Localisation of antifreeze proteins in Rhagium mordax using immunofluorescence

A Picture on the front page: 400x magnification of a spiny bristle on a winter-collected Rhagium mordax larvae. The scene is captured using only reflected autofluorescence at 580 nm. Larva is 4if of the 22112011 series (See appendix 6). Foreword This report is the product of a master thesis project made by Johannes Lørup Johnsen at Roskilde University, fall of 2011. It is an integrated master thesis in the subjects Environmental-and Molecular Biology, worth 30 ECTS. The experimental nature of this project is the author's own choice, as a 30 ECTS integrated master thesis does not require experimental work. The experimental work herein is closely related to that of a previous study of type III antifreeze proteins in the European eelpout, Zoarces viviparus (Johnsen 2011). Were it not for the limitations of the ECTS system, these two studies would be included in the same larger project. The report is built around the model of a scientific paper, with the addition of expanded introduction and results sections. The goal of this report is to make a tight introduction to the field of antifreeze proteins, that any biologist should be able to comprehend. As a result, a detailed method is included as an appendix. It also serves as a starting point for new experiments and studies of antifreeze proteins in Rhagium mordax. Abbreviations used in this paper: AFP(s) = Antifreeze protein(s) TH = Thermal hysteresis Ih = Hexagonal ice crystals DAPI = Tissue samples at 461 nm light (4',6-diamidino-2-phenylindole or Alexa Fluor 350) FITC = Tissue samples at 518 nm light (Fluorescein isothiocyanate or Alexa Fluor 488) TRITC = Tissue samples at 580 nm light (Rhodamine) 4 Summary Larvae of the blackspotted pliers support beetle, Rhagium mordax, express antifreeze proteins in their haemolymph during temperate climate winter. It is believed that they also express antifreeze proteins in their cuticle as a means of preventing inoculative freezing. Larvae of Rhagium mordax were collected during winter (March) and summer (May) of 2011. Larvae were fixated, embedded in paraffin wax, sectioned on a microtome, incubated with custom made anti-AFP antibodies and visualised on a fluorescence microscope. The larvae of both winter and summer showed AFP activity in their cuticle, gut lumen and-epithelium. Due to the long synthesis process of AFPs, the larvae contain them all year round. The distribution of these AFPs change during summer, possibly relocating to vesicles in the cuticle and gut lumen/epithelium.