Abstract
BackgroundThe study of the mechanisms by which larvae of the Culex quinquefasciatus mosquito survive exposure to the entomopathogen Lysinibacillus sphaericus has benefited substantially from the generation of laboratory-selected colonies resistant to this bacterium. One such colony, RIAB59, was selected after regular long-term exposure of larvae to the L. sphaericus IAB59 strain. This strain is characterized by its ability to produce the well known Binary (Bin) toxin, and the recently characterized Cry48Aa/Cry49Aa toxin, able to kill Bin-resistant larvae. Resistance to Bin is associated with the depletion of its receptor, Cqm1 α-glucosidase, from the larvae midgut. This study aimed to identify novel molecules and pathways associated with survival of the RIAB59 larvae and the resistance phenotype.MethodsA transcriptomic approach and bioinformatic tools were used to compare the profiles derived from the midguts of larvae resistant and susceptible to L. sphaericus IAB59.ResultsThe RNA-seq profiles identified 1355 differentially expressed genes (DEGs), with 673 down- and 682 upregulated transcripts. One of the most downregulated DEGs was cqm1, which validates the approach. Other strongly downregulated mRNAs encode the enzyme pantetheinase, apolipoprotein D, lipases, heat-shock proteins and a number of lesser known and hypothetical polypeptides. Among the upregulated DEGs, the top most encodes a peroxisomal enzyme involved in lipid metabolism, while others encode enzymes associated with juvenile hormone synthesis, ion channels, DNA binding proteins and defense polypeptides. Further analyses confirmed a strong downregulation of several enzymes involved in lipid catabolism while the assignment of DEGs into metabolic pathways highlighted the upregulation of those related to DNA synthesis and maintenance, confirmed by their clustering into related protein networks. Several other pathways were also identified with mixed profiles of down- and upregulated transcripts. Quantitative RT-PCR confirmed the changes in levels seen for selected mRNAs.ConclusionsOur transcriptome-wide dataset revealed that the RIAB59 colony, found to be substantially more resistant to Bin than to the Cry48Aa/Cry49Aa toxin, developed a differential expression profile as well as metabolic features co-selected during the long-term adaptation to IAB59 and that are most likely linked to Bin resistance.
Highlights
The study of the mechanisms by which larvae of the Culex quinquefasciatus mosquito survive expo‐ sure to the entomopathogen Lysinibacillus sphaericus has benefited substantially from the generation of laboratoryselected colonies resistant to this bacterium
Fourth-instar larvae from the resistant RIAB59 colony, as well as from a susceptible Cx. quinquefasciatus colony from which it was derived (CqSLab), were harvested and had their midguts dissected and subjected to RNA extraction followed by whole transcriptome shotgun sequencing (RNA-seq)
differentially expressed genes (DEG) in pathways associated with lipid catabolism, DNA metabolism, apoptosis and immune response were regulated and are likely to be associated with the response to L. sphaericus and with the stable resistance phenotype
Summary
The study of the mechanisms by which larvae of the Culex quinquefasciatus mosquito survive expo‐ sure to the entomopathogen Lysinibacillus sphaericus has benefited substantially from the generation of laboratoryselected colonies resistant to this bacterium. One such colony, RIAB59, was selected after regular long-term exposure of larvae to the L. sphaericus IAB59 strain. Culex quinquefasciatus is one of the most abundant mosquito species associated with urban areas, those which are characterized by precarious sanitation This antropophilic species is a known vector of human pathogens and it has been the target of control programmes worldwide. The major challenge for L. sphaericus utilization is to overcome the resistance that can be selected against the Bin toxin, the active ingredient of the commercially available biolarvicides
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