Discovery Of Molluscacide Novel Target And Creation Of Molluscicidal Compounds Based On The Target

Schistosomiasis japonica is a major infectious disease that seriously endangers human health.Over the past decades,China has made great advancements in schistosomiasis prevention.However,Oncomelania hupensis continues to exist in epidemic areas in the middle and lower reaches of the Yangtze River.The factors of the spread and prevalence of schistosomiasis still exist.Hence,the threat of recurrence of the schistosomiasis epidemic persists.Oncomelania hupensis is the only intermediate host of Schistosoma japonicum,and snail control can fundamentally block the spread of schistosomiasis,which is also a critical measure for the prevention and control of schistosomiasis.Currently,molluscicide is mainly used to kill snails,with niclosamide being the commonly used drug（molluscicide）globally,including China.Niclosamide is the only molluscicide recommended by the World Health Organization（WHO）.The disadvantage of niclosamide is high toxicity to aquatic animals.At the recommended dosage,niclosamide can also be fatal for aquatic organisms（e.g.,frogs,snakes,fish,and crabs）.Consequently,the wide application of niclosamide is limited in the background of the Yangtze River Protection Policy.Additionally,long-term repeated use of a single drug increases the risk of drug resistance,although no decrease has been observed in the sensitivity of Oncomelania hupensis to niclosamide.Therefore,more molluscicides with a novel structure and unique action mechanism is urgently required for the prevention and control of schistosomiasis.In this study,new molluscicide was searched from the secondary metabolites of actinomycetes.The novel molluscicide target was discovered and verified by investigating their action mechanism.Based on the novel target,2-[（4-chlorophenyl）methyl]-5-（trifluoromethyl）-1H-pyrrole-3-nitrile was used as the lead compound.Through structural modification,a new structure of molluscicidal activity compounds was created.The main results of the study are as follows:（1）A total of 203 actinomycete strains with different morphologies and colors were isolated from collected soil samples by spread plate method.The molluscicidal activity of metabolites extracted from these 203 actinomycete strains was investigated by immersion test.Among them,the metabolites of 28 actinomycete strains had a molluscicidal activity against Oncomelania hupensis,and the positivity rate was 13.79%.The fermentation supernatant of strains A024,A091,A154,A183,and A199 could kill all Oncomelania hupensis for 72 h.Herein,the A183 actinomycete exhibited the highest molluscicidal activity,and its fermentation supernatant could cause 100%death of Oncomelania after 72 h of treatment even when the supernatant was diluted 50 times.Based on the results of culture characteristics,physiological and biochemical reactions,and 16S r RNA gene sequencing,A183 actinomycete was named Streptomyces nigrogriseolus A183.（2）S.nigrogriseolus A183 metabolites were initially separated using a macroporous resin.Then,to obtain an isolated purified product,a silica gel column was used for the second separation,followed by a octadecylsilyl silica gel column for the third separation and a silica gel column for the fourth separation.At the isolated product concentration of 1.25 mg/L,the mortality rate of Oncomelania hupensis after 72 h immersion was 100%,indicating the high molluscicidal activity of the product.The ATP and ADP content of Oncomelania hupensis tissue was determined by high performance liquid chromatography（HPLC）.After treatment with 0.10 mg/L extract,the ATP content of the tissue significantly decreased from 198.15 to168.47μg/g.After treatment with 1 mg/L extract,the ATP content further reduced to 131.73μg/g.On the contrary,no significant difference in the ADP content was observed before and after the treatment.Effects of the fermentation supernatant on the activities of the mitochondrial respiratory chain complexesⅠ,Ⅱ,Ⅲ,andⅣand mitochondrial membrane potential were investigated.The extract could inhibit the activity of complex I and reduce mitochondrial membrane potential of Oncomelania hupensis.Thus,we preliminarily extrapolated that the mitochondrial respiratory chain complex I and mitochondrial membrane potential are possibly the molluscicide targets.The molluscicidal activities of the mitochondrial respiratory chain complexⅠinhibitor and three mitochondrial uncouplers were determined using the immersion method.LC₅₀ of the three uncouplers after immersion for 24 h was 0.065,0.135,and 0.110 mg/L;LC₅₀ of the three uncouplers after immersion for48 h was 0.064,0.124,and 0.082 mg/L;and LC₅₀ of the three uncouplers after immersion for72 h was 0.063,0.129,and 0.061 mg/L.It is confirmed that mitochondrial uncoupling can be a target for screening new molluscicide.（3）Considering mitochondrial respiratory chain uncoupling as the action mechanism,starting from 2-[（4-chlorophenyl）methyl]-5-（trifluoromethyl）-1H-pyrrole-3-nitrile,54compounds were designed and synthesized,and their molluscicidal activities were determined.The relationship between the structures of these compounds and their molluscicidal activities was preliminarily discussed.Benzene and pyrrole rings were directly connected,and molluscicidal activities were significantly improved.Br at the 4-position of the pyrrole ring was necessary for molluscicidal activity.The molluscicidal activity of the unsubstituted group at the 1-position of the pyrrole ring was the highest.Substitution on the benzene ring was necessary to maintain molluscicidal activity.Para and meta substitutions led to higher molluscicidal activities than the ortho substitution,and the ortho substitution led to higher molluscicidal activities than nonsubstitutions.Herein,C02 and F02 had high molluscicidal activities.LC₅₀ of C02 and F02 after immersion for 24 h was 0.225 and 0.286 mg/L,respectively.LC₅₀ after 48 h immersion was 0.111 and 0.149 mg/L,respectively.LC₅₀ after 72h treatment was 0.087 and 0.078 mg/L,respectively,which was equivalent to the efficacy of niclosamide,the only molluscicide recommended by WHO.A new method to screen molluscicides based on the target was explored.