Changes in gut and Malpighian tubule transport during seasonal acclimatization and freezing in the gall flyEurosta solidaginis

Abstract

SUMMARY Since few studies have examined cold tolerance at the organ level in insects, our primary objective was to characterize the functional responses of the gut and Malpighian tubules (MT) to seasonal acclimatization, chilling and freezing in larvae of the goldenrod gall fly Eurosta solidaginisFitch (Diptera, Tephritidae). From September to December, hemolymph osmolality(455-926 mOsmol kg l-1) and freezing tolerance increased markedly in field-collected larvae. Chlorophenol Red was readily transported into the lumen of the foregut, the posterior portion of the midgut, the ureter, the proximal region of the anterior pair of MT, and entire posterior pair of MT. Ouabain and KCN inhibited transport of Chlorophenol Red in the gut and MT. Transport was readily detected at 0°C and the rate of transport was directly related to temperature. The rate of fluid transport by the MT decreased steadily from a monthly high in September (10.7±0.8 nl min-1 for the anterior pair; 12.7±1.0 nl min-1for the posterior pair) until secretion was no longer detectable in December;this decrease parallels entry into diapause for this species. Even in larvae that died following freezing for 40 days at -20°C, individual organ function was retained to a limited extent. Through the autumn, cholesterol concentrations in the hemolymph increased nearly fourfold. In contrast, the ratio of cholesterol to protein content (nmol mg l-1) in the MT membrane remained relatively constant (22∼24 nmol mg l-1protein) during this period. Freezing of larvae for 20 days at -20°C caused a significant decrease in cholesterol levels in the hemolymph and the MT membranes compared to unfrozen controls. These results suggest that cholesterol plays a role in seasonal cold hardening and freeze tolerance in insects.

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

“…Concomitantly, the ratio of cholesterol to protein in the membrane fraction of fat body decreased more than 6.5-fold (from 105 to 16 nmol mg −1 protein). These results contrast with data from a similar study of seasonal acclimatization in a freeze-tolerant gall fly larvae (E. solidaginis) in which hemolymph cholesterol increased four-fold during cold-hardening from September to November, and freezing at −20°C or −80°C decreased cholesterol levels in the hemolymph (Yi and Lee, 2005). Thus, contrary to our prediction that cholesterol levels would increase during cold acclimation, patterns of cholesterol regulation varied markedly among tissues and no simple pattern emerged (Fig.…”

“…The cholesterol was measured in hemolymph and both the supernatant and membrane fractions of FB, MG and MT as described by Yi and Lee (2005). Briefly, in separate 1.5 ml Eppendorf tubes, 1.0 ml of reagent (Cholesterol Assay Kit, Diagnostic Chemicals Limited, Oxford, CT, USA) and 10 µl of sample preparations were mixed.…”

Seasonal accumulation of acetylated triacylglycerols by a freeze-tolerant insect

“…Other neutral lipids likely present in E. solidaginis, such as cholesterol and diacylglycerol, were not detected because of the dilution required to resolve the storage lipid peaks (e.g. cholesterol concentration is three orders of magnitude lower than that of storage lipids in E. solidaginis) (Yi and Lee, 2005), and we spiked samples with cholesterol to confirm that the unknown lipid did not coelute with cholesterol (supplementary material Fig. S1B).…”

The influence of freeze duration on postfreeze recovery by caterpillars ofPyrrharctia isabella (Lepidoptera: Arctiidae): when is survival enough to qualify as recovery?

“…Substantial cellular disruption occurs in larvae of E. solidaginis at very low freeze temperature (À801C), with hemocytes and tracheae experiencing the greatest damage and gut tissues having the best resiliency (Yi and Lee, 2003). At less extreme freeze temperatures, Eurosta larvae show better retention of cell integrity and only modest reduction in epithelial transport function (Yi and Lee, 2005). Moreover, lethal freezing of Eleodes blanchardi beetles at high subzero temperatures does not compromise post-thaw aerobic respiration, indicating integrity of the tracheal system and cells, such that most of the damage is probably restricted to the nervous system (Zachariassen et al,'79).…”

“…This approach has a separate level of ecological relevance because some insects experience uninterrupted freezes lasting weeks or even months at a time. The cause for mortality in this situation may emanate from disturbances in cell metabolism and homeostasis (Layne and Kennedy, 2002 (Hanec and Beck, '60;Miller, '69;Somme, '74;Fields and McNeil, '88;Yi and Lee, 2005).…”