Mitochondrial metabolism and oxidative stress in tropical cockroach under fluctuating thermal regimes

International audienceThe cockroach Gromphadorinha coquereliana can survive under low temperature and extensive periods of cold stress. To assess energy management and insect adaptation in response to cold, we measured mitochondrial activity and oxidative stress in muscle and fat body tissues from G. coquereliana -under a fluctuating thermal regime (FTR, stressed at 4°C for 3-h on 3 consecutive days, with or without 24-h recovery after last exposure). Compared to our earlier work showing that a single exposure to cold significantly affects mitochondrial parameters, here, repeated exposure to cold triggered an acclimatory response, resulting in unchanged mitochondrial bioenergetics. Immediately after cold exposure, we observed an increase in the overall pool of ATP and a decrease in typical antioxidant enzyme activity. We also observed decreased activity of uncoupling protein 4 in muscle mitochondria. After 24-h of recovery, we observed an increase in antioxidant enzymes expression in muscle and fat body and a significant increase in the expression of UCP4 and HSP in the latter. This indicates that processes related to energy conversion and disturbance under cold stress may trigger different protective mechanisms in these tissues, and these mechanisms must be activated to restore insect homeostasis. In conclusion, the measured mitochondrial parameters and the enzymatic assays results suggest that mitochondria are not affected during FTR but oxidative stress markers are decreased, and a 24-h recovery period allows for the restoration of redox and energy homeostasis, especially in the fat body. This confirms the crucial role of the fat body in intermediary metabolism and energy management in insects and in the response to repeated thermal stress

Thermal stress causes DNA damage and mortality in a tropical insect

International audienceCold tolerance is considered an important factor determining the geographic distribution of insects. We have previously shown that despite its tropical origin, the cockroach is capable of surviving exposures to cold. However, the freezing tolerance of this species had not yet been examined. Low temperature is known to alter membrane integrity in insects, but whether chilling or freezing compromises DNA integrity remains a matter of speculation. In the present study, we subjected the adults to freezing to determine their supercooling point (SCP) and evaluated whether the cockroaches were capable of surviving partial and complete freezing. Next, we conducted single cell gel electrophoresis (SCGE) assays to determine whether heat, cold and freezing altered hemocyte DNA integrity. The SCP of this species was high and around -4.76°C, which is within the typical range of freezing-tolerant species. Most cockroaches survived to 1 day after partial ice formation (20% mortality), but died progressively in the next few days after cold stress (70% mortality after 4 days). One day after complete freezing, most insects died (70% mortality), and after 4 days, 90% of them had succumbed. The SCGE assays showed substantial levels of DNA damage in hemocytes. When cockroaches were heat-stressed, the level of DNA damage was similar to that observed in the freezing treatment, though all heat-stressed insects survived. The present study shows that can be considered as moderately freeze-tolerant, and that extreme low temperature stress can affect DNA integrity, suggesting that this cockroach may possess an efficient DNA repair system

Bioenergetic parameters of mitochondria isolated from the fat body tissue of <i>G</i>. <i>coquereliana</i>.

<p>(A-B) Respiration was measured in the presence of 10 mM succinate and in the absence (state 4 respiration) or presence of 400 μM ADP (state 3 respiration). C) RCR refers to respiratory control ratio. D) Changes in the UCP activity of the fat body of <i>G</i>. <i>coquereliana</i> cockroaches following cold exposure. UCP activity was measured in isolated mitochondria in the presence of succinate palmitic acid (PA, an activator of UCP) and GTP (an inhibitor of UCP). Data represent mean value ±SD. Statistical significance is indicated by either <i>p</i>≤0.05 (*) or <i>p</i>≤0.01 (**), or <i>p</i>≤0.001 (***). Student's <i>t</i>-test.</p

Changes in HSP 70 levels in the fat body and leg muscles of <i>G</i>. <i>coquereliana cockroaches</i>.

<p>Control—insect not exposed to cold stress. Cold—insect stressed for 3 h by cold (4°C). A) Immunological detection of HSP 70. Each lane had 50 μg of the soluble protein fraction loaded. The results are representative of at least 4 independent experiments. B) The average (±SD) HSP 70 protein expression level in tested tissues. The protein bands were digitally quantified using Biostep GelixOne G230 software. Significant differences from the respective control value are indicated by asterisks: <i>p</i>≤0.05 (*), <i>p</i>≤0.01 (**), or <i>p</i>≤0.001 (***). Student's <i>t</i>-test.</p

The physiological role of fat body and muscle tissues in response to cold stress in the tropical cockroach <i>Gromphadorhina coquereliana</i>

