Principal Investigator
Researcher
Email:erjunling@sippe.ac.cn
Personal Web:

Key Laboratory of Insect Developmental and Evolutionary Biology, CAS

Education: 

1999-2003, Shinshu University, Japan, Ph.D. 

1994-1997, Anhui Agricultural University, Sericulture, M.S. 

1990-1994, Anhui Agricultural University, Sericulture, B.S. 

Working experiences: 

2008-present, Institute of Plant Physiology and Ecology, Professor 

2007- 2008, University of Missouri-Columbia, USA, Post-doc 

2004-2007, University of Missouri-Kansas City, USA, Post-doc 

2003-2004, Shinshu University, Japan, Post-doc 

1997- 1999, Anhui Agricultural University, Researcher 

Insect PPO has been studying for over 100 years. In the history of insect PPO study, it can be divided into three stages: to understand the melanization phenomenon, to purify melanization-induced protein PPO, and to understand the progress of PPO activation. Insect PPO is produced by hemocytes and released into hemolymph as one of immunity proteins to fight against pathogens. However, none questions whether there are PPO proteins in other tissues and whether the insect PPO has some unknown functions. For the last five years, our group focused on finding the new functions of insect PPO in different tissues or places. We think we answer some enigmatic questions in the field of entomology. The details on those works are described as below. 

Enigma to protect insects BEFORE ecdysis. Not like mammalians, the insects experience several times of ecdysis during the whole life stage. To prepare for each ecdysis, the insects stop feeding and moving around. Due to it, the insects are easily attacked by pathogens at these stages. So how can insects protect themselves before ecdysis? This question is an enigma in the field of entomology. We found that there is PPO in the molting fluids that are secreted and accumulated among the old and newly-produced cuticles during each time of ecdysis. In the molting fluids, there are many immunity proteins including PPO. Molting fluids have direct antibacterial activities. When the larvae under ecdysis were infected by fungi, PPO was activated to induce the molting fluids into melanization, by which to stop fungal further infection. In addition, we detected that many proteins in the molting fluids can regulate ecdysis. Therefore, we demonstrate that there is PPO in the molting fluids and melanization induced in the molting fluids protects pathogenic infection before ecdysis for the first time (J Biol Chem, 2014). 

Enigma to protect insects AFTER ecdysis. After ecdysis, the newly-molted larvae are very weak since the cuticle is thin. Theoretically, the newly-molted larvae should be easily infected by pathogens (especially fungi) since the skins are still under developed. However, very few newly-molted larvae are infected in the field. How insects can protect themselves after ecdysis is another enigma not answered. After carefully studying, we found that proteins in the molting fluids stayed on the new cuticle when the old cuticle was shed. The molting fluids became dry very quickly. Fungal spores were found to interact with PPO. During germination, the fungal spores secret protease BPS8 that is hijacked by insect PPO for activation, by which to induce melanization around the fungi spores to inhibit spore germination. We, therefore, demonstrate that PPO in the retained molting fluids continues to protect the newly-molted larvae from pathogenic infection after ecdysis. This work tells us the reason why the pests infected by fungi explode after the rainfall season on the field. We now understand that the rain water washes the remained molting fluids including PPO from the skin, and then the insects are easily infected by fungi. Unexpectedly, the remained molting fluids act exactly like bullet-proof coat to protect the insects against pathogens infection after ecdysis (Front Immunol, 2017). 

Detoxification of plant phenolic metabolites by foregut PPO. Besides the hindgut, we also found that there is PPO in the insect foregut. Normally plant phenolic metabolites are healthy to human being. However, most phenolic materials are toxic to insects. Insects living on plant leaves can grow and develop normally, which means that the insects have novel functions to detoxify the phenolic metabolites in foods. However, so far, we know nothing about the mechanism. We found that there is PPO in the foregut. The activated phenoloxidase (PO) can oxidize phenolics, which reminds us that the foregut PPO may have close relationship with the phenolic detoxification. Eventually our work shows that the foregut PPO can metabolize phenolic materials into non-toxic intermediates that are either transferred into hemocoel or the hindgut for PPO to melanize feces. When PPO was deleted, Drosophila larvae became dead quickly if feeding on food containing L-DOPA. With this work, we answer another confusing question on how the insects to detoxify the phenolic metabolites produced in plant foods (Sci Rep, 2015).  

Novel mechanism to activate insect PPO. Insect active phenoloxidase (PO) has an active pocket that is occupied by a place holder. For PPO activation, the place holder must be removed from the active site pocket. Traditionally, it was thought that insect PPO was cleaved by serine protease one time for activation. Although a complicated activation pathway has been studied, there is no proof to show the direct cleavage on PPO that can remove the place holder. Our previous works disclose the influences of PPO protein structure on activation by expressing recombinant PPO in vitro. We realize that this system can also be used to understand PPO activation. By adding a His-tag to either N-terminus or C-terminus of the recombinant PPO and using the commercial serine protease for activation, we show that insect PPO is activated after being cleaved at N-terminus and C-terminus at the same time in the beginning, and followed by another cleavage at N-terminus of the large fragment to remove the place holder. Therefore, eventually insect PPO must be cleaved at least three sites before removing the place holder from the active site pocket if we neglect all other regulation factors (Biochimie, 2014). 

PPO is an important immunity protein to induce melanization around the invading bacteria and parasites. This protein is also very important to wound healing, by which to cause melanization and coagulation of hemolymph (ex vivo) to plug the wounds. Using the sterilized needle, wounds were made on the skin and midgut respectively. Starting from the wound-induced melanization, we carefully studied the mechanism of wound regeneration in the midgut (Sci Rep, 2016) and skin (Cell Cycle, 2017). Further works were performed to disclose the development and differentiation of insect epidermis from larva to adult stages, which is also helpful for understanding the immune defense in the epidermis.  

The above works disclose that insect PPO has many novel functions, which are neglected for almost one century. Now we understand that PPO acts exactly like a bullet-proof coat to protect insects from toxic phenolics in the foregut (Sci Rep, 2015), and to remove bacteria in feces to avoid spreading out in the living environment (J Biol Chem, 2012), and to stay in the molting fluids to defend against bacteria and fungi infection before (J Biol Chem, 2014) and after (Front Immunol, 2017) ecdysis. Due to creative works on the insect PPO, we were invited to write a review on the functions of insect PPO recently (Front Physiol, 2014), and it has been cited a lot of time since publication.