Jack bean urease modulates neurotransmitter release at insect neuromuscular junctions

Thiago Carrazoni; Christine Nguyen; Lucas F Maciel; Andres Delgado-Cañedo; Bryan A Stewart; Angela B Lange; Chariston A Dal Belo; Celia R Carlini; Ian Orchard

doi:10.1016/j.pestbp.2018.02.009

Jack bean urease modulates neurotransmitter release at insect neuromuscular junctions

Pestic Biochem Physiol. 2018 Apr:146:63-70. doi: 10.1016/j.pestbp.2018.02.009. Epub 2018 Mar 2.

Authors

Thiago Carrazoni¹, Christine Nguyen², Lucas F Maciel³, Andres Delgado-Cañedo³, Bryan A Stewart², Angela B Lange², Chariston A Dal Belo³, Celia R Carlini⁴, Ian Orchard⁵

Affiliations

¹ Universidade Federal do Rio Grande do Sul, Graduate Program in Cell and Molecular Biology, Center of Biotechnology, Porto Alegre, RS, Brazil; University of Toronto Mississauga, Department of Biology, Mississauga, ON, Canada; Pontifícia Universidade Católica do Rio Grande de Sul, Brain Institute, Porto Alegre, RS, Brazil. Electronic address: thi.carrazoni@gmail.com.
² University of Toronto Mississauga, Department of Biology, Mississauga, ON, Canada.
³ Universidade Federal do Pampa, Campus São Gabriel, São Gabriel, RS, Brazil.
⁴ Universidade Federal do Rio Grande do Sul, Graduate Program in Cell and Molecular Biology, Center of Biotechnology, Porto Alegre, RS, Brazil; Pontifícia Universidade Católica do Rio Grande de Sul, Brain Institute, Porto Alegre, RS, Brazil. Electronic address: celia.carlini@pucrs.br.
⁵ University of Toronto Mississauga, Department of Biology, Mississauga, ON, Canada. Electronic address: ian.orchard@utoronto.ca.

PMID: 29626993
DOI: 10.1016/j.pestbp.2018.02.009

Abstract

Background: Plants have developed a vast range of mechanisms to compete with phytophagous insects, including entomotoxic proteins such as ureases. The legume Canavalia ensiformis produces several urease isoforms, of which the more abundant is called Jack Bean Urease (JBU). Previews work has demonstrated the potential insecticidal effects of JBU, by mechanisms so far not entirely elucidated. In this work, we investigated the mechanisms involved in the JBU-induced activity upon neurotransmitter release on insect neuromuscular junctions.

Methods: Electrophysiological recordings of nerve and muscle action potentials, and calcium imaging bioassays were employed.

Results and conclusion: JBU (0.28 mg/animal/day) in Locusta migratoria 2nd instar through feeding and injection did not induce lethality, although it did result in a reduction of 20% in the weight gain at the end of 168 h (n = 9, p ≤ 0.05). JBU (0.014 and 0.14 mg) injected direct into the locust hind leg induced a dose and time-dependent decrease in the amplitude of muscle action potentials, with a maximum decrease of 70% in the amplitude at the highest dose (n = 5, p ≤ 0.05). At the same doses JBU did not alter the amplitude of action potentials evoked from motor neurons. Using Drosophila 3rd instar larvae neuromuscular preparations, JBU (10^-7 M) increased the occurrence of miniature Excitatory Junctional Potentials (mEJPs) in the presence of 1 mM CaCl₂ (n = 5, p ≤ 0.05). In low calcium (0.4 mM) assays, JBU (10^-7 M) was not able to modulate the occurrence of the events. In Ca²⁺-free conditions, with EGTA or CoCl₂, JBU induced a significant decrease in the occurrence of mEPJs (n = 5, p ≤ 0.05). Injected into the 3rd abdominal ganglion of Nauphoeta cinerea cockroaches, JBU (1 μM) induced a significant increase in Ca²⁺ influx (n = 7, p ≤ 0.01), similar to that seen for high KCl (35 mM) condition. Taken together the results confirm a direct action of JBU upon insect neuromuscular junctions and possibly central synapses, probably by disrupting the calcium machinery in the pre-synaptic region of the neurons.

Keywords: Calcium influx; Insects; Neuromuscular junction; Neurotoxicity; Plant ureases.

MeSH terms

Acetylcholinesterase / genetics*
Animals
Lepidoptera / genetics*
Mutation*
Spain

Substances

Acetylcholinesterase