First insights into insecticidal activity against Aedes aegypti and partial biochemical characterization of a novel low molecular mass chymotrypsin-trypsin inhibitor purified from Lonchocarpus sericeus seeds.

BACKGROUND: Arboviroses such as dengue, Zika and chikungunya represent a serious public health issue as a consequence of the absence of approved vaccines or specific antiviral drugs against the arboviruses that cause them. One way to prevent these diseases is by combating the vector mosquito, Aedes aegypti (Diptera), which has serine proteases in the midgut. Protease inhibitors are molecules that can block enzyme activity, impairing digestion and nutrition, which can lead to death. Thus, we purified and characterized a novel chymotrypsin-trypsin inhibitor (LsCTI) from Lonchocarpus sericeus seeds and investigated its effect upon Ae. aegypti egg hatching, larval development and digestive proteases. RESULTS: LsCTI showed a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the molecular mass determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was 8870.45 Da. Kinetics analyses revealed a noncompetitive type of inhibition and low inhibition constant (Ki ) for chymotrypsin (8.24 x 10-8 m). The thermal resistance was remarkable, even at 100 °C for 180 min. The inhibitor concentration required for 50-percent enzyme inhibition (IC50 ) of LsCTI was 4.7 x 10-7 m for Ae. aegypti midgut larval enzymes. LsCTI did not affect egg hatchability at 0.3 mg mL-1 , but caused a high larval mortality rate (77%) and delayed development (37%). CONCLUSIONS: LsCTI is a novel protease inhibitor with remarkable biochemical characteristics and is a potential tool to control Ae. aegypti development. © 2017 Society of Chemical Industry.

Trypsin inhibitor from Leucaena leucocephala seeds delays and disrupts the development of Aedes aegypti, a multiple-disease vector.

BACKGROUND: Nowadays, the Aedes aegypti mosquito represents a serious public health issue in view of the large outbreaks of the arboviral diseases zika, dengue, chikungunya and yellow fever. This holometabolous insect has midgut digestive enzymes that are trypsin- and chymotrypsin-like proteins. Protease inhibitors are able to bind to proteolytic enzymes and promote a blockage in digestion and nutrition, leading to death. Thus, we investigated the effect of trypsin inhibitor of Leucaena leucocephala (LTI) seeds on egg hatching, larval development and digestive midgut proteases. RESULTS: LTI was obtained by trichloroacetic acid precipitation followed by a single chromatography step on anhydrous trypsin sepharose. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate showed a single protein band with a molecular mass close to 20 kDa. After exposure of Ae. aegypti eggs to LTI (0.3 mg mL-1 ), egg hatching was reduced (50%). LTI did not show acute toxicity on newly hatched larvae incubated under the same conditions, but after 10 days of exposure a high mortality rate (86%) was observed and the surviving larvae had a 25% delay in development. LTI was able to inhibit in vitro the midgut enzymatic activity (70%), and when larvae were incubated with LTI solution we observed an inhibition of 56%. CONCLUSIONS: LTI is a promising new tool to control critical points of Ae. aegypti development. © 2016 Society of Chemical Industry.

Insecticidal activity against Aedes aegypti of m-pentadecadienyl-phenol isolated from Myracrodruon urundeuva seeds.

BACKGROUND: Myracrodruon urundeuva Fr. Allemao is a common tree in the Caatinga that has been widely used for various medical purposes. Previous studies showed that the ethanol seed extract of M. urundeuva has potent activity against the larval stage of the dengue vector Aedes aegypti. Given this potential insecticidal activity, bioguided separation steps were performed in order to isolate the active compound(s). RESULTS: The isolation process resulted in only one active chemical compound, identified by infrared spectroscopy and mass spectrometry as m-pentadecadienyl-phenol. This compound presented potent larvicidal and pupicidal activity (LC50 10.16 and 99.06 µg mL(-1) respectively) and great egg hatching inhibitory activity (IC50 49.79 µg mL(-1)). The mode of action was investigated through observations of behavioural and morphological changes performed in third-instar larvae treated with m-pentadecadienyl-phenol solution after 1, 6, 12, 16 and 20 h of exposure. Some changes were observed as flooding of the tracheal system, alterations in siphonal valves and anal gills and lethargy, probably caused by the strong anticholinesterasic activity reported previously. CONCLUSION: The compound isolated from M. urundeuva seeds, m-pentadecadienyl-phenol, showed potent activity against immature stages of dengue vector, Ae. aegypti, being considered the main larvicidal principle.

