Factors Contributing to Seasonal Increases in Inoculative Freezing Resistance in Overwintering Fire-Colored Beetle Larvae Dendroides Canadensis (Pyrochroidae)

Abstract

The insects and microarthropods that vary seasonally in susceptibility to cross-cuticular inoculation by external ice (inoculative freezing) represent a phylogenetically diverse group; however, few studies have explored possible mechanisms experimentally. This study documents seasonally variable inoculative freezing resistance in Dendroides canadensis beetle larvae and combines immunofluorescence, in vivo removal of epicuticular lipids and in vitro chamber studies to explore the roles of seasonal modification in the cuticle and in epidermal and hemolymph antifreeze proteins (AFPs). Seasonal cuticular modifications contribute to the inhibition of inoculative freezing since more cold-hardy larvae froze inoculatively when epicuticular waxes were removed with hexane and, in in vitro chamber experiments, cuticle patches (with the underlying epidermis removed) from winter larvae provided greater protection from inoculative freezing than did cuticle patches from summer larvae. The results indicate that seasonal modifications in epidermal and hemolymph AFPs contribute most strongly to the inhibition of inoculative freezing. Subcuticular epidermal AFPs were present in immunocytochemically labeled transverse sections of winter larvae but were absent in summer ones. Winter integument patches (cuticle with epidermis) were more resistant to inoculative freezing than were summer integument patches. Integument patches resisted inoculative freezing as well as live winter-collected larvae only when hemolymph AFP was added. The results also suggest that some integumentary ice nucleators are removed in cold-hardy larvae and that AFP promotes supercooling by inhibiting the activity of these nucleators.

Antifreeze Proteins of the Beetle Dendroides canadensis Enhance One Another's Activities

“…Overwintering larvae of the beetle Dendroides canadensis produce antifreeze proteins, which are an important component of their freeze avoiding capabilities ( − ). A family of 13 AFPs are known from D. canadensis ( , ).…”

Thermal biology of aquatic insects in alpine lakes: Insights from diving beetles

“…All species studied here survived 3 h of ice enclosure after 24‐h acclimation at 4°C. This suggest they are able to avoid or resist such damage that direct contact with ice poses, at least during relatively short exposure times, something which could be accounted for protective mechanisms such as a thick and physically resistant cuticle or cryoprotectant molecules (such as antifreeze proteins or epicuticular waxes) (Danks, 2007; Olsen et al., 1998). Such capacity to survive ice enclosure seems to be common (although not universal; see Hotaling et al., 2021) among other aquatic alpine insects.…”

Cold Tolerance of Transgenic Tobacco Carrying Gene Encoding Insect Antifreeze Protein

“…发生而且可通过抑制虫体体液和肠道的冰核剂以降 低过冷却点 [23][24][25] 。新疆准噶尔小胸鳖甲属于避冻昆 虫, 推测它们可能也是通过表达一系列抗冻蛋白基 因家族成员阻止外源冰晶通过体表而引发的初始结 冰, 同时以过冷却的方式度过北方寒冷的冬季。 所有的植物都易受冷冻和霜冻的危害。当温度 降低时, 由于冰核细菌的存在而引发植物表皮表面 冰晶的形成 [26] 。在体外实验中, 抗冻蛋白通过降低 异源冰核位点的效率而稳定液体的过冷却状态, 降 低结冰温度 [27] 。同时抗冻蛋白也能抑制冰晶的生长 和重结晶。最终仅需要比依数性复合物较低浓度的 抗冻蛋白就可有效阻止霜冻的伤害 [28][29] 和保护细胞 免受超过结冰温度时的伤害 [30] 。昆虫和植物属于不 [31] 键。Duman 等 [32] 研究表明利用增强剂甘油和柠檬酸 可提高昆虫抗冻蛋白对冰核剂的抑制功能。赤翅甲 的抗冻蛋白(DAFPs)需要一些增强剂例如某种蛋白 质或甘油、柠檬酸等小分子量可溶物以产生最适水 平的热值活性 [33][34]…”