Evaluation of candidate genes expression in RNA interfering system (RNAi) in Diaphorina citri (Kuwayama) (Hemiptera: Liviidae), vector of citrus huanglongbing

Author(s)

Diogo Manzano Galdeano

• Document type: Doctoral Thesis
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2016-00-00

Examining board members

Henrique Marques Souza; Katlin Brauer Massirer; Ricardo Harakava; Tiago Campos Pereira

• Advisor: Marcos Antonio Machado

Abstract

In early years, the Asian citrus psyllid, Diaphorina citri (Kuwayama) has become one of the most important vectors in citrus, due to the increasing damage caused to plants by bacteria transmission, Candidatus Liberibacter americanus and Candidatus Liberibacter asiaticus, causal agents of citrus huanglongbing (HLB). Healthy seedlings, eradication of infected plants and the chemical control of the vector have been used as HLB control strategies. However, the use constant and intensive of insecticides is not a good strategy, not only to the financial costs but also to the environmental problems. To reduce the high costs in development the new insecticides and making the control more efficient and specific, the gene knockdown by RNA interference (RNAi) is one of the promises of new technologies used in the control of pests and vectors. The aims of this study were to development double-stranded RNA (dsRNAs) delivery systems, searching candidate genes to be knockdown, evaluate the mortality and gene expression of D. citri. Thus, an in vitro feeding system composed of different sucrose concentrations was used to evaluate the nymph and adult D. citri survival for five days. After that, sixteen candidate genes were searching in transcriptome and genome D. citri. After confirmation of gene sequences, inhibitor of apoptosis, trehalase, cathepsin D, cathepsin L, chitin synthase, Na+/K+ ATPase subunit-A e ATP synthase subunit-D like mitochondrial genes were selected to be transcripts in vitro, and three different dsRNAs concentrations (200, 500 e 1000 ng.?L-1) of each gene were put in artificial diet to adult insects. Moreover, a dsRNAs (inhibitor of apoptosis, cathepsin D chitin synthase) delivery system by Murraya spp. leaves was development to evaluate the RNAi effects in nymphs. Mortality rates and gene expression of D. citri were evaluated after feeding dsRNAs via artificial diet and Murraya spp., and also, the dsRNAs stability in both delivery systems were evaluated. Fifteen and thirty percent sucrose in vitro feeding system showed the highest survival rates of adult D. citri populations from Citrus Center 'Sylvio Moreira' /IAC and University of California-Davis, respectively. After five days, the D. citri mortality rates in the dsRNAs delivery system of target genes on artificial diets ranged from 21-48%, 28-51% and 37-63% at concentrations of 200, 500 and 1000 ng.?L-1, respectively. Nymphs survival rates in the delivery system of inhibitor of apoptosis, cathepsin D and chitin synthase dsRNAs via plant leaves were 40, 71 and 50%, respectively. DsRNAs molecules were stable in the delivery systems evaluated. Furthermore, reduction in target gene expression levels of D. citri in the RNAi assays were confirmed by real time PCR. These datas suggest methods of providing dsRNAs, artificial diet and orange jasmine leaves, are useful tools for gene function studies and possible control of D. citri (AU)