Elucidating the biochemical overwintering adaptations of larval Cucujus clavipes puniceus and Cucujus clavipes clavipes, non-model organisms, via high throughput proteomics.

摘要

Beetles form the largest group of organisms on the planet and exhibit numerous interesting phenotypes. One in particular, Cucujus clavipes , is a freeze avoiding beetle that has two unique traits which are linked; the ability to vitrify (or form a glass-like transition state) and survive temperatures as low as -100°C. There are two sub-species, Cucujus clavipes puniceus (Western, C.c.p.) and Cucujus clavipes clavipes (Eastern, C.c.c.). Previous work has shown C.c.p. undergoes dehydration, diapause, produces anti-freeze proteins (AFPs), glycerol, antifreeze glycolipid (AFGL) to successfully overwinter. C.c.c. produces AFGL, glycerol, and AFPs to overwinter, though they generally produce less of each compound than C.c.p. To characterize biochemical adaptations, we applied high-throughput proteomics to ascertain proteins that may contribute to overwintering. To facilitate our study, especially data analysis, we compiled a compendium of low temperature responsive proteins.;We generated a database containing 2,030 low temperature responsive protein/gene product entries, of which 1,353 were up-regulated and 549 were down-regulated in response to various cold exposures across 34 different species.;Using a tandem MS based approach, we compared the proteomes of winter and summer collected C.c.p. to identify proteins that may play functional roles in successful overwintering. Using Gene Ontology (GO) analysis and manual interpretation, we identified 104 proteins in winter and 128 proteins in summer samples. We found evidence to indicate a cytoskeletal rearrangement between seasons, with Winter NDSC possessing unique actin and myosin isoforms while summer larvae up-regulated a actinin, tubulin, and tropomyosin. We also detected a fortification of the cuticle in winter via unique cuticle proteins, specifically larval/pupal rigid cuticle protein 66 precursor and larval cuticle protein A2B. Also, of particular interest in the winter larvae, was an up-regulation of proteins related to silencing of genes, proteins involved with metabolism of amines, and immune system process, among others.;Previous studies showed that some individuals typically supercool to mean values of approximately -40°C, with some individuals supercooling to as low as -58°C, but these non-deep supercooling (NDSC) individuals eventually freeze if temperatures drop below this. However, other larvae, especially if exposed to very cold temperatures, supercool even further. These deep supercooling (DSC) individuals do not freeze even if cooled to -100°C. In addition, the body water of the DSC larvae vitrifies (turns to a glass) at glass transition temperatures of -58 to -70°C. This study examines the proteomes of DSC and NDSC larvae to assess proteins that may contribute to or inhibit the DSC trait. Using high throughput proteomics, we identified 138 proteins and 513 Gene Ontology categories in the DSC group and 104 proteins and 573 GO categories in the NDSC group. GO categories up-regulated in DSC include alcohol metabolic process, cellular component morphogenesis, monosaccharide metabolic process, regulation of biological quality, extracellular region, structural molecule activity, and antioxidant activity. Proteins unique to DSC include alpha casein precursor, alpha-actinin, vimentin, tropomyosin, beta-lactoglobulin, immunoglobulins, tubulin, cuticle proteins and endothelins.;Winter temperatures are lower in Alaska than Indiana, and previous studies showed that Alaska C.c.p. has somewhat different overwintering adaptations compared to Indiana C.c.c. This study examines the proteome differences between winter and summer acclimatized C.c.c. larvae. We identified 84 proteins in winter C.c.c., and 50 proteins in summer C.c.c. Winter larvae enrich the following GO categories; binding, organelle, metabolic process, and biosynthetic process. Proteins up-regulated in winter relative to summer larvae include cytoskeletal elements, muscle proteins, ATP synthesis proteins, glycolysis proteins, binding proteins, anti-microbial agents, and calcium binding proteins.;Anti-freeze proteins (AFPs) are produced by a variety of species and confer cryoprotection during exposure to low temperature. Insect AFPs are among the most active and the highest activity was recorded in the beetle Cucujus clavipes, who produces the beetle type AFPs. This study applied tandem MS to ice purified protein samples from the two Cucujus clavipes subspecies. We also created the first database containing published AFPs and other ice binding proteins in NCBI, resulting in 1,111 entries; 84 beetle type AFPs from 7 different species and 1,027 non-beetle type AFPs from 240 different species. AFPs from homogenates of C. clavipes larvae were isolated by ice-binding procedures. The resulting AFPs were trypsinized and the peptides applied to a tandem MS analyzer. We identified both beetle and non-beetle type AFPs and non-AFP proteins using our database and a cross species approach. In Cucujus clavipes puniceus (Western subspecies) from Alaska, we identified 18 total AFPs, including a protein in the gut similar to the plant Daucus carota AFP. In Cucujus clavipes clavipes (Eastern subspecies) from Indiana, we identified 25 AFPs, including one plant AFP. There were four non-AFP proteins detected in both sub-species: actin, lysozyme C, myosin, and tropomyosin. Both subspecies possessed a plant type AFP, identified previously in Populus suaveolens. (Abstract shortened by UMI.).