evaluated its biological function. The T. brasiliensis intestinal thrombin inhibitor, termed brasiliensin, was sequenced and primers were designed to synthesize double strand RNA (dsRNA). Gene knockdown (RNAi) was induced by two injections of 15 μg of dsRNA into fourth instar nymphs. Forty-eight hours after the second injection, bugs from each group were allowed to feed on hamsters. PCR results showed that injections of dsRNA reduced brasiliensin expression in the anterior midgut by approximately 71% in knockdown nymphs when compared with controls. The reduction in gene expression was confirmed by the thrombin inhibitory activity assay and the citrated plasma coagulation time assay which showed activity reductions of ∼18- and ∼3.5-fold, respectively. Knockdown nymphs ingested approximately 39% less blood than controls. In order to confirm the importance of brasiliensin in blood ingestion, fourth instar nymphs were allowed to ingest feeding solution alone or feeding solution containing 15 U of thrombin prior to blood feeding. Fifty-five percent less blood was ingested by nymphs which were fed thrombin prior to blood feeding. The results suggest that anticoagulant activity in the midgut is an important determinant of the amount of blood taken from the host. The role of anticoagulants during blood ingestion is discussed in the light of this novel insight.

The role of salivary nitrophorins in the ingestion of blood by the triatomine bug Rhodnius prolixus (Reduviidae: Triatominae)

“…This difference in NP profile, in addition to other mechanical parameters of the mouthparts and cibarial pump, could help to explain the differential feeding performance observed between species such as why R. prolixus is more effective in ingesting blood in mammals and birds than other species such as Rhodnius robustus, Rhodnius nasutus and Rhodnius neglectus (Sant'Anna et al, 2001). The intestinal environment could also be involved in these differences in efficiency as our group has demonstrated the influence of intestinal anticoagulant in determining the efficiency of blood ingestion by Triatoma brasiliensis (Araujo et al, 2007).…”

The challenge of RNAi-mediated control of hemipterans

“…Reduction in blood feeding [66] recorded, despite lethal effects in earlier injection experiments and similar knockdown levels in both experiments [20].…”

Kazal-type inhibitors in the stomach of Panstrongylus megistus (Triatominae, Reduviidae)

“…In T. infestans, the activator of the intrinsic pathway of coagulation factor XII is inhibited by a double-domain Kazal-type inhibitor that also seems to interact with coagulation factor X (Campos et al, 2004). Thrombin, the key enzyme of the coagulation cascade, is inhibited by similar double-domain Kazal-type inhibitors in the stomach of D. maxima, T. infestans and T. brasiliensis Campos et al, 2002;Araujo et al, 2007). The stomach contents of P. megistus prolonged the coagulation time after extrinsic as well as intrinsic activation, but most strongly after the addition of thrombin.…”

“…The deduced full-length amino acid sequences of Kazal-type inhibitors from D. maxima, T. infestans and T. brasiliensis possess six, seven and eight Kazal-like domains, respectively (Mende et al, 1999;Lovato et al, 2006;Araujo et al, 2007) and that of P. megistus seven domains. These proteins are suggested to be expressed as precursors and processed posttranslationally to individual inhibitors (Lovato et al, 2006).…”

“…The inhibitor of R. prolixus, initially named prolixin G and then rhodniin, belongs to the class of Kazaltype inhibitors, possessing two Kazal-like domains (Hellmann and Hawkins, 1965;Friedrich et al, 1993). In triatomines, Kazal-type inhibitors are further characterized from the stomach of Dipetalogaster maxima, T. infestans and Triatoma brasiliensis Mende et al, 1999;Campos et al, 2002;Lovato et al, 2006;Araujo et al, 2007).…”