Phenoloxidases take part in key insect physiological processes including pigmentation, cuticle tanning, wound healing, and defense responses. Some mosquito phenoloxidases generate active chemicals and eumelanin to kill and sequester malaria parasites. While reverse genetic analyses have revealed components of this enzyme system, important knowledge gaps still exist due to lack of biochemical analysis. In studies of the Manduca sexta prophenoloxidase (PPO) activation system, we accumulated wealthy knowledge on PPOs, PPO activating proteases, and their regulators. We cloned cDNAs of all nine Anopheles gambiae PPOs, expressed them as functional proteins in E. coli, crystallized three, and solved one PPO structure. Initial kinetic analysis revealed striking differences in substrate preference and catalytic efficiency. We validated the proteomic approach to semi-quantify PPOs and other proteins in the mosquito and detected PPOs in tissues using polyclonal antibodies. By injecting into female adults dsRNA encoding a conserved region in PPOs, we substantially reduced their mRNA and protein levels. Based on these advances, we propose to study the structures, functions, and expression of A. gambiae PPO1-9. Acquired knowledge and reagents will be useful for exploring how parasites evade or suppress the host immune mechanism in the future.

Public Health Relevance

Mosquitoes transmit pathogens that kill approximately half a million people each year. The transmission success is in part determined by the insect immune system. This project aims at elucidating structures, functions, and expression of prophenoloxidase-1 through 9 in a major vector species, which participate in melanotic encapsulation of parasites and nematodes. Multiple phenoloxidases with different expression profiles and biochemical properties generate reactive compounds to kill and entrap the pathogens and to perform other physiological functions. Acquired knowledge and mutated genes have potentials to be applied for disrupting human disease transmission in mosquitoes.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Exploratory/Developmental Grants (R21)
Project #
Application #
Study Section
Vector Biology Study Section (VB)
Program Officer
Costero, Adriana
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Oklahoma State University Stillwater
Other Basic Sciences
Schools of Earth Sciences/Natur
United States
Zip Code
He, Yan; Wang, Yang; Hu, Yingxia et al. (2018) Manduca sexta hemolymph protease-2 (HP2) activated by HP14 generates prophenoloxidase-activating protease-2 (PAP2) in wandering larvae and pupae. Insect Biochem Mol Biol 101:57-65
Cao, Xiaolong; Jiang, Haobo (2018) Building a platform for predicting functions of serine protease-related proteins in Drosophila melanogaster and other insects. Insect Biochem Mol Biol 103:53-69
Yang, Fan; Wang, Yang; Sumathipala, Niranji et al. (2018) Manduca sexta serpin-12 controls the prophenoloxidase activation system in larval hemolymph. Insect Biochem Mol Biol 99:27-36
Feng, Congjing; Zhao, Ya; Chen, Kangkang et al. (2018) Clip domain prophenoloxidase activating protease is required for Ostrinia furnacalis Guenée to defend against bacterial infection. Dev Comp Immunol 87:204-215
Li, Miao; Christen, Jayne M; Dittmer, Neal T et al. (2018) The Manduca sexta serpinome: Analysis of serpin genes and proteins in the tobacco hornworm. Insect Biochem Mol Biol 102:21-30
Cao, Xiaolong; Gulati, Mansi; Jiang, Haobo (2017) Serine protease-related proteins in the malaria mosquito, Anopheles gambiae. Insect Biochem Mol Biol 88:48-62
He, Xuesong; Cao, Xiaolong; He, Yan et al. (2017) Hemolymph proteins of Anopheles gambiae larvae infected by Escherichia coli. Dev Comp Immunol 74:110-124
He, Yan; Wang, Yang; Zhao, Picheng et al. (2017) Serpin-9 and -13 regulate hemolymph proteases during immune responses of Manduca sexta. Insect Biochem Mol Biol 90:71-81
Cao, Xiaolong; Jiang, Haobo (2017) An analysis of 67 RNA-seq datasets from various tissues at different stages of a model insect, Manduca sexta. BMC Genomics 18:796
Wang, Yang; Jiang, Haobo (2017) Prophenoloxidase activation and antimicrobial peptide expression induced by the recombinant microbe binding protein of Manduca sexta. Insect Biochem Mol Biol 83:35-43

Showing the most recent 10 out of 19 publications