Gene Family Novelty and Signatures of Cold Specialization in Beetles
Diverse insect lineages have evolved to cope with cold conditions, and strategies vary; insect taxa are frequently described as freeze avoidant or freeze tolerant, with further subdivision of the freeze avoidant species into chill susceptible or chill tolerant species (Bale 2002).Nebria (Catonebria ) appears to be chill tolerant (Slatyer & Schoville 2016), as it maintains neuromuscular activity below freezing, has modest mortality as it approaches its supercooling point, and yet recovers within minutes from its critical thermal minimum. However, this categorization fails to capture the fact that N. riversi is specialized to perform at cold temperatures, as it forages nocturnally at sub-zero temperatures in high elevation riparian habitats that are fed by ice and snowmelt. Nebria riversi must grow, develop and reproduce under constantly cold environmental conditions, and thus its fitness depends on permanently altering molecular pathways to function efficiently at cold temperature. A number of key physiological adaptations are expected (Clark & Worland 2008; Teets & Denlinger 2013), including 1) shifts to maintain cellular ion homeostasis (ion transport mechanisms), 2) regulation of cellular metabolism (mitochondrial performance and ATP synthesis), 3) efficient removal of toxic byproducts (detoxification of reactive oxygen species), 4) homeoviscous adaptation to maintain the liquid crystalline phase of cell membranes (an increase in unsaturated fatty acid content in membrane lipids), 5) regulation of protein denaturation (heat shock response, ubiquitination and proteolysis), and 6) neuromuscular adaptation. Despite an extensive literature and considerable knowledge of the physiological strategies insects employ to tolerate cold (see reviews in: Overgaard & MacMillan 2017; Teets & Denlinger 2013), comparative genomic and transcriptomic analyses of cold specialized insects remains limited (Cicconardi et al. 2020; Dennis et al. 2015; Kelley et al. 2014; Kim et al. 2017; Schovilleet al. 2020).
One fundamental strategy for ectotherms to maintain the physical function in cold environments is to increase metabolic rate (Addo‐Bediako et al. 2002; Williams et al. 2016). This involves both energy generation and management of subsequent oxidative stress caused by the accumulation of reactive oxygen species (ROS), which cause significant damage to nucleic acids, lipids, and proteins, as well as cellular structures like the plasma membrane (Storey & Storey 2010). The detoxification of ROS is thus an important requirement in coping with cold environmental conditions (Lalouette et al.2011). Several studies have shown that many cellular organelles are involved in dealing with this oxidative stress, including the endoplasmic reticulum, Golgi apparatus, and the lysosome (Butler & Bahr 2006; Jiang et al. 2011; Malhotra & Kaufman 2007; Pascua-Maestroet al. 2017). In N. riversi , we found enriched GO terms for expanding gene families associated with oxidative stress, including endoplasmic reticulum (GO:0005783), intracellular membrane-bounded organelle (GO:0043231), regulation of cellular metabolic process (GO:0031323), Golgi apparatus (GO:0005794), lysosomal membrane (GO:0005765), anterograde synaptic vesicle transport (GO:0048490), and carbohydrate phosphorylation (GO:0046835).
