Studies On The Mechanism Of Cold Hardiness In The Desert Beetle Microdera Punctipennis Dzungarica In Xinjiang

The desert beetle Microdera punctipennis dzungarica (Coleoptera: Tenebriondae) is a special species in Gurbantonggut Desert in Central Asia. M. punctipennis adults were nocturnal; they burrowed in the soil during the day but were active at night. They mate to lay eggs with high cold tolerance when remaining snow and ice are still there in the early spring. Production of antifreeze proteins (AFPs) is an antifreeze mechanism widely adopted by desert tenebrionid insects. Freeze-avoiding insects are often in a state of supercooling, which make a higher body vapor pressure than the ambient ice vapor pressure in winter conditions, so insect body often faces the problem of losing water. Therefore, the balance between the degree of supercooling and ability to resistance dehydration determines insect's cold tolerance. To investigate the possible strategy and physiological mechanism that M. punctipennis employed for cold survival, the seasonal changes of supercooling point (SCP), body water content, body fluid osmolality and the expression of antifreeze protein gene (Mpafp) were measured over 13 months. The relationships among these parameters were analyzed by correlation analysis. In order to further study the effect of low temperature and drought stress on the above cold-related parameters, cold and dry stress treatment were conducted on the adult insects, respectively, and the measurements were performed accordingly. The results showed that SCP of M. punctipennis adult changed from -8.0°C in summer to lower than -18.7°C in winter, demonstrate that M. punctipennis adult became deep supercooling state in winter by the depression of supercooling point, therefore this insect was freezing avoidance. During winter, M. punctipennis adult endured moderate water loose which was only about 10%, suggesting that desert beetle M. punctipennis could effectively prevent the body water from losing. The mechanism may mainly attribute to the highly chitinized body wall of the desert insect which has very low water permeability. Thus, water loss or ingress in insect body could be greatly inhibited during winter. Real-time quantitative PCR revealed that Mpafp mRNA was actively synthesized in winter. The synthesis of antifreeze protein began as early as at the beginning of autumn, and the peak value was reached in winter. The Mpafp mRNA level was increased by 13.1 fold from mid-summer to early winter, meanwhile the body fluid osmolality increased accordingly from 550 mOsm to 1486 mOsm. The correlation coefficient of Mpafp mRNA level and SCP was -0.8080 (R2=0.6528), showed that the higher the level of Mpafp mRNA, the lower supercooling points of insect, and Mpafp mRNA explained 65.28% of the variation in SCPs, demonstrating that antifreeze protein as the major factor caused body supercooling in the early winter. On the other hand, the significant correlation (r=-0.8256, R2=0.6816) between antifreeze protein mRNA level and the total water reflected the indirect influence of antifreeze protein on water content via decreasing SCP of M. punctipennis, which in turn stimulated water evaporation, thus reduced the water content.The results showed that the level of Mpafp mRNA is up-regulated after treated by low temperature(4℃) to some degrees. The maximum mRNA level is 8.23- fold of the control after treated at low temperature for 20 days. Meanwhile, changes of body fluid osmolality was in consistent with the changes of mRNA levels in that the maximum concentration of body fluid osmolality (756 mOsm) is 1.35 fold of the control, very significantly higher than that of control insect. Determination of water content showed that total water content decreased rapidly within 3 days after cold treatment, then maintained at about 50%. However, the content of free water within 3 days after cold treatment was increased from 8.87% of the control to 13.76%, and maintained at 15%. Correlation analysis showed certain correlation between the mRNA level of Mpafp and the free water (r=0.7616, R2=0.5800). Cold treatment at 4℃showed less effect on the supercooling point of M. punctipennis adult.Our results showed that dry stress could up-regulated the relative transcription level of Mpafp. The Mpafp mRNA levels reached the peak after treated by dry stress for 15 days, which was 3.53 fold of the control. However, changes of body fluid osmolality was not so fast as the level of Mpafp mRNA, and the maximum value of body fluid osmolality (646 mOsm) was 1.14 fold of the control, very significantly higher than control. There were significant correlations between M. punctipennis antifreeze protein mRNA level and the total water or absolute body water content (for total water, r=-0.8161, R2=0.6661, P=0.0476; for absolute body water, r=-0.8174, R2=0.6681, P=0.0470). Dry stress showed less effect on the supercooling point of M. punctipenni adult. In order to further study whether the production of antifreeze protein was also regulated during larvae development under laboratory conditions, 10 different developmental period of M. punctipennis were reared and the mRNA levels were measured compared to the egg. The results showed that antifreeze protein in Microdera punctipennis was developmentally regulated. The mRNA level of Mpafp was high in the final larvae and newly emerged adult, and higher than summer with the lowest level of Mpafp mRNA, but low in young larvae and pupae. The peak value was in adult, which was 2.1 fold of the egg. Meanwhile, the body fluid osmolality changed in consistent with the Mpafp mRNA level, though the increament was not so large as the Mpafp mRNA level. Correlation analysis showed no correlation between antifreeze protein mRNA level and the total water or absolute body water content, but significant correlationship (r=-0.8595, R2=0.7387) between antifreeze protein mRNA level and SCP. Pupae reared under laboratory conditions produced low level of antifreeze protein. M. punctipennis reared under constant temperatures would not suffer severe winter cold, so there may not need for abundant antifreeze protein. But certain low level of antifreeze protein was needed in order to protect the insects from cold damage when the environment temperature falls suddenly.In conclusion, the expression of Microdera punctipennis antifreeze protein gene (Mpafp) changed with the seasonal temperatures. It was high in late autumn and eraly winter, and low in summer. Meanwhile, osmotic concentration of body fluid changed in accordance with the level of Mpafp mRNA. The seasonal supercooling point appeared the contrast trend in that it was low in winter and high in summer. The Mpafp mRNA level and total body water content was significantly correlated. It is suggested that the antifreeze protein may have influence on water content via decreasing SCP of M. punctipennis, which in turn stimulated water evaporation, thus reduced the water content. Low temperature and drought stress increased expression of Mpafp at different levels, but cold stress could increase largely the expression level of Mpafp. There were no significant correlation between Mpafp mRNA level and SCP in the cold treated insect. Microdera punctipennis reared under laboratory conditions also produced antifreeze proteins, while the Mpafp mRNA level was higher in old larvae and adults than in young larvae and pupae. The osmotic concentration of body fluid showed same pattern as the level of Mpafp mRNA during the development. There was significant correlation between Mpafp mRNA level and SCP.It is concluded from the above conclusion that the desert insects Microdera punctipennis with freezing avoidance type cold hardiness mechanism: when the environment encountered at low temperature, body produce a certain amount of antifreeze protein, there by increasing the concentration of body fluid osmolality (and the increasing of body fluid osmolality is mainly caused by the accumulation of antifreeze proteins), while the insect lower supercooling points (meanwhile the decreaseing of the supercooling point further promoted the accumulation of antifreeze proteins), stimulate the water content of body to evaporate with the form of gradient, so insect lower body water content (at the same time the decreasing body water content could further promote the accumulation of antifreeze proteins), the interaction of four parameters above, resulting in insect without ice in a state of supercooling to have cold hardiness, avoiding damage from freezing.