The neurotoxic mechanism of Jack Bean Urease in insects involves the interplay between octopaminergic and dopaminergic pathways.

In the last decades, the entomotoxicity of JBU and its derived peptides became an object of study, due mainly to the ubiquitous interaction of these compounds with different species of insects and their potential as natural insecticides. In this work, we investigated the neurotoxic effects of JBU in Nauphoeta cinerea cockroaches by dissecting pharmacologically the monoaminergic pathways involved. Selective pharmacological modulators for monoaminergic pathways in in vivo and ex vivo experimental models were employed. Thus, the analysis of N. cinerea neurolocomotory behavior demonstrated that JBU (1.5 and 3 µg/g) induces a significant decrease in the exploratory activity. In these assays, pretreatment of animals with phentolamine, SCH23390 or reserpine, interfered significantly with the response of JBU. Using in vivo abductor metathoracic preparations JBU (1.5 µg/g) induced progressive neuromuscular blockade, in 120 min recordings. In this set of experiments, the previous treatment of the animals with phentolamine, SCH23390 or reserpine, completely inhibited JBU-induced neuromuscular blockade. The recordings of spontaneous compound neural action potentials in N. cinerea legs showed that JBU, only in the smallest dose, significantly decreased the number of potentials in 60 min recordings. When the animals were pretreated with phentolamine, SCH23390, or reserpine, but not with mianserin, there was a significant prevention of the JBU-inhibitory responses on the action potentials firing. Meanwhile, the treatment of the animals with mianserin did not affect JBU's inhibitory activity. The data presented in this work strongly suggest that the neurotoxic response of JBU in N. cinerea involves a cross talking between OCTOPAMIN-ergic and DOPAMIN-ergic nerve systems, but not the SEROTONIN-ergic neurotransmission. Further molecular biology studies with expression of insect receptors associated with voltage clamp techniques will help to discriminate the selectivity of JBU over the monoaminergic transmission.

Neurotoxic effects of sublethal concentrations of cyanobacterial extract containing anatoxin-a(s) on Nauphoeta cinerea cockroaches.

The detection of cyanotoxins, such as the anatoxin-a(s), is essential to ensure the biological safety of water environments. Here, we propose the use of Nauphoeta cinerea cockroaches as an alternative biological model for the biomonitoring of the activity of anatoxin-a(s) in aquatic systems. In order to validate our proposed model, we compared the effects of a cyanobacterial extract containing anatoxin-a(s) (CECA) with those of the organophosphate trichlorfon (Tn) on biochemical and physiological parameters of the nervous system of Nauphoeta cinerea cockroaches. In brain homogenates from cockroaches, CECA (5 and 50 µg/g) inhibited acetylcholinesterase (AChE) activity by 53 ±â€¯2% and 51 ±â€¯7%, respectively, while Tn (5 and 50 µg/g) inhibited AChE activity by 35 ±â€¯4% and 80 ±â€¯9%, respectively (p < 0.05; n = 6). Moreover, CECA at concentrations of 5, 25, and 50 µg/g decreased the locomotor activity of the cockroaches, diminishing the distance travelled and increasing the frequency and duration of immobile episodes similarly to Tn (0.3 µg/g) (p < 0.05, n = 40, respectively). CECA (5, 25 and 50 µg/g) induced an increase in the leg grooming behavior, but not in the movement of antennae, similarly to the effect of Tn (0.3 µg/g). In addition, both CECA (50 µg/200 µl) and Tn (0.3 µg/200 µl) induced a negative chronotropism in the insect heart (37 ±â€¯1 and 47 ±â€¯8 beats/min in 30 min, respectively) (n = 9, p > 0.05). Finally, CECA (50 µg/g), Tn (0.3 µg/g) and neostigmine (50 µg/g) caused significant neuromuscular failure, as indicated by the monitoring of the in vivo neuromuscular function of the cockroaches, during 100 min (n = 6, p < 0.05, respectively). In conclusion, sublethal doses of CECA provoked entomotoxicity. The Tn-like effects of CECA on Nauphoeta cinerea cockroaches encompass both the central and peripheral nervous systems in our insect model. The inhibitory activity of CECA on AChE boosts a cascade of signaling events involving octopaminergic/dopaminergic neurotransmission. Therefore, this study indicates that this insect model could potentially be used as a powerful, practical, and inexpensive tool to understand the impacts of eutrophication and for orientating decontamination processes.

Central and peripheral neurotoxicity induced by the Jack Bean Urease (JBU) in Nauphoeta cinerea cockroaches.

BACKGROUND: Ureases of Canavalia ensiformis are natural insecticides with a still elusive entomotoxic mode of action. We have investigated the mechanisms involved in the neurotoxicity induced by Jack Bean Urease (JBU) in Nauphoeta cinerea (Olivier). METHODS: To carry out this study we have employed biochemical and neurophysiological analysis of different cockroach organ systems. RESULTS AND CONCLUSIONS: The injection of the insects with JBU (0.75-6µg/g animal), although not lethal within 24h, caused significant inhibition of the brain acetylcholinesterase activity (60±5%, p<0.05, n=6). JBU (1.5µg/200µL), acetylcholine (0.3µg/200µL) or neostigmine (0.22µg/200µL), induced a positive cardiac chronotropism (∼25%) in the cockroaches (p<0.05, n=9). JBU (6µg/g) increased the insects' grooming activity (137±7%), similarly to octopamine (15µg/g) (p<0.05, n=30, respectively). Pretreating the insects with phentolamine (0.1µg/g) prevented the JBU- or octopamine-induced increase of grooming activity. JBU (6µg/g) caused 65±9% neuromuscular blockade in the cockroaches, an effect prevented by bicuculline (5µg/g) (p<0.05, n=6). JBU (6µg/g) decreased the frequency whilst increasing the amplitude of the spontaneous neural compound action potentials (1425±52.60min-1, controls 1.102±0.032mV, p<0.05, n=6, respectively). Altogether the results indicate that JBU induces behavioral alterations in Nauphoeta cinerea cockroaches probably by interfering with the cholinergic neurotransmission. The neuromuscular blocking activity of JBU suggests an interplay between acetylcholine and GABA signaling. GENERAL SIGNIFICANCE: The search for novel natural molecules with insecticide potential has become a necessity more than an alternative. Understanding the mode of action of candidate molecules is a crucial step towards the development of new bioinsecticides. The present study focused on the neurotoxicity of Canavalia ensiformis urease, a natural insecticide, in cockroaches and revealed interferences on the cholinergic, octopaminergic and GABA-ergic pathways as part of its entomotoxic mode of action.