Cold acclimated insects also need to cope with damage to proteins, cells and tissues, and often compensate by modification of canonical temperature stress pathways (Lyzenga & Stone 2012). In N. riversi , we found numerous enriched GO terms that are linked to degradation of damaged proteins and DNA repair, including response to heat (i.e . heat shock proteins involved in protein folding; GO:0009408), protein polyubiquitination (GO:0000209), ubiquitin protein ligase binding (GO:0031625), proteasome-mediated ubiquitin-dependent protein catabolic process (GO:0043161), and protein K48-linked ubiquitination (GO:0070936). This finding is concordant with other studies that focus on other cold adapted insect species (Dennis et al. 2015; Kim et al. 2017; Schoville et al. 2020). The most severe damage to cells results from intracellular freezing, which results in unrepairable damage to cellular membranes in freeze-intolerant species (Bale 1987). One of the common strategies insects use to overcome freezing temperature is to increase the concentration of solutes in hemolymph and thereby lower the ice nucleation point (Storey & Storey 2012; Teets et al. 2011; Teetset al. 2019). In N. riversi , we found that genes associated with trehalose transport have rapidly expanded, and note that this sugar is widely implicated in cold tolerance among insects as it readily increases blood sugar content (Khani et al. 2007; Sinclair et al. 2003). Similarly, the enriched GO term phosphatidylinositol binding (GO:0035091) is involved in ice nucleation (Neven et al. 1989), which limits supercooling and prevents intracellular damage from ice formation. This pathway often includes proteins (i.e. protein ice nucleators, PINS, and antifreeze proteins, AFPs) that are known to cooperate with trehalose in freeze avoidance in beetles (Duman 2001).
Finally, for an insect species like N. riversi to be active in a constantly subzero environment, an important challenge is to maintain neuromusculatory activity for foraging, mating, and predator avoidance behaviors. Indeed, several studies have shown that species distributed in cooler areas have lower critical thermal minimum (CTmin) compared to closely related species living in warmer environments (Bishop et al. 2017; Slatyer et al.2016). In N. riversi , we found rapid expansion of troponin I genes compared to other coleopteran species, which is a cardiac and skeletal muscle protein that serves an inhibitory function to actomyosin ATPase and muscular contraction, but also prevents muscle damage during exercise (Farah et al. 1994; Fishilevich et al. 2019). Troponin I is typically single copy in most insects, and increasing the number of protein isoforms is expected to increase muscle fiber type specificity and/or developmental regulation of muscle development. Amino acid changes in troponin I (and other members of the troponin complex) have been linked to improved muscle performance under cold conditions (Shaffer & Gillis 2010).
Among the expected changes, two categories were not enriched in gene family expansion events. Cellular ion homeostasis, although it is also crucial for ensuring the function of neuronal signal transduction and muscle contraction (Lubawy et al. 2020; MacMillan et al.2014; Pivovarov et al. 2019), was not associated with gene family expansions in N. riversi . Similarly, changes to the structure of phospholipid bilayer, which is a common strategy to avoid damage to the cytoplasmic membrane and internal membranes (Enriquez & Colinet 2019; Holmstrup et al. 2002), was not found among the gene expansion events. There are three possible reasons we do not find evidence for these physiological changes. First, both mechanisms are often seen in overwintering insects or following laboratory acclimation treatments (Danks 2005; Enriquez & Colinet 2019; Parker et al. 2018), so may instead be associated with plastic gene regulatory responses to cold instead of turnover in gene families. Second, additional evidence of cold adaptation might be discovered in N. riversi with improved taxon sampling. As N. riversi sits on a long branch in our Coleoptera tree, the power to distinguish smaller gene expansion events is limited, because gene expansion tests must adjust for the rate of evolutionary change. Third, it is notable that insect species that lack signatures of these two physiological responses include the two Antarctic midges (Kelley et al. 2014; Kim et al. 2017), and like N. riversi , they live in constantly cold habitats. Another mechanism of cold specialization, which we did not evaluate here, likely arises from selection on protein-coding genes. Structural changes to protein coding genes could act to permanently alter cellular properties involving homeostasis and membrane viscosity in these species, and has been seen in several cold-specialized insects (Denniset al. 2015; Parker et al. 2018; Schoville et al.2020). Since N. riversi sits on a long branch in our Coleoptera tree, tests for positive selection, such as the branch-site test, will lack power due to saturation of synonymous sites and the occurrence of multi-nucleotide substitutions (Gharib & Robinson-Rechavi 2013; Venkatet al. 2018). Therefore, sampling the genomes of species of Carabidae or Adephaga, especially groups with contrasting cold and warm specialized species, are needed to increase our ability to detect signatures of temperature adaptation.