Participation Of Hemocytes In Fat Body Degradation Via Cathepsin L Expression

Background and significantHolometabolous insects undergo several molting including larvae-larvae, larvae-pupa and pupa-adult molting. Insect molting and metamorphosis are mainly regulated by two hormones:ecdysone (20E) and juvenile hormone (JH). The interaction between20E and JH signaling pathways determines the different physiological activities and morphologies of the insects. During metamorphosis, larval tissues change dramatically. The larval tissues undergo degradation and adult tissues begin to form. Hemocytes had been proved to participate in this process while the mechanism was unknown.The purpose of this paper is to find the relationship between hemocytes and degrade of larval tissues. The function of cathepsin L which specially expressed in hemocytes was also studied.Research progressInsect hemocytes were considered to mainly take part in the innate immunity of insect. They can form capsule and nodule around the microorganism and foreign dodies. The bacteria can be enfulfed by hemocytes. While other funtions of insect hemocytes also be reported. Hemocytes are important for insect development and metamorphosis. Insect hemocytes can synthesis and secrete many important protein, one of which is Cathepsin. In the previous tudies, knock down of cathepsin L, which specially expressed in hemocytes, can block the decomposition of fatbodies and induce the larval dead and chimerical pupae formation.Scientific questionPrevious study indicated that Cathepsin L can be up-regulated by20E. Cathepsin L is considered to be related to fat body degradation by affecting the function of granulocytes. However, the role of hormonal regulation and mechanism of cathepsin L in granulocytes for fat body degradation is still not completely understood.Methods and resultsBy using molecular and cellular techniques, we have investigated the function of cathepsin L in hemocytes and tracked the hemocytes during metamorphosis.1. The numbers and morphological of hemocytes change in insect development. The numbers of granulocytes increased obviously accompanied by the volume increase during metamorphosis. Besides, more granules can be found. The granulocytes in this period were called macrogranulocytes. However, the number of plasmatocytes decreased.2. Western blot were used to show the expression profile of cathepsin L in hemocytes from5th feeding to6th-120h larvea. The expression of cathepsin L was up-regulated during metamorphosis. Immunocytochemistry showed that cathepsin L is located in the cytoplasm of granulocytes and plasmatocytes.3.20E was added into the hemocytes cultured in vitro to study the morphological change of hemocytes and the expression of cathepsin L. The results showed that20E can up-regulated the expression of cathepsin Lin addition,20E can promotes the conversion of the granulocytes to macrogranulocytes, which can attach on the expanded plasmatocytes.20E also can promote the spread of plasmatocytes. Western blot showed that Cathepsin L was up-regulated by20E.4. The number of granulocytes was decreased obviously by knocking down cathepsin L by RNAi and numerous granulocytes can be found in supernatant, most of which were dead. The conversion of granulocytes to macrogranulocytes was also blocked. The spread of plasmatocytes is suppressed, too.5. The hemocytes stained by CFDA SE (Carboxyfluorescein diacetate, succinimidyl ester) were found in the fatbodies of6th-96h larvae. The fatbodies were undergoing degradation and the fat cells detached from each other.6. The cells were apoptosis after overexpression of protease domain CIA of cathepsin L in HaEpi celllines.7. Hemocytes underwent apoptosis during metamorphosis. Most hemocytes were granulocytes with a ratio of about30%-40%. Some plasmatocytes also undergo apoptosis in sixth-96h larvae.8. Cathepsin L is detected not only in hemocytes, but also in hemolymph plasma and fat body of sixth-96h to sixth-120h larvae. Hence, hemocytes release cathepsin L into plasma by apoptosis or entrance into the fat body during late-stage metamorphic molting.Conclusion and significanceWe present further evidence in the current work to demonstrate the mechanism of hemocyte participation in fat body remodeling. Granulocytes are transformed into MGrs and cathepsin L is expressed during metamorphosis. The steroid hormone20E promotes cathepsin L expression and the transformation of granulocytes into MGrs. Cathepsin L is necessary for the transformation of granulocytes into MGrs. The CIA proteinase domain of cathepsin L is able to induce apoptosis. Hemocytes, especially granulocytes, undergo apoptosis during metamorphosis and release cathepsin L into hemolymph plasma and fat body for its degradation. Hence, hemocytes participate in fat body degradation by expressing cathepsin L under the regulation of20E.