An Acad Bras Cienc (2020) 92(suppl.1): e20190053 DOI 10.1590/0001-3765202020190053 Anais da Academia Brasileira de Ciências | Annals of the Brazilian Academy of Sciences Printed ISSN 0001-3765 I Online ISSN 1678-2690 www.scielo.br/aabc | www.fb.com/aabcjournal AGRARIAN SCIENCES Embryo and larval development of the Running title: EMBRYO AND LARVAL yellow clam Mesodesma mactroides (Reeve, DEVELOPMENT OF THE Mesodesma mactroides 1854) (Mesodesmatidae) in laboratory Academy Section: AGRARIAN SCIENCES JUAN J.S. SANTOS, JULIANA P. BERNARDES, JUAN R.B. RAMÍREZ, CÁSSIO O. RAMOS, CARLOS HENRIQUE A. DE MIRANDA GOMES & LUIS ALBERTO ROMANO Abstract: The yellow clam Mesodesma mactroides (Reeve, 1854) is a sand mollusc e20190053 with historical and socioeconomic importance in Brazil, Uruguay and Argentina. A 92 guaranteed form to access a successful reestablishment of the species in their natural (suppl.1) environment is directly linked to their reproduction biology. Then, our report introduces 92(suppl.1) the embryonic and larval development of the yellow clam reared in laboratory for such purposes. M. mactroides broodstock were selected as specimens who possess a mean total shell length and weight of 66 ± 3.82 mm and 27.15 ± 4.07 g for an afterwards spawn induction through stripping technique. Regarding the embryonic development, newly fertilized oocytes exhibited a mean diameter of 51.20 ± 6.64 μm. The first polar corpuscle, trochophores and D-veliger appeared at 20 min, 18 and 24 h after fertilization, respectively. Umbonate and pediveliger larvae were noticed on the 8th and 25th day, respectively, with complete metamorphosis occurring only at the 27th day, when all larvae were retained in a 200 μm nylon mesh. Therefore, with that basic understanding of the embryonic and larval development of M. mactroides in the laboratory, forwards studies will focus in establish a technological package for this species. Key words: broodstock, D-veliger, M. mactroides, pediveliger, sand mollusk, stripping. INTRODUCTION 1959, McLachlan 2018). Excessive extraction and massive mortalities drastically reduced their Mesodesmatidae is a socioeconomically populations (Carvalho et al. 2013a, b, Santos et important family of sand molluscs throughout al. 2016), but the actual causes of mortalities are the world, including promising species for unknown. In the face of a threatening status, it aquaculture (Santos et al. 2016) such as toheroa is important to develop strategies to manage (Paphies ventricosa) (Redfearn 1982, Gadomski and increase their natural stocks (Gianelli et al. et al. 2015) and pipi (Paphies australis) (Hooker 2015). 1997) in New Zealand, macha (Mesodesma The cultivation of yellow clam in the donacium) (Uriarte 2008, Ayerbe et al. 2017) in laboratory has been considered an option to Chile and Peru, and the yellow clam Mesodesma foster repopulation programs and aquaculture mactroides (Reeve, 1854) in southern South activities, as it has already occurred with America. M. mactroides is distributed from the M. donacium in Peru (Ayerbe et al. 2017). coast of Rio de Janeiro, Brazil to Buenos Aires, However, the knowledge about the cultivation Argentina (Rios 1994), standing out for its of M. mactroides, as many other tropical native historical fishing value in these regions (Coscarón species with commerce potential, is still lacking. An Acad Bras Cienc (2020) 92(suppl.1) JUAN J.S. SANTOS et al. EMBRYO AND LARVAL DEVELOPMENT OF THE Mesodesma mactroides Developing culture methods for M. mactroides The animals were acclimatized in 5-liter would be beneficial, as reduce the pressure on buckets containing sand (3 kg) and transferred natural populations and would increase coastal to 40-liter tanks with a temperature of 18 °C, productivity (Cáceres-Martínez & Vásquez- aeration and continuous flow of seawater Yeomans 2008, López et al. 2008). (35 ppt). The diet of the broodstock was a Previous studies on pathogen prevalence, microalgae mix composed of Chaetoceros anthropogenic influence, seasonality (Carvalho muelleri, Isochrysis galbana and Rhodomonas et al. 2013a, b, Santos et al. 2016), salinity salina (1:1:1) at the concentrations of 15 to 20 tolerance, histopathology and immunology x 104 cellsmL-1 daily. The average total shell (Carvalho et al. 2015a, b, 2016) have been carried length and weight of the broodstock specimens out to comprehend the environmental behavior selected for this experiment were 66 ± 3.82 mm of M. mactroides. To cultivate a new species, it and 27.15 ± 4.07 g, respectively. is important to understand its biological and ecological requirements and provide adequate Spawning induction simulation of environmental conditions in At the end of the third week of conditioning, the laboratory for a better performance in spawning inductions were attempted thorough subsequent cultivation systems (Urban 2000, three different methodologies: two thermal Madrones-Ladja 2002, Huo et al. 2014). In this inductions with a gradually raising temperature sense, little is known about the embryonic and an abrupt thermal shock, as well