View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Biblioteca Digital do IPB RESEARCH ARTICLE Reproductive Cycle and Strategy of Anodonta anatina (L., 1758): Notes on Hermaphroditism MARIANA HINZMANN1,2, MANUEL LOPES‐LIMA1,2*, AMILCAR TEIXEIRA3, SIMONE VARANDAS4, RONALDO SOUSA2,5, ANABELA LOPES1,2, ELSA FROUFE2, 1,2 AND JORGE MACHADO 1ICBAS‐UP—Abel Salazar Biomedical Sciences Institute, University of Porto, Porto, Portugal 2CIMAR‐LA/CIIMAR—Centre of Marine and Environmental Research, University of Porto, Porto, Portugal 3CIMO‐ESA‐IPB—Mountain Research Centre, School of Agriculture, Polytechnic Institute of Bragança, Campus de Santa Apolónia, Bragança, Portugal 4CITAB‐UTAD—Centre for Research and Technology of Agro‐Environment and Biological Sciences, University of Trás‐os‐Montes and Alto Douro, Vila Real, Portugal 5CBMA—Centre of Molecular and Environmental Biology, University of Minho, Campus de Gualtar, Braga, Portugal ABSTRACT Freshwater mussels have decreased dramatically in Iberia over the last decades. These animals are responsible for important ecosystem services such as recycling nutrients and improving water clarity. Under this view a better knowledge on the biological features of these animals is extremely important for future conservation and management actions. In this study the reproductive and gametogenic cycle of Anodonta anatina were studied during 2 years in one population as well as the sex ratio and hermaphroditism in six distinct populations, using standard histology. Gametogenesis was continuous in both sexes and germinal epithelium in early stages of development. Gametes were present throughout the reproductive cycle. Oogenesis and spermatogenesis occurred mainly between January and May. Larvae brooding occurred between September and March and main glochidia discharge occurred over a short period (2–3 weeks) in March. For the sex‐ratio and hermaphroditism assessments a variable number of individuals were collected from several populations from lakes and rivers. Previous studies described A. anatina as mainly dioecious with only a few populations presenting occasional hermaphroditism. However, the present study indicates that A. anatina sexual behavior is influenced by environmental conditions, being mainly dioecious in rivers with increased hermaphroditism in standing waters. Although self‐ fertilization was not confirmed, additional studies with molecular characterization of larvae using fast evolving markers should be used in future studies to enlighten this process. Overall, this study indicates that for more efficient conservation actions and management plans, freshwater mussel reproductive biology should be studied at the population level mainly in the subfamily Anodontinae. J. Exp. Zool. 9999A: 1–13, 2013. © 2013 Wiley Periodicals, Inc. How to cite this article: Hinzmann M, Lopes‐Lima M, Teixeira A, Varandas S, Sousa R, Lopes A, Anodonta anatina J. Exp. Zool. Froufe E, Machado J. 2013. Reproductive cycle and strategy of (L., 1758): 9999A:1–13, 2013 Notes on hermaphroditism. J. Exp. Zool. 9999:1–13. © 2013 WILEY PERIODICALS, INC. 2 HINZMANN ET AL. Freshwater mussels (Bivalvia, Unionoida) are among the most several parts of the continent and is listed as threatened and critically threatened faunal groups globally (Neves et al., '97; protected in Germany (Zettler et al., 2006). In Iberia, all species of Lydeard et al., 2004; Simberloff, 2012). The consequences of this native unionoid mussels are decreasing both in number of catastrophic decline go far beyond the loss of species per se since populations and individuals (Araujo et al., 2009). These animals freshwater mussels are responsible for critical trophic and non‐ are extremely important ecologically serving important ecosystem trophic functions (Vaughn, 2010; Allen and Vaughn, 2011). In functions (e.g., biomass transfer from the water column to the most European species very few studies have been done, either on benthos, reduction of water turbidity, control of the concentration the distribution and population structure but also on the study of and composition of suspended particles, nutrient cycling and their basic life cycle traits. This situation may be related with provision of habitat for other organisms; Vaughn, 2010; Sousa the complex and distinct life‐history patterns of each species. et al., 2011, 2012). All of these facts turn the research on the Recently, biologists have made the first attempts to organize biological traits (mainly the reproductive characters) of these the life‐history strategies of freshwater mussels into conceptual mussel species crucial for future conservation actions and frameworks (Bauer and Wächtler, 2000; Dillon, 2000). However, management. Additionally, recent findings suggest