Spermatophore replacement of pink shrimp Farfantepenaeus brasiliensis after manual extrusion: Effect of molting André Braga ⁎, Diogo L.A. Lopes, Luís H. Poersch, Wilson Wasielesky Marine Station of Aquaculture, Oceanography Institute, Federal University of Rio Grande, C.P. 474, Rio Grande, RS 96 201-900, Brazil abstract article info Article history: Received 6 April 2014 Received in revised form 4 June 2014 Accepted 25 June 2014 Available online 1 July 2014 Keywords: Shrimp Farfantepenaeus brasiliensis Males Extrusion Spermatophore replacement Molting This study aimed to evaluate Farfantepenaeus brasiliensis spermatophore replacement after manual extrusion and analyze the effect molting has on this process. Three trials were conducted. In the first trial, the replacement of spermatophores after extrusion was macroscopically evaluated via an analysis of morphological changes in the terminal ampoule during the formation of new spermatophores. In the second trial, the sperm quality in the different stages of spermatophore replacement identified in the first trial was compared. In the third trial, the replacement time of spermatophores after extrusion was determined, with and without molting. In all trials, 30 manually extruded wild males were individually stocked in 0.49 m 2 tanks using different samples of randomly selected animals for each trial. The results were obtained by daily visual examination of the gonopore and coxae regions of the fifth pereiopod pair and sperm quality analyses. In trial 1, three successive macroscopic stages of spermatophores were observed during replacement: unformed, partially formed and formed. In trial 2, the sperm count in formed spermatophores was significantly higher than that in partially formed spermatophores, whereas the spermatophore weight was not significantly different. Therefore, spermatophore replacement most likely comprises the following phases: (1) deposition of a primitive spermatophore with all structural com- ponents in each terminal ampoule; (2) gradual deposition of spermatozoa; and (3) stiffening of the spermato- phore into typical elongate form. Trial 3 demonstrated that after extrusion, spermatophores are completely formed in 16 days without molting and in 24 h with molting. Spermatophores formed after molting have sperm quality similar to that of gradually formed spermatophores. © 2014 Elsevier B.V. All rights reserved. 1. Introduction The primary biological functions of penaeid spermatophores are to protect and avoid the loss of spermatozoa in the transfer to females during copulation. Additionally, in closed thelycum species, spermato- phores seal the thelycum, preventing the need for replacement of the spermatophore and insemination by other males (Bauer, 1991; Subramoniam, 1991). Penaeids have spermatophores of varying complexity depending on the thelycum type. For example, females with open thelycum, i.e., Litopenaeus, receive morphologically complex spermatophores composed of accessory structures, such as wing, germinate body and/or flanges. In the closed thelycum penaeid, males produce simple spermatophores that are divided into a main body and an appendage, which is the structure that internally seals the thelycum (Bauer, 1991; Bauer and Cash, 1991). Independent of spermatophore morphological complexity, penaeids usually replace their spermatophores after ejaculation via copulation or extrusion (Heitzmann et al., 1993; Malek and Bawab, 1974a,b). When ejaculation does not occur, spermatophores degenerate; melanin is deposited extracellularly and new healthy spermatophores are deposited in the terminal ampoules. This process of deterioration has been described for domesticated Litopenaeus vannamei, but there is no evidence that it occurs in wild males (Alfaro and Lozano, 1993; Alfaro-Montoya, 2010; Diamond et al., 2008; Parnes et al., 2006). Some authors have reported that, when spermatophore is experi- mentally ejaculated, the replacement process may be affected by popu- lation differences, temperature and molting (Heitzmann et al., 1993; Pascual et al., 1998; Rosas et al., 1993). The molting process in crusta- ceans is divided into four stages: intermolt, premolt, molt and postmolt (Brusca and Brusca, 2003). The following events of spermatophore replacement have been proposed for L. vannamei throughout the molting stages: (1) intermolt, when a spermatophore is present in each terminal ampoule; (2) premolt, when the spermatophore is degenerated via degradation of the extracellular matrix and phagocyto- sis of spermatozoa; (3) molt, when the spermatophores are absent; and (4) postmolt, when two new spermatophores are present, one in each terminal ampoule (Parnes et al., 2006). Spermatophore replacement time and effect of molting have been evaluated for open thelycum species (Chow et al., 1991; Heitzmann Aquaculture 433 (2014) 313–317 ⁎ Corresponding author. Tel.: +55 53 32368132; fax: +55 53 32368042. E-mail address: andrebraga_pa@yahoo.com.br (A. Braga). http://dx.doi.org/10.1016/j.aquaculture.2014.06.032 0044-8486/© 2014 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Aquaculture journal homepage: www.elsevier.com/locate/aqua-online