Molecular studies in the genus Bombuspopulation analyses and defence mechanisms in conditions of thermal stress and infection by the parasite Crithidia bombi

Laburpena

Bumblebees are hymenoptera species belonging to the genus Bombus and characterised for being eusocial, particularly large and hairy. Most are distributed in temperate, alpine and arctic zones of America, Europe and Asia, and like other bees, they have suffered a global decline since the middle of the XX century. Given their ecological and economic importance as commercial pollinators, different molecular studies have been carried out in this thesis with the general aim of contributing to their conservation. In the first part of this thesis, the diversity of the cryptic species that conform the B. lucorum-complex in the Iberian Peninsula, B. lucorum and B. magnus, has been characterised. Its distribution in the peninsula is not clear because of the difficulty in distinguishing them morphologically and the lack of molecular studies. For this reason, in the first chapter the species of the B. lucorum complex were identified in a sampling throughout the Iberian Peninsula, using a fragment of the cytochrome oxidase subunit 1 (cox1) gene, used as a DNA barcode to differentiate species. Results confirmed that B. lucorum is a relatively abundant species limited to the northern half of the Iberian Peninsula. Additionally, two individuals of B. magnus were found indicating that this species is infrequent and enlarging its distribution to the Northern Plateau. In the second chapter of the thesis, Iberian individuals of B. lucorum were characterised genetically and morphologically together with individuals from France and Belgium. Results from mitochondrial cox1 showed a differentiation of bumblebees in the center of the peninsula that could be explained with the existence of a glacial refugium during the Pleistocene. However, no differentiation was detected with nuclear STRs (microsatellite) and the geometric morphometrics analysis of the wings, pointing to the existence of current genetic flow between the populations north of the Pyrenees and those in the center of the Iberian Peninsula. In the second part of the thesis the immune activity of B. terrestris was studied in stress conditions. In the third chapter, the expression of five immune and two heat shock genes was measured in bumblebees under heat (38 °C) and cold (9 °C) conditions. The heat treatment increased the expression of heat shock genes but did not alter significantly the immune gene expression. On the other hand, the cold treatment only caused a significant upregulation of the gene BGRP1 related to the cellular immune response. In addition, when bumblebees were also submitted to starvation, expression of all genes increased, possibly mirroring a synergy effect between the responses to both starvation and temperature stressors. In the fourth chapter of the thesis, I analysed whether the microbiota of B. terrestris protects against the gut parasite Crithidia bombi through the stimulation of three antimicrobial peptide (AMP) genes and Mucin-5AC, a gene with a putative role in gut epithelium protection. Although the supplemented microbiota increased the resistance against the parasite in comparison with the bumblebees that had reduced microbiota, there was not a significant change in AMP nor Mucin-5AC genes. However, a significant interaction was detected among the presence of Crithidia and the microbiota treatment, where AMP gene expression was higher when bumblebees were infected and received supplemented microbiota. Therefore, the microbiota could interact with the parasite stimulating the immune response in the host, although this mechanism alone does not explain the protective effect observed.