Study On The Mechanism Of Spinosad To Yellow Fever Mosquito,Aedes Aegypti

The yellow fever mosquito,Aedes aegypti transmits a number of vector-borne diseases such as Zika and Dengue.Due to the lack of a vaccine and effective antiviral drugs,curbing the spread of vector borne diseases relies on the integrated vector management.Application of ecologically friendly biological insecticides,such as spinosad could extremely improve the efficiency of mosquito control and address the issue of resistance against conventional insecticides.Spinosad is a novel,naturally-occurring insecticide found in the metabolites of the soil actinomycete Saccharopolyspora spinosa.Spinosad is strikingly effective against mosquitos,especially strains that have developed resistance to conventional insecticides.In this study,we use Ae.aegypti as a model to investigate the mode of action,metabolic enzymes,and immunoassay of spinosad.1)Target site of spinosadWe identified 10 candidate n ACh R subunits in Ae.aegypti as homologues to Anopheles gambiae and Drosophila melanogaster using a reciprocal best hit BLAST.As Aaeα6 was shown to be conserved across insect species,we focused our effort on Aaeα6 for identifying spinoad target in the current study.A 32-bp nucleotides deletion at the 5’ end of Aaeα6 gene(N-terminal Loop D in Ae.aegypti n ACh R α6 protein)was generated using CRISPR/Cas9,resulting in two pre-mature stop codons leading to a complete loss of function in the target receptor,n ACh R α6.Aaeα6-KO strain showed 59.5-fold resistance to spinosad.The degree of dominance was determined to be-0.91,indicating a recessive mode of inheritance for spinosad resistance in Aaeα6-KO strain.2)Putative metabolic enzymes of spinosadThe in vivo and in vitro tests demonstrated that Ae.aegypti is able to degrade spinosad..To investigate which protein metabolizes spinosad,a spinosad-sepharose was prepared to screen spinosad binding proteins.2-D electrophoresis and LC-MS were used to identify 21 proteins interacted with spinosad.Gene ontology enrichment analysis showed that the identified proteins were involved in translation,protein metabolic process,and cellular macromolecule metabolic process,etc.The in vitro metabolism validation of a serine protease(AAEL002624)using its recombinant protein indicated it could interact with spinosad and metabolize spinosad.3)Transcriptome changes of Ae.aegypti ovary and midgut after exposure to spinosadHigh dose of spinosad in blood meal dramatically decreased the mosquito survival rate.Although the lower dose of spinosad in blood meal did not kill most females,the exposure caused lower egg production and decreased egg hatching rate.Transcriptome analysis was used to dig into the underlying mechanism of the affects induced by spinosad.Spinosad blood meal induced the up regulation of ABC transporter pathway,indicated the role of ABC transporters involving the transportation and degradation of spinosad.The results showed that the down regulation of metabolism pathway of essential amino acids,such as isoleucine in the midgut could cause the reduction of amino acid nutrient during vitellogenesis.Up regulation of glutaminase could use ammonia to produce glutamate for the further synthesis of essential amino acid,which recovery the waste ammonia and rescue the decrease of essential amino acid caused by spinosad-blood meal.Spinosad also results in the down regulation of enzymes involved in the insect hormone biosynthesis,such as juvenile hormone and ecdysone in the ovary,further led to block of the initiation of vitellogenin synthesis and egg development.4)Monoclonal antibody of spinosadA novel succinic derivative of spinosyn A as a hapten was synthesized,and an indirect competitive enzyme-linked immunosorbent assay(ic ELISA)was developed using a new monoclonal antibody(designed as m Ab3E6)specifically against spinosyn A.The m Ab3E6 showed less than 0.1% cross reactions with other macrolides.A m Ab3E6-based immunoaffinity column was developed for enrichment extraction of spinosyn A and reduction of matrix interference.The limit of detection of the ic ELISA is 0.76 ng/ml.The ic ELISA had average fortified recoveries ranging from 78.1% to 103.2% and the validation results correlated well with high-performance liquid chromatography.The developed immunoaffinity chromatography-assisted ic ELISA is potentially a sensitive and convenient tool for determining spinosad residues and others.