<div><p>Protective mechanisms against cold stress are well studied in terrestrial and polar insects; however, little is known about these mechanisms in tropical insects. In our study, we tested if a tropical cockroach <i>Gromphadorhina coquereliana</i>, possesses any protective mechanisms against cold stress. Based on the results of earlier studies, we examined how short-term (3 h) cold (4°C) influences biochemical parameters, mitochondrial respiration activity, and the level of HSPs and aquaporins expression in the fat body and leg muscles of <i>G</i>. <i>coquereliana</i>. Following cold exposure, we found that the level of carbohydrates, lipids and proteins did not change significantly. Nevertheless, we observed significant changes in mitochondrial respiration activity. The oxygen consumption of resting (state 4) and phosphorylating (state 3) mitochondria was altered following cold exposure. The increase in respiratory rate in state 4 respiration was observed in both tissues. In state 3, oxygen consumption by mitochondria in fat body was significantly lower compared to control insects, whereas there were no changes observed for mitochondria in muscle tissue. Moreover, there were cold-induced changes in UCP protein activity, but the changes in activity differed in fat body and in muscles. Additionally, we detected changes in the level of HSP70 and aquaporins expression. Insects treated with cold had significantly higher levels of HSP70 in fat body and muscles. On the other hand, there were lower levels of aquaporins in both tissues following exposure to cold. These results suggest that fat body play an important role in protecting tropical insects from cold stress.</p></div

The physiological role of fat body and muscle tissues in response to cold stress in the tropical cockroach <i>Gromphadorhina coquereliana</i>

<div><p>Protective mechanisms against cold stress are well studied in terrestrial and polar insects; however, little is known about these mechanisms in tropical insects. In our study, we tested if a tropical cockroach <i>Gromphadorhina coquereliana</i>, possesses any protective mechanisms against cold stress. Based on the results of earlier studies, we examined how short-term (3 h) cold (4°C) influences biochemical parameters, mitochondrial respiration activity, and the level of HSPs and aquaporins expression in the fat body and leg muscles of <i>G</i>. <i>coquereliana</i>. Following cold exposure, we found that the level of carbohydrates, lipids and proteins did not change significantly. Nevertheless, we observed significant changes in mitochondrial respiration activity. The oxygen consumption of resting (state 4) and phosphorylating (state 3) mitochondria was altered following cold exposure. The increase in respiratory rate in state 4 respiration was observed in both tissues. In state 3, oxygen consumption by mitochondria in fat body was significantly lower compared to control insects, whereas there were no changes observed for mitochondria in muscle tissue. Moreover, there were cold-induced changes in UCP protein activity, but the changes in activity differed in fat body and in muscles. Additionally, we detected changes in the level of HSP70 and aquaporins expression. Insects treated with cold had significantly higher levels of HSP70 in fat body and muscles. On the other hand, there were lower levels of aquaporins in both tissues following exposure to cold. These results suggest that fat body play an important role in protecting tropical insects from cold stress.</p></div

Changes in glycogen, lipid and protein content in the fat body and leg muscles of <i>G</i>. <i>coquereliana</i> cockroaches.

<p>Control—insect not exposed to cold stress. Cold—insect stressed for 3 h by cold (4°C). Data represent mean value ±SD. Significant differences from the respective control value are indicated by asterisks: <i>p</i>≤0.05 (*), <i>p</i>≤0.01 (**), or <i>p</i>≤0.001 (***). Student's <i>t</i>-test.</p

Bioenergetic parameters of mitochondria isolated from the fat body tissue of <i>G</i>. <i>coquereliana</i>.

<p>(A-B) Respiration was measured in the presence of 10 mM succinate and in the absence (state 4 respiration) or presence of 400 μM ADP (state 3 respiration). C) RCR refers to respiratory control ratio. D) Changes in the UCP activity of the fat body of <i>G</i>. <i>coquereliana</i> cockroaches following cold exposure. UCP activity was measured in isolated mitochondria in the presence of succinate palmitic acid (PA, an activator of UCP) and GTP (an inhibitor of UCP). Data represent mean value ±SD. Statistical significance is indicated by either <i>p</i>≤0.05 (*) or <i>p</i>≤0.01 (**), or <i>p</i>≤0.001 (***). Student's <i>t</i>-test.</p

Bioenergetic parameters of mitochondria isolated from the muscle tissue of <i>G</i>. <i>coquereliana</i>.

<p>(A-B) Respiration was measured in the presence of pyruvate (10 mM) plus malate (10 mM) and in the absence (state 4 respiration) or presence of 400 μM ADP (state 3 respiration). C) RCR refers to respiratory control ratio. D) Changes in the UCP activity in the fat body and muscles of <i>G</i>. <i>coquereliana</i> following cold exposure. UCP activity was measured in isolated mitochondria in the presence of pyruvate, palmitic acid (PA, an activator of UCP) and GTP (an inhibitor of UCP). Data represent mean value ±SD. Statistical significance is indicated by either <i>p</i>≤0.05 (*) or <i>p</i>≤0.01 (**), or <i>p</i>≤0.001 (***). Student's <i>t</i>-test.</p