that even the development of these frameworks is hampered by a lack of host generalists such as Anodontinae mussels may be increasingly life‐history information for most species, particularly for traits limited in their reproductive cycles by the host availability (Douda such as age at maturity, growth rate, longevity and fecundity et al., 2013). Hence, precise data on the timing of glochidia (Strayer et al., 2004). This information is vital for the generation of production and the capability of hermaphroditism may be highly testable hypotheses about the evolutionary, ecological, and important for assessing the risk of host limitation in the field. management consequences of life‐history variation. So, it is of Under this view, to increase the knowledge on the reproductive primordial importance to increase fundamental knowledge on biological traits of A. anatina, the aim of this study was to describe basic biology and habitat requirements of each species so that in detail the histology of the sexual (spermatogenesis plus managers can more effectively conserve and manage the mussel oogenesis) development of A. anatina. In addition, we verified the fauna. sex ratio and hermaphroditism from distinct habitats such as The reproductive biology of unionoids is distinctive: fertiliza- rivers, lagoons or impoundments to allow a more detailed tion is internal and the fertilized eggs form larvae (glochidia) evaluation on the sexual strategy of this species. that are brooded in the gills of females until they are released in to the water column. To complete their development into juvenile mussels, they need to parasitize certain host fishes until METHODS complete metamorphosis into young adult mussels (Kat, '84). Sampling Recent studies within the subfamily Anodontinae have been For the seasonal sexual development, from March 2009 to carried on the reproduction development on related species March 2011 six A. anatina were collected monthly in Tâmega (Sereflisan et al., 2009; Lima et al., 2012) to fill that gap of River near Ribeira de Pena, Portugal (N 41°3203000; W 7°4701500; knowledge. Fig. 1); all of these animals were also checked to determine the sex The present study is focused on Anodonta anatina, a freshwater ratio. The mussels were transported to the laboratory on ice in a mussel that is still widespread in Europe. Current distribution cooler box and processed within 24 hr. In the laboratory, bivalves encompasses the Iberian Peninsula in the Southwest, the were anesthetized in 2‐phenoxyethanol solution 0.4% (v/v) for Scandinavia in the North and Russia in the East (Graf, 2007). about 30 min and dissected for histology. To minimize eventual Although widespread, this species already disappeared from negative impacts, the number of animals sacrificed was reduced and the seasonal study was made in the larger population of A. anatina in Portugal. Grant sponsor: Portuguese Foundation for Science and Technology (FCT). Histological Procedure grant numbers: PTDC/AAC‐AMB/117688/2010 CONBI, PTDC/MAR/098066/ Samples of gonad and gill tissue were excised from each animal 2010 SYNERG. and preserved in Bouin's solution (Panreac, Barcelona, Spain). Ã ‐ Correspondence to: Manuel Lopes Lima, Abel Salazar Biomedical After being preserved in Bouin's solution for a week, transverse Sciences Institute, Applied Physiology Laboratory, Aquatic Production Department, University of Porto, Rua de Jorge Viterbo Ferreira No. 228, sections of the gonads were dehydrated in graded ethanol, 4050‐313 Porto, Portugal. embedded in paraffin, sectioned at 5–8 mm and stained with E‐mail: [email protected] hematoxylin and eosin for histological examination. Received 15 June 2012; Revised 10 April 2013; Accepted 16 April 2013 The sex was determined visually by the presence of male and DOI: 10.1002/jez.1801 female gonad tissue. The maturation level of the gonads was Published online XX Month Year in Wiley Online Library fi (wileyonlinelibrary.com). established using the classi cation stage table of the gamete development index (GDI) adapted from Barber ('96). Gills were J. Exp. Zool. SEXUAL STRATEGY AND CYCLE OF Anodonta anatina 3 river and lagoon/reservoir populations were analyzed by one‐way ANOVA followed by a Duncan multiple range test. Both tests were carried using JMP statistical package (SAS Institute, Cary, NC, USA). Genetic Analysis To avoid taxonomic uncertainties due to the usual misidentifica- tion between A. cygnea and A. anatina, identities were confirmed by genetic analysis. For this, foot tissue samples were collected and placed directly into 96% ethanol. The whole genomic DNA was extracted from small tissue samples pieces (2 mm3) using a standard high‐salt protocol (Sambrook et al., '89). A fragment of approximately 700 bp of