Gene expression patterns in model and non-model insect speciesthe use of cell lines and immune transcriptome analysis

Abstract

This thesis focuses on the investigation of different types of insects, with gene expression analysis as a common methodology. Thus, the study of model, non-model, and economical important species needs to be assessed differently based on the information available and the one that remains unknown. The first part includes Chapter 1 and 2, centered in the study of Tribolium castaneum and Ceratitis capitata cell lines, two model and economic important species, and the effect of several stimuli/treatments in gene expression patterns. The second part is focused on the study of tiger beetles transcriptome, first with the validation of reference gene for quantitative PCR in two different species and conditions in Chapter 3, and finally by performing an analysis studying the immune transcriptome of Calomera littoralis and characterising two novel antimicrobial peptides in the last chapter. Chapter 1 is focused on the rust red flour beetle, Tribolium castaneum, one of the most studied insect model species. We characterize the impedance profile with the xCELLigence Real-Time Cell Analyzer and study the effect of increased temperature in cell growth in the TcA T. castaneum cell line. Additionally, cells are exposed to heat shock, increased salinity, acidic pH and UV-A light with the aim of measuring the expression levels of Hsp27, Hsp68a and Hsp83 genes. Results show a high thermotolerance of TcA in terms of cell growth and viability, and a significant up-regulation of Hsp genes after one hour of exposure to 40 ºC and UV light. The second chapter focus on the Mediterranean fruit fly, Ceratitis capitata, known to be among the most dangerous pest species in the world. Some mechanisms for controlling its presence in agriculture include the insect growth regulators (IGRs), centered in part in juvenile hormone analogs, acting as a repressor of development and metamorphosis. In this work, the medfly CCE/CC128 cell line was used to assess lipopolysaccharide (LPS) and Juvenile Hormone III treatments to evaluate cell growth variation and changes in gene expression covering different functions. Results showed a slight retardation in cell growth for those cells treated with JH and an overexpression of Shade after the treatment, one of the Halloween genes member of the cytochrome p450 family, involved in development and the synthesis of 20-hydroxyecdysone (20E). Chapter 3 focus on reference genes, used as normalizers for expression studies. Considering the interest that tiger beetles have generated in the past years, it is crucial to dispose of a validated reference gene panel for expression studies. Nine candidate genes were tested in Cicindela campestris and Calomera littoralis across several conditions and their stabilities were assessed with geNorm, NormFinder, BestKeeper and ?CT method algorithms. No universal gene is found to be suitable for all situations, but NADH, B-t, Vatpase and ArgKin seem to present the most constant expression stability, indicating their suitability as reference genes in most conditions. This is the first report evaluating the stability of housekeeping genes in adephagan beetles. Last chapter focuses on the innate immune system in tiger beetles, which could provide useful information about humoral immunity of predatory insects. SSH method was performed in Calomera littoralis beetles to obtain those genes that were overexpressed after an infection with Escherichia coli. Two cecropin antimicrobial peptides were characterized and identified as CliCec-A and CliCec-B2. CliCec-B2 confirmed cecropins antimicrobial activity against some Gram (+) and Gram (-) bacteria, but not against yeast. Expression of both cecropins showed increases after an infection and highlighted their involvement in adult beetle mandibles, which could be related to their alimentary habits.