Acta Tropica 168 (2017) 21–28 Contents lists available at ScienceDirect Acta Tropica journal homepage: www.elsevier.com/locate/actatropica Cloning, characterization and transmission blocking potential of midgut carboxypeptidase A in Anopheles stephensi V. VenkatRao a , Surendra K. Kumar a , P. Sridevi b , Vijaykumar Yogesh Muley c , R.K. Chaitanya d,∗ a Department of Animal Biology, School of Life Sciences, University of Hyderabad, Hyderabad 500046, India b Department of Biotechnology, Indira Gandhi National Tribal University, Amarkantak, 484224, India c Centre for Computational Science, School of Basic & Applied Sciences, Central University of Punjab, Bhatinda 151001, India d Centre for Animal Sciences, School of Basic & Applied Sciences, Central University of Punjab, Bhatinda, 151001, India a r t i c l e i n f o Article history: Received 6 July 2016 Received in revised form 27 December 2016 Accepted 27 December 2016 Available online 10 January 2017 Keywords: Carboxypeptidase A Plasmodium berghei Anopheles stephensi Midgut Transmission blocking vaccine (TBV) a b s t r a c t Transmission-blocking vaccines (TBV) interrupt malaria parasite transmission and hence form an impor- tant component for malaria eradication. Mosquito midgut exopeptidases such as aminopeptidase N & carboxypeptidase B have demonstrated TBV potential. In the present study, we cloned and characterized carboxypeptidase A (CPA) from the midgut of an important malarial vector, Anopheles stephensi. ClustalW amino acid alignment and in silico 3-dimensional structure analysis of CPA predicted the presence of active sites involved in zinc and substrate binding that are conserved among all the known mosquito species. Real-time PCR analysis demonstrated that CPA is predominantly expressed in the midgut throughout the mosquito life cycle and that this gene is significantly elevated in P. berghei-infected mosquitoes compared to uninfected blood-fed controls. The high midgut CPA activity correlated with the prominent mRNA lev- els observed. Peptide-based anti-CPA antibodies were raised that cross-reacted specifically to ∼48 kDa and ∼37 kDa bands, which correspond to zymogen and active forms of CPA. Further, the addition of CPA- directed antibodies to P. berghei-containing blood meal significantly reduced the mosquito infection rate in the test group compared to control and blocked the parasite development in the midgut. These results support further development of A. stephensi CPA as a candidate TBV. © 2016 Elsevier B.V. All rights reserved. 1. Introduction Global efforts such as long-lasting insecticidal bed-nets, indoor residual spraying with insecticides, rapid diagnostic testing and artemisinin-based combination therapy has reduced mortality rates and malaria disease burden. Despite these measures, 214 million malaria cases were recorded in the previous year (WHO, World Malaria Report, 2015). The size and genetic complexity of the malarial parasite make development of malaria vaccine a cum- bersome effort. The life cycle of the malaria parasite is well characterized in the human host whereas its survival strategies and transmission in the mosquito remain a challenging scientific problem. The malaria parasite has to undergo a series of obligatory developmental pro- cesses inside the mosquito vector. Male and female gametocytes fertilize within the lumen of the midgut. The resultant ookinete ∗ Corresponding author. E-mail addresses: chaitanyark@gmail.com, chaitanyark@cup.ac.in (R.K. Chaitanya). breaches through the peritrophic matrix and laminar surface of the midgut and differentiates at the basal lamina to form an oocyst. Within each oocyst, asexual multiplications produce thousands of sporozoites which are disseminated into the hemocoel. These sporozoites chemotactically invade salivary gland epithelial cells and are delivered to a new vertebrate host during the succeed- ing blood meal (Greenwood et al., 2008). Ookinete traversal of the midgut is considered a critical process during transmission that involves specific interactions between the parasite and midgut epithelial surface (Vega-Rodríguez et al., 2014). With respect to the dual-host life cycle and multiple forms of malaria parasite, an effective vaccine is a combination of pre-erythrocytic, asexual, erythrocytic, and sexual stage com- ponents. According to the 2013 update to the Malaria Vaccine Technology Roadmap laid down by World Health Organisation, one of the criteria to be considered in development of the malaria vaccine is the reduction in transmission of the parasite. Transmission-blocking vaccine (TBV) interrupts malaria transmis- sion by targeting the sexual or mosquito stages of the parasite antigens. Mosquito midgut invasion by ookinete is a promising target for transmission-blocking approach as parasite numbers http://dx.doi.org/10.1016/j.actatropica.2016.12.035 0001-706X/© 2016 Elsevier B.V. All rights reserved.