Embryo and larval development of the yellow clam Mesodesma mactroides (Reeve, 1854) (Mesodesmatidae) in laboratory.

The yellow clam Mesodesma mactroides (Reeve, 1854) is a sand mollusc with historical and socioeconomic importance in Brazil, Uruguay and Argentina. A guaranteed form to access a successful reestablishment of the species in their natural environment is directly linked to their reproduction biology. Then, our report introduces 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.


INTRODUCTION
Mesodesmatidae is a socioeconomically important family of sand molluscs throughout the world, including promising species for aquaculture ) such as toheroa (Paphies ventricosa) (Redfearn 1982, Gadomski et al