Effects Of Periplocin T On Sensory-CNS-motor Circuit Of Drosophila Melanogaster Larvae

In the process of nerve signal transduction,V-ATPase provides kinetic energy for the synthesis,secretion,uptake and storage of neurotransmitters in synaptic vesicles,and also participates in the release of neurotransmitters in vesicles.Periplocoside T（PST）is a major component with higher insecticidal activity in Periploca sepium.In the previous study,it was found PST could inhibit the activity of V-ATPase in the brush border membrane vesicle（BBMV）of the midgut of Mythimna separata larvae in a concentration-dependent manner.In view of this,the toxicity and behavioral characteristics of four Drosophila melanogaster larvae-w¹¹¹⁸（Wild strain from Oregon-R）、para^ts1（Mutant strain of pyrethroid-resistant insecticides produced by point mutation of sodium channel I265N of Oregon-R）、DSC1^-/-(Mutant strain of w¹¹¹⁸knockout DSC1 channel)、para^ts1;DSC1^-/-(Mutant strain of para^ts1 knockout DSC1channel)with different genetic backgrounds were studied in this study with Drosophila larvae as the tested insect and PST as the tested reagent.The effects of PST on sensory-CNS-motor circuit potential of four Drosophila larvae were compared by intracellular microelectrode technique,and it was clear whether periplocin compounds affected the excitatory conduction of neural circuit by acting on synapse V-ATPase,thus clarifying its insecticidal mechanism.The main results are as follows:1.The toxicity test showed that the sensitivity of larvae of four Drosophila strains to PST from high to low was as follows:para^ts1、w¹¹¹⁸、para^ts1;DSC1^-/-、DSC1^-/-.DSC1^-/-strain with DSC1 channel knocked out has the lowest sensitivity to PST,which is significantly different from w¹¹¹⁸,and its LC₅₀ is 1.66 times that of w¹¹¹⁸;Compared with para^ts1;DSC1^-/-strain,the sensitivity of larvae to PST decreased after knocking out DSC1 channel in para^ts1strain.In the behavior test,there is no significant difference in response to light mechanosensory touch and body wall movement ability of four Drosophila strains under normal conditions,but there is significant difference in mouth hook movement ability,which indicates that the difference of nervous system gene background has different effects on behavior.After feeding 500 mg/L PST for 48 hours,the sensitivity to mechanical stimulation decreased,but there was no significant difference before and after feeding.Wall movement and mouth hook movement ability are significantly reduced and the larvae of w¹¹¹⁸ and para^ts1 strains with no DSC1channel knocked out decreased their body wall movement ability and mouth hook movement ability significantly more than para^ts1;DSC1^-/-and DSC1^-/-with DSC1channel knocked out.2.The measurement of sensory-CNS-motor circuit potential activity of larvae of four Drosophila strains showed that the EJP frequency of w¹¹¹⁸ and para^ts1 strains without knocking out DSC1 channel was significantly lower than that of para^ts1;DSC1^-/-and DSC1^-/-strains with knocking out DSC1 channel under normal conditions.Within the recorded 20 minutes,there was no significant difference in the attenuation trend of excitability among four Drosophila strains,but the attenuation trend of the two strains with the DSC1 channel knocked out was smaller than that of the two strains without knocking out,which indicated that the DSC1 channel played a negative role in regulating the activity of larval neural circuits to maintain the stability of the nervous system under normal conditions.3.4μM PST was directly used to treat the isolated samples of larval sensory-CNS-motor circuit.PST accelerated the decay of EJP frequency of neural circuit and inhibited the transmission of excitability from sensory-CNS-motor circuit of Drosophila larvae.There was no significant difference in the degree of inhibition among the four Drosophila strains.In vitro samples of larval sensory-CNS-motor circuit were treated with 2μM BA1,which also accelerated the attenuation of EJP frequency of neural circuit and inhibited the transmission of excitability of larval sensory-CNS-motor circuit of Drosophila.There was also no significant difference in the inhibition degree among the four Drosophila strains.After PST treatment,the decreasing trend of EJP frequency of sensory-CNS-motor circuit was basically the same as that of BA1 treatment,which indicates that PST and BA1 have similar effects,which may inhibit the transmission of sensory-CNS-motor circuit excitability of Drosophila larvae by inhibiting the activity of V-ATPase.4.After PST and BA1 treatment,although there was no significant difference in the inhibition amplitude of nervous system excitability transmission among four Drosophila strains,the EJP frequency of two strains with DSC1 channel knocked out decreased slightly.and this result is consistent with the results of toxicity test and behavior test.It is speculated that DSC1 channel may play a positive role in maintaining the stability of neural circuit and enhance excitatory conduction under adverse conditions,but PST or BA1 mainly inhibits excitatory conduction by acting on V-ATPase in presynaptic membrane in neural circuit.Therefore,due to the lack of this regulation mechanism,the sensitivity of Drosophila strains with DSC1 channel knocked out decreased,which shows a slight decrease in EJP frequency.The above results further confirmed that PST,like BA1,inhibited the excitability transmission of Drosophila larvae from the sensory-CNS-motor circuit by inhibiting the activity of V-ATPase and the difference of sensitivity of four strains of Drosophila melanogaster larvae to PST is related to the role of DSC1 channel in regulating the stability of nervous system,which may be due to the difference of genetic background of nervous system.