Ichthyol Res DOI 10.1007/s10228-011-0224-4 SHORT REPORT The annual cycle of spermatogenesis in Lipophrys pholis (Blenniidae), a recently proposed sentinel species for pollution monitoring Filipa Ferreira • Miguel Machado Santos • Maria Armanda Reis-Henriques • Maria Natividade Vieira • Nuno Miguel Monteiro Received: 3 November 2010 / Revised: 15 April 2011 / Accepted: 18 April 2011 Ó The Ichthyological Society of Japan 2011 Abstract Since reproductive parameters are common Keywords Lipophrys pholis Á Sentinel species Á endpoints in pollution monitoring, the description of the Spermatogenesis Á Gonadal development Á Reproduction spermatogenesis of Lipophrys pholis, a recently proposed sentinel species, is of critical importance. Mature males were used to determine the annual maturation cycle, using Introduction a stereological approach. The spermatogenic cycle was classified into three stages: early spermatogenesis During the last few decades, special attention has been (May), mid spermatogenesis (September), and spawning devoted to the abnormal presence of natural or synthetic (November and January). The testes were asynchronously chemicals in the wild, particularly in aquatic ecosystems arranged with various cell types observable during all that tend to receive and concentrate many of these mole- seasons, although in different proportions. Gonadosomatic cules, some of which are able to act as endocrine disruptors index and spermatozoa proportions confirmed the estab- (Colborn et al. 1993; Sumpter 2005; Holbech et al. 2006). lished reproductive season. Differences between the sam- To assess the toxic effects of environmental contaminants, pled populations and those from septentrional locations are inclusion of fish in monitoring programs is now a common discussed, namely the absence of ‘‘resting’’ and ‘‘spent’’ approach. The development of biological early warning periods. systems, able to signal the presence of contaminants in aquatic environments, emphasized the need for validation of sentinel species, intrinsically sensitive to chemical pol- lution. Although several fish species have been increas- ingly used as sentinels in marine ecosystem biomonitoring F. Ferreira Á M. M. Santos Á M. A. Reis-Henriques Á programs, some problems often arise due to limitations M. N. Vieira CIMAR/CIIMAR, Interdisciplinary Centre of Marine imposed by the particular ecology of some of the most and Environmental Research, University of Porto, well-established model species (Ferreira et al. 2009). Thus, Rua dos Bragas 177, 4050-123 Porto, Portugal there is still space for the introduction of previously unused model species whose biology is tightly connected with the M. N. Vieira Department of Biology, Faculty of Sciences of the University environment being assessed. of Porto, Rua do Campo Alegre, 4169-007 Porto, Portugal Lipophrys pholis is a common intertidal Blenniidae whose distribution ranges from Mauritania to Norway, & N. M. Monteiro ( ) including the Azores Islands and parts of the Mediterra- CIBIO, Research Centre in Biodiversity and Genetic Resources, Campus Agra´rio de Vaira˜o, R. Padre Armando Quintas, nean (Zander 1986) (Fig. 1). Given its importance in the 4485-661 Vaira˜o, Portugal dynamics of rocky intertidal communities (Silva et al. e-mail: [email protected] 2010), the species ecology and behavior have been inten- sively studied since the beginning of the twentieth century N. M. Monteiro CEBIMED, Faculty of Health Sciences of the Fernando Pessoa (Ford 1922; Qasim 1956, 1957; Shackley and King 1977; University, Rua Carlos da Maia 296, 4200-150 Porto, Portugal Faria et al. 1996; Monteiro et al. 2005). Considering that 123 F. Ferreira et al. gaps that still exist on the spermatogenic cycle are addressed by detailed description of the annual gonad maturation cycle, using a stereological approach. Materials and methods Four sampling campaigns were conducted during 2006 (January, May, September, and November). A total of 191 adults males were sampled, whose sex was determined using genital papilla morphology (Ferreira et al. 2010), from 7 intertidal rocky shores along the Portuguese coast [Fig. 1; Vila Praia de Aˆ ncora (n = 26); Viana do Castelo (n = 27); Sa˜o Bartolomeu do Mar (n = 26); Cabo do Mundo (n = 31); Praia da Boa Nova (n = 27); Foz (n = 25); and Castelejo (n = 29)]. Fish were collected in rocky pools, with a hand net, during low tides. Given that Lipophrys pholis experiences a microhabitat shift, which coincides with the onset of reproduction, when both males and females attain 8 cm length (Faria et al. 1996; Monteiro et al. 2005), special care was taken to collect only large, Fig. 1 Lipophrys pholis geographical distribution, according to mature, individuals. Collected individuals were rapidly Zander (1986). Letters indicate sampling locations. A Vila Praia de transported to the laboratory and immersed in cold sea- Aˆ ncora, 41°4804700N, 8°5105500W; B Viana do Castelo, 41°4106100N, water to ensure partial immobilization. All fish were 0 00 0 00 0 00 8°51 02 W; C Sa˜o Bartolomeu do Mar, 41°34 25 N, 8°47 54 W; measured (total length in mm; T ) and weighed (total body D Cabo do Mundo, 41°1204800N, 8°4205000W; E Praia da Boa Nova, L 41°1105600N, 8°4204100W; F Foz, 41°0902900N, 8°4005600W; G Castele- wet weight in g; TW). After the initial measuring proce- jo, 37°505900N, 37°505900W dures, fish were quickly sacrificed by spinal transection. The gonads were excised and weighed (WG; g), and the mean value of the gonadosomatic index calculated for each -1 L. pholis is generally abundant and easy to catch, inhabits sample site [GSI: 100 WG (WT) ]. Since the GSI showed a the intertidal zone during the majority of its lifecycle while similar pattern of variation in all the sampled rocky bea- displaying a restricted home range, and exhibits wide ches (Fig. 2a), two locations were selected for subsequent geographical distribution, it may be assumed that this histological analysis. A total of 41 mature L. pholis males blenniid combines some of the basic characters expected in were used (Cabo do Mundo, n = 21; Castelejo, n = 20) a sentinel species. There is now evidence that L. pholis is and their gonads macroscopically and microscopically also highly responsive to a wide variety of pollutants, from observed. The whole testicular tissue was preserved in those present in oil spills (Lyons et al. 1997; Harvey et al. Bouin’s solution for 8–12 h and then transferred to 70% 1999), to neurotoxic compounds (Sole´ et al. 2008), organic ethanol. Paraffin sections (3–5 lm thick) were stained with contaminants (Lima et al. 2008) or estrogenic chemicals hematoxylin–eosin and mounted with EntellanÒ (Merck). (Ferreira et al. 2009). The stereological approach was designed based on point Since reproductive-related parameters, such as fertility, counting (Freere and Weibel 1967) using a light microscope embryonic development or gonadal development, are equipped with a charge-coupled device (CCD) camera able common endpoints addressed in monitoring programs, to record 4.9-megapixel digital images. Our methodology detailed knowledge on the reproductive biology of sentinel was similar to the one proposed by Matta et al. (2009), with species, such as L. pholis, is of great importance. Although minor alterations. Briefly, in each male, 5 images were taken some data exist on the spawning season (based upon direct in each of two different testis sections (10 fields per indi- observation of the presence of eggs masses and/or parental vidual). A grid formed by 8 horizontal 9 6 vertical lines, guarding males; Almada et al. 1990a, b, 1992), reproduc- creating a total of 48 interceptions, was overlaid on each tive cycle (Qasim 1957; Shackley and King 1977)or image (recorded at 4009 magnification), and the cell types ultrastructural characterization of sperm cells (Silveira encountered below the intersection points were registered (if et al. 1990), detailed knowledge on the annual cycle of no cell was present bellow the intersection, a zero was reg- sperm production is lacking. In this study, available data on istered). The percentage of each observed cell types [type A L. pholis male reproduction are gathered and knowledge spermatogonia (Spga), type B spermatogonia (Spgb), 123 Annual spermatogenesis cycle in Lipophrys pholis Fig. 2 a GSI variation in the sampled rocky beaches (vertical bars e type A spermatogonia (Spga) and type B spermatogonia (Spgb), depict standard errors) and photomicrographs of the histological f primary spermatocytes (SpcI) and secondary spermatocytes (SpcII), architecture of Lipophrys pholis testis: (b early spermatogenesis, and g spermatids (Spt) and spermatozoa (Spz). The scale for b, c, and c mid spermatogenesis, and d spawning). Additionally, the micro- d represents 200 lm while for e, f, and g, it represents 20 lm scopic morphology of the spermatogenic cells are also depicted: primary spermatocytes (SpcI), secondary spermatocytes May (0.33 ± 0.02%) (Fig. 2a). Several stages of sper- (SpcII), spermatids (Spt), and spermatozoa (Spz)] was cal- matogenic cells were visible throughout the year, although culated for the four sampling events. The description of in distinct proportions (Fig. 2d–f). Spermatogonia, primary L. pholis spermatogenesis was evaluated based on the spermatocytes, secondary spermatocytes, and spermatids scheme of Weltzien et al. (2002), OECD guidance document were usually located at the periphery of the lobules, while for the diagnosis of endocrine-related histopathology of fish spermatozoa were seen, as free elements, in the center of gonads (2009), and Schulz et al. (2010). the lobule (Fig. 2f). The seasonal changes in the testes were defined based on the histological characteristics (Fig. 2) and on the relative abundance of spermatogenic Results cell types (Fig. 3). According to the frequency of testicular components, The highest GSI values were recorded in January three stages of gonadal maturation were defined. Early (1.13 ± 0.03%), whilst the lowest values were observed in spermatogenesis (May): the seminiferous wall was thick, 123 F. Ferreira et al. Fig. 3 Percentage of spermatogenic cell during the gonadal development of Lipophrys pholis, at Cabo do Mundo and Castelejo.