Contents lists available at ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene Research paper Molecular cloning, characterization and gene expression analysis of aminolevulinic acid synthase in Litopenaeus vannamei Ivane R. Pedrosa-Gerasmio a,b , Hidehiro Kondo a , Ikuo Hirono a, ⁎ a Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, Tokyo, Japan b Department of Marine Sciences, College of Science and Mathematics, Mindanao State University-Iligan Institute of Technology, Iligan City, Philippines ARTICLE INFO Keywords: Aminolevulinic acid synthase Heme pathway Molting Pacific white shrimp Gene silencing Hemoprotein ABSTRACT 5-Aminolevulinic acid synthase (ALAS) is the rate-limiting enzyme in the biosynthesis of heme, a prosthetic group that is found in hemoproteins, including those involved in molting. To better understand the roles of ALAS in L. vannamei (LvALAS), we analyzed its sequence and tissue distribution, the effects of age and bacterial infection on its gene expression, and the effects of LvALAS gene silencing. We also examined the expressions of three hemoproteins, the cytochrome oxidase subunit I (COX I) and subunit IV (COX IV) and catalase. Three LvALAS splicing variants were found in the hepatopancreas, with the main splicing variant having an open reading frame that encodes 532 aa. LvALAS transcripts were found in each of the eleven tissues tested in this study, with the highest gene expression in the intestine. The transcript abundances of LvALAS, COX I and COX IV in the hepatopancreas and stomach tended to decrease with age. LvALAS and catalase gene expressions sig- nificantly increased in the stomach after V. parahaemolyticus infection. LvALAS gene expression in the hepato- pancreas, stomach and intestine (12- and 24-hours post-injection) was relatively lower in dsALAS-injected shrimp than in PBS-injected shrimp. All the PBS-injected shrimp molted after 8–10 days while no molting ac- tivity was observed in the dsALAS-injected shrimp group within the 14 days post-injection period. Our results provide evidence that (1) only the housekeeping form of ALAS exists in L. vannamei; LvALAS gene expression (2) decreases with age and (3) increases after bacterial infection; and (4) an ALAS-dependent pathway is necessary for proper molting in L. vannamei. 1. Introduction 5-Aminolevulinic acid synthase (ALAS; EC 2.3.1.37) is the first and rate-limiting enzyme of the heme biosynthesis pathway that catalyzes the condensation of glycine and succinyl-CoA to form 5-Aminolevulinic acid (5-ALA) in the mitochondria (Hunter and Ferreira, 2009). 5-ALA then continues in a series of enzymatic cascades in the cytosol and after coproporphyrinogen III is formed, this will enter back to the mi- tochondria to form protoporphyrinogen IX, then protoporphyrin IX (PPIX). Ferrochelatase (EC 4.99.1.1) inserts ferrous ion into PPIX to form heme (or ferroprotoporphyrin IX) (Yang et al., 2016). Heme is required in nearly all living organisms, acting as a prosthetic group of hemoproteins (heme-containing proteins) having important functions, e.g. oxygen transport (hemoglobin), respiration (mitochondrial cyto- chromes), antioxidant defenses (catalase), ecdysone synthesis (nuclear receptor E75) and detoxification (cytochrome P450) (Reinking et al. 2005; Tsiftsoglou et al. 2006; Annalora et al. 2017). Most of the heme is synthesized in developing red blood cells in the bone marrow to be incorporated in hemoglobin and in the liver for the formation of other heme-containing enzymes (Ajioka et al., 2006). In crustaceans, hemo- cyanin (copper-containing blood pigment; not a hemoprotein) is used instead of hemoglobin for oxygen transport (Wheal et al., 2016). Al- though it is generally known that hemoglobin is not utilized by crus- taceans, mRNA transcripts of other hemoproteins like catalase (Tavares-Sánchez et al., 2004; Arockiaraj et al., 2012) and nitric oxide synthase (Yao et al., 2010; Inada et al., 2010), are expressed in various https://doi.org/10.1016/j.gene.2020.144421 Received 18 September 2019; Received in revised form 29 January 2020; Accepted 29 January 2020 Abbreviations: LvALAS, aminolevulinic acid synthase in Litopenaeus vannamei; ALAS, 5-Aminolevulinic acid synthase; COX I, cytochrome oxidase subunit I; COX IV, cytochrome oxidase subunit IV; 5-ALA, 5-Aminolevulinic acid; PPIX, protoporphyrin IX; RNA, ribonucleic acid; Cdna, DNA complementary to RNA; PCR, polymerase chain reaction; RT-PCR, reverse transcription polymerase chain reaction; qPCR, quantitative reverse transcription polymerase chain reaction; ORF, open reading frame; HRM, Heme regulatory motif; SV, splice variant; ssRNA, single-stranded ribonucleic acid; dsRNA, double-stranded ribonucleic acid; SEM, standard error of the mean; ANOVA, analysis of variance; PBS, phosphate-buffered saline; UTR, untranslated region; Aa, amino acid(s); Bp, base pair(s); IRE, iron responsive element; Hpi, hours post infection; AHPND, acute hepatopancreatic necrosis disease ⁎ Corresponding author. E-mail addresses: h-kondo@kaiyodai.ac.jp (H. Kondo), hirono@kaiyodai.ac.jp (I. Hirono). Gene 736 (2020) 144421 Available online 01 February 2020 0378-1119/ © 2020 Elsevier B.V. All rights reserved. T