Short-Term Estivation and Hibernation Induce Changes in the Blood and Circulating Hemocytes of the Apple Snail Pomacea canaliculata

Abstract

States of natural dormancy include estivation and hibernation. Ampullariids are exemplary because they undergo estivation when deprived of water or hibernation when exposed to very low temperatures. Regardless of the condition, ampullariids show increased endogenous antioxidant defenses, anticipating the expected respiratory burst during reoxygenation after reactivation, known as "Preparation for Oxidative Stress (POS)". In this work, we tested the POS hypothesis for changes in the blood and hemocytes of the bimodal breather Pomacea canaliculata (Ampullariidae) induced at experimental estivation and hibernation. We described respiratory (hemocyanin, proteins, lactate), antioxidant (GSH, uric acid, SOD, CAT, GST), and immunological (hemocyte levels, ROS production) parameters. We showed that, although the protein level remains unchanged in all experimental groups, hemocyanin increases in response to estivation. Furthermore, lactate remains unchanged in challenged snails, suggesting an aerobic metabolism during short-term challenges. Blood uric acid increases during estivation and arousal from estivation or hibernation, supporting the previously proposed antioxidant role. Regarding hemocytes, we showed that the total population increases with all challenges, and granulocytes increase during hibernation. We further showed that hibernation affects ROS production by hemocytes, possibly through mitochondrial inhibition. This study contributed to the knowledge of the adaptive strategies of ampullariids to tolerate adverse environmental conditions.

Keywords: Oxidative stress defenses; hemocyanin; hypometabolism; immune system.

Figure 1

Biochemical compounds and antioxidant enzymes in the blood of P. canaliculata during activity–dormancy–arousal cycles. (a) Total protein concentration; (b) hemocyanin; (c) lactate; (d) uric acid; (e) reduced glutathione; (f) superoxide dismutase; (g) catalase; and (h) glutathione S-transferase activity. Mean ± SEM. Asterisks (*) indicate significant differences with the control group (p < 0.05, Kruskal-Wallis, Dunn’s test).

Figure 2

Changes in the circulating hemocytes of challenged animals. (a) Total hemocyte levels; (b) relative composition of circulating hemocytes. Asterisks (*) indicate significant differences with the control group (p < 0.05, Kruskal-Wallis, Dunn’s test).

Figure 3

Production of ROS by circulating hemocytes. (a) Flow cytometry histograms showing distinct peaks of DCF fluorescence corresponding to different ROS levels for active, est, and arousal-est hemocytes; (b) changes in the mean fluorescence intensity (MFI) of DCF between groups; (c) histograms showing the fluorescence peaks of active, hib and arousal-hib hemocytes. Gray histograms represent autofluorescence controls. Asterisks (*) indicate significant differences with the control group (p < 0.05, Kruskal-Wallis, Dunn’s test).

Figure 4

Inhibition of ROS production by CCCP. (a) Inhibition ratio for total hemocytes; (b) inhibition ratio for separate hemocyte subpopulations. Abbreviations: agr, agranulocytes; gra, granulocytes; hya, hyalinocytes. Asterisks (*) indicate significant differences with the control group (p < 0.05, Kruskal-Wallis, Dunn’s test).