Potential of laticifer fluids for inhibiting Aedes aegypti larval development: evidence for the involvement of proteolytic activity.

It has been shown previously that the laticifer fluid of Calotropis procera (Ait.) R.Br. is highly toxic to the egg hatching and larval development of Aedes aegypti L. In the present study, the larvicidal potential of other laticifer fluids obtained from Cryptostegia grandiflora R.Br., Plumeria rubra L. and Euphorbia tirucalli L. was evaluated. We attempted to correlate larvicidal activity with the presence of endogenous proteolytic activity in the protein fraction of the fluids. After collection, the fluids were processed by centrifugation and dialysis to obtain the soluble laticifer protein (LP) fractions and eliminate water insoluble and low molecular mass molecules. LP did not visibly affect egg hatching at the doses assayed. LP from Cr. grandiflora exhibited the highest larval toxicity, while P. rubra was almost inactive. E. tirucalli was slightly active, but its activity could not be correlated to proteins since no protein was detected in the fluid. The larvicidal effects of LP from C. procera and Cr. grandiflora showed a significant relationship with the proteolytic activity of cysteine proteinases, which are present in both materials. A purified cysteine proteinase (papain) from the latex of Carica papaya (obtained from Sigma) was similarly effective, whereas trypsin and chymotrypsin (both serine proteinases) were ineffective. The results provide evidence for the involvement of cysteine proteinase activity in the larvicidal action of some laticifer fluids. C. procera is an invasive species found in areas infested with Ae. aegypti and thus could prove useful for combating mosquito proliferation. This is the first report to present evidence for the use of proteolytic enzymes as chemical agents to destroy Ae. aegypti larvae.

Potential of laticifer fluids for inhibiting Aedes aegypti larval development: evidence for the involvement of proteolytic activity

It has been shown previously that the laticifer fluid of Calotropis procera (Ait.) R.Br. is highly toxic to the egg hatching and larval development of Aedes aegypti L. In the present study, the larvicidal potential of other laticifer fluids obtained from Cryptostegia grandiflora R.Br., Plumeria rubra L. and Euphorbia tirucalli L. was evaluated. We attempted to correlate larvicidal activity with the presence of endogenous proteolytic activity in the protein fraction of the fluids. After collection, the fluids were processed by centrifugation and dialysis to obtain the soluble laticifer protein (LP) fractions and eliminate water insoluble and low molecular mass molecules. LP did not visibly affect egg hatching at the doses assayed. LP from Cr. grandiflora exhibited the highest larval toxicity, while P. rubra was almost inactive. E. tirucalli was slightly active, but its activity could not be correlated to proteins since no protein was detected in the fluid. The larvicidal effects of LP from C. procera and Cr. grandiflora showed a significant relationship with the proteolytic activity of cysteine proteinases, which are present in both materials. A purified cysteine proteinase (papain) from the latex of Carica papaya (obtained from Sigma) was similarly effective, whereas trypsin and chymotrypsin (both serine proteinases) were ineffective. The results provide evidence for the involvement of cysteine proteinase activity in the larvicidal action of some laticifer fluids. C. procera is an invasive species found in areas infested with Ae. aegypti and thus could prove useful for combating mosquito proliferation. This is the first report to present evidence for the use of proteolytic enzymes as chemical agents to destroy Ae. aegypti larvae.