The Research Of Hemocyanin’s Oxygen-carrying Capacity In The Migratory Locust Locusta Migratoria

Animals need great amount oxygen to maintain aerobic metabolism. Apart from oxygen diffusion through oxygen concentration gradient, there are other two ways for oxygen transport in animal kingdom:respiratory protein and tracheal system. The respiratory protein can reversibly bind oxygen for transport to the deep tissue. Three types of respiratory proteins are known:hemocyanins(Hc), hemoglobins(Hb) and hemerythrins.In the insects, the presence of specialized oxygen-transport proteins in the insects has been considered generally unnescessary because of the tracheal system with extremely high efficiency for oxygen transporting in the last decades.However, the respiratory proteins have been found in more and more insect species, hemoglobins have been confirmed to have the significant capability of carrying oxygen in homometabolous insects. On the contrary, very few types of hemocyanins were found in hemimetabolous insects, and we lacked the evidence for these insect hemocyanins’function of carrying oxygen.In this study, we investigated the oxygen binding properties of hemolymphs of locusla migratoria, a typical kind of hemimetabolous insects, with an improved polarographic-fluorescence method which using enzymatic way to change the concentration of dissolved oxygen, measure the oxygen binding properties of locust egg’s and adult’s hemolymph. When the dissolved oxygen concentration was reduced, the fluorescence intensity of the egg hemolymph increased12%, but that of the adult male hemolymph did not change. It indicated that the egg’s hemolymph have the capacity of oxygen-binding, but not of the male adult’s hemolymph. This result was similar with the expressing pattern of hemocyanin through the development procedure from embryo to adult.Then, in order to verify the oxygen-canying function of hemocyanin, we extracted the RNA of locust eggs, and get cDNA by reverse transcription, designed primers with restriction sites by the known cDNA sequences of locust hemocyanin subunit1(HC1) and2(HC2). The target fragments of HC1and HC2which were amplified by PCR, connected with the expression vector pET-28(a) respectively. After this, transformed the vector pET-28(a)-HCl and pET-28(a)-HC2into E.coli BL21, we obtained the hemocyanin subunitl and subunit2by prokaryotic expression. After the activity detection of oxygen binding, we found that the subunt2have the capacity which the fluorescence intensity increased about19%under reduced oxygen condition, but subunt1no response. This showed that the hemocyanin subunit2involved in the oxygen-carrying process.In order to further confirm the hemocyanin with oxygen-carrying function is involved in the process of locust embryo’s development, we used the RNA interference method with an improved method of microinjection (survival90%after injected), to surpress the expression of hemocyanin subunit2outstanding in two days after inject dsRNA. Embryonic development showed that the locust egg’s development slowed down under hypoxia. By comparision with the control group, the treatment group slowed down one development period, some eggs of treatment group stayed in the early blastokinesis period. This indicated that the hemocyanin may participate the embryonic development of locust.In this study, we discovered that the hemocyanin with oxygen-carrying capacity in the terrestrial insect locusta migratoria for the first time, and provided the preliminary proof of the hemocyanin participated in the process of locust embryo’s development, and lay the foundation to further research about the respiratory protein and trachea formation, embryonic development and insect hypoxia adaptation.