Elucidating the Biochemical Overwintering Adaptations of Larval Cucujus clavipes puniceus and Cucujus clavipes clavipes, Non-Model Organisms, via High Throughput Proteomics

Beetles form the largest group of organisms on the planet and exhibit numerous interesting phenotypes. One in particular, Cucucus 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 -100å¡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, anti-freeze 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; including bacteria (9 species), yeast (1 species), animals (including nematodes (1 species), collembola (2 species), insects (5 species), fish (1 species), amphibians (1 species), reptiles (1 species), mammals (2 species)), and plants (moss (1 species), gymnosperms (1 species) and angiosperms (9 species)). There were 39 studies using 12 different cold treatments; 20 used proteomics and 18 used transcriptomics. Concerning our purpose of identifying specific temperature responsive proteins/gene products across species, we found 113 shared proteins/gene products groups, each of which was found in at least two species. Of these shared proteins/gene products groups, 58 proteins/gene products (including protein/gene product families) that were consistently regulated, meaning always either up- or down-regulated, across species. Another 23 proteins/gene products were inconsistently regulated, meaning that the proteins/gene products were up-regulated in some species and treatments while being down-regulated in other species and treatments. An additional 32 proteins/gene products that are part of larger family headings and are difficult to separate from related member proteins (such the ribosomal proteins, 30S, 50S, and others) were inconsistently regulated.

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. This represents the first high throughput MS/MS analysis of a non-model, cold-tolerant organism without a concurrent microarray analysis. 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 γ 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 (bromodomain adjacent to zinc finger domain 2A and anti-silencing protein 1), proteins involved with metabolism of amines (2-isopropylmalate synthase and dihydrofolate reductase), and immune system process (lysozyme C precursor), among others.

Previous studies showed that some individuals typically supercool to mean values of approximately -40å¡C, with some individuals supercooling to as low as -58oC, 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 -100oC. In addition, the body water of the DSC larvae vitrifies (turns to a glass) at glass transition temperatures of -58 to -70oC. 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 (DNA binding, chaperones, protein-protein binding proteins), organelle (which includes the cytoskeleton), metabolic process (including ATP synthesis proteins, general enzymes, and expression proteins), and biosynthetic process (expression proteins, ATP synthesis proteins, and kinases). Proteins up-regulated in winter relative to summer larvae include cytoskeletal elements (including myosin, actin, paramyosin, tropomyosin, and actinin), muscle proteins (myosin, actin, paramyosin, and tropomyosin), ATP synthesis proteins (ATP synthase), glycolysis proteins (glycogen phosphorylase), binding proteins (HSP70, nucleolin, DNA gyrase, and myosin) anti-microbial agents, and calcium binding proteins (alpha casein precursors).

Anti-freeze proteins (AFPs) are produced by a wide 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.