as a and larval development of M. mactroides stripping technique. in laboratory. Thus, our study describes the The thermal induction with gradually morphology and growth during the embryonic increasing temperature method consisted of an and larval development of the yellow clam acclimatization of 40 broodstock specimens in M. mactroides under laboratory conditions, a tank (200 L) with continuous water flow and contributing to develop the technological salinity of 35 ppt to a temperature gradually package for this species. increasing from 18 to 26 °C during 9 hours. The second method of thermal induction was a shock method that consisted in exposing 40 MATERIALS AND METHODS broodstock specimens acclimated at 18 °C in Laboratory collection and conditioning a 2,500 L tank with mild aeration to an abrupt procedures increase in the temperature (23.5 °C) for 15 hours Fifty adult specimens of M. mactroides were (overnight). The tanks contained a mesh sieve collected in April 2017 (autumn season), during of 18 µm at the water outlet to retain female a low tide in the tidal zone of Mar Grosso beach, gametes. in the municipality of São José do Norte, Rio The spawning was performed using the Grande do Sul, Brazil (32°3’10”S 51°59’26”W). The stripping method, using eight specimens of M. animals were stored in 20-liter containment mactroides evaluated under optical microscopy basins at room temperature and transported to certify the presence of viable gametes (Helm to the Laboratory of Marine Molluscs at the et al. 2004). The proportion of males and Universidade Federal de Santa Catarina, Brazil females used in this procedure was 1:1. Prior to (LMM - UFSC). the fertilization, the pooled eggs (10,000,000) An Acad Bras Cienc (2020) 92(suppl.1) e20190053 2 | 11 JUAN J.S. SANTOS et al. EMBRYO AND LARVAL DEVELOPMENT OF THE Mesodesma mactroides were sieved and mixed in 15 L of seawater with observation verifying survival and motility, sperm added (1:7). samples were fixed in 4% buffered formaldehyde The analysis of embryogenic and larval for later analysis under optical microscopy. development was proceeded after fertilization. When all larvae reached the pediveliger stage The zygotes obtained with the most efficient and were retained at 200-μm meshes, they were spawning method were selected for ready to settle and the larviculture phase was morphological evaluation. considered to be completed Embryonic development Morphometric analyses After fecundation, the embryos were transferred Growth averages and standard deviation (± to a 2,500-liter flat-bottomed tank at the density SD) (embryonic phase: diameter, larval phase: of 4,000 embryos L-1, with seawater previously larval height and length) and morphology (cell filtered and UV-sterilized, temperature of 25 ± 1 divisions and structural development) were °C, salinity of 32 ppt and mild aeration. Embryos evaluated in living and preserved specimens were monitored through their development under the x40 objective on a Carl Zeiss® and morphological analyses determining all microscope (Axio Imager A2) and Leica® DM500 embryonic phases lasted until they reached (connected with the Leica LAZ EZ software) for the D-veliger larva stage. Samples were taken embryonic and larval stages, respectively. at 5-minute intervals in the first hour after fertilization, 30-minute intervals for 8 hours and Statistics every 3 hours until completing 24 hours, using a Height and length of embryos and larvae 2 mL pipette and observed under a Sedgewick- were analyzed through Generalized Linear Rafter chamber. Model after the assumptions of normality and homoscedasticity were confirmed. Regression Larval development analyses were made to evidence patterns of the The D-veliger larvae were transferred to a data as a function of time. The regression model 4,000-liter flat-bottomed tank, with salinity of was selected based on the determination factor 32 ppt and mild aeration. Everyday, the water (r2). Growth was calculated as a function of the was completely renewed, the temperature was accumulated mean temperature values for each recorded before and after water exchanges, and experimental day to evaluate the influence of the larvae were sieved with mesh sizes ranging temperature changes over time on the larval from 35 μm at the beginning to 200 μm at the end development (Degrees/Day). The analyses were of cultivation, with samples being collected for performed using the software GraphPad Prism 6. morphometric analysis. The larvae were initially fed with 1x104 cellsmL-1 of Chaetoceros calcitrans, Isochrysis galbana and Pavlova lutheri (ratio: RESULTS 0.30: 0.35: 0.35x104 cellsmL-1, respectively), Spawning induction 4 -1 increasing to 2x10 cellsmL with the addition of From the three techniques used to obtain 4 Chaetoceros muelleri (ratio: 0.6: 0.7: 0.35: 0.35x10 embryos, it was observed that both thermal -1 cellsmL , respectively). The larvae growth was induction with gradual increase of temperature monitored daily by measuring shell length and and exposure to thermal shock were height of 10 to 20 specimens.