Seasonal accumulation of acetylated triacylglycerols by a freeze-tolerant insect

Abstract

Most animals store energy as long-chain triacylglycerols (lcTAGs). Trace amounts of acetylated triacylglycerols (acTAGs) have been reported in animals, but are not accumulated, likely because they have lower energy density than lcTAGs. Here we report that acTAGs comprise 36% of the neutral lipid pool of overwintering prepupae of the goldenrod gall fly, Eurosta solidaginis, while only 17% of the neutral lipid pool is made up of typical lcTAGs. These high concentrations of acTAGs, present only during winter, appear to be synthesized by E. solidaginis and are not found in other freezetolerant insects, nor in the plant host. The mixture of acTAGs found in E. solidaginis has a significantly lower melting point than equivalent lcTAGs, and thus remains liquid at temperatures at which E. solidaginis is frozen in the field, and depresses the melting point of aqueous solutions in a manner unusual for neutral lipids. We note that accumulation of acTAGs coincides with preparation for overwintering and the seasonal acquisition of freeze tolerance. This is the first observation of accumulation of acTAGs by an animal, and the first evidence of dynamic interconversion between acTAGs and lcTAGs during development and in response to stress. INTRODUCTIONLipids fulfill diverse biological functions including cell membrane structure, signalling and energy storage (Canavoso et al., 2001;Reue, 2011;Yen et al., 2008). Long-chain triacylglycerols (lcTAGs) are generally used as fuel stores by animals because of their high energy density, and are consequently the most abundant lipid class in animals (Williams et al., 2011;Yen et al., 2008). No other neutral lipid has been reported as a storage molecule in terrestrial animals.Instead of having three long-chain fatty esters (as in lcTAGs), acetylated triacylglycerols (acTAGs) have an acetyl group esterified at the 3-position of the glycerol backbone. This distinct structure results in the reduced viscosity and the significantly lower energy density of acTAGs compared with lcTAGs (Durrett et al., 2010). Trace amounts of acTAGs have been described in a broad range of organisms, including the antlers of Japanese deer (Cervus nippon) (Yang et al., 2004), the udders of domestic cows (Bos primigenius) (Limb et al., 1999) and the seeds of the burning bush plant (Euonymus alatus) (Durrett et al., 2010). acTAGs constitute a very small proportion of the total lipid pool in the two mammal species investigated [<0.1% by mass Limb et al., 1999;Yang et al., 2004)], but predominate (92% by moles) in E. alatus seed oils (Durrett et al., 2010). Euonymus alatus synthesises acTAGs via an unusual diacylglycerol acyltransferase (DGAT) by esterifying an acetyl group, rather than a third long-chain fatty acid, to diacylglycerol (DAG) (Durrett et al., 2010). Although trace acTAGs are present in a few animals, we are not aware of any clear hypotheses for a physiological role of acTAGs in biological systems. Here we report that the goldenrod gall fly, Eurosta solidaginis (Fitch 1855), accumulates significan...

Cold-hardening during long-term acclimation in a freeze-tolerant woolly bear caterpillar, Pyrrharctia isabella

“…Sample means were analyzed by ANOVA using SigmaPlot 12.5 (Systat Software, Richmond, CA, USA), followed by either Bonferroni-Dunn or Tukey's post hoc tests for multiple comparisons (Statview 5.1, SAS Institute, Cary, NC, USA). To evaluate cellular viability in the live/dead vital dye assays, we used R for one-way ANCOVA analysis Marshall et al, 2014). A value of P<0.05 between groups was considered a statistically significant difference.…”

“…In addition to thermal hysteresis proteins and glycoproteins, recent work has identified novel classes of glycolipid antifreezes and acetylated triacylglycerols that may play important roles in seasonal cold tolerance and winter survival (Walters et al, 2011;Marshall et al, 2014). Seasonal remodeling of membranes often includes a decrease in saturated lipids that helps maintain membrane fluidity and allows continued function at low temperatures (Crockett, 1998;Yeagle, 1991).…”

Diapause induces remodeling of the fatty acid composition of membrane and storage lipids in overwintering larvae of Ostrinia nubilalis, Hubn. (Lepidoptera: Crambidae)

“…Recent work has established that the neutral lipid of overwintering freeze tolerant larvae of Eurosta solidaginis is dominated by acetylated triacylglycerols rather than as previously thought, the energydense long-chain triacylglycerols (Marshall et al, 2014), generally used by animals. We found no evidence of acetylated triacylglycerols in O. nubilalis which would have been visible on the HPTLC plates if present.…”

The many roles of fats in overwintering insects

“…3B) form the majority of the neutral lipid pool (by mass) of overwintering E. solidaginis (Marshall et al, 2014). These acTAGs differ from the more common long-chain triacylglycerols (lcTAGs) in that they have an acetyl group rather than a fatty moiety at the third position on the glycerol backbone.…”

“…there is stoichiometric balance between acTAGs, lcTAGs and nonesterified fatty acids, and acTAGs appear to be rapidly converted back into lcTAGs in a stoichiometric ratio in the spring (Marshall et al, 2014). acTAGs contain one fewer fatty moiety than lcTAGs and, therefore, have lower energy density (Durrett et al, 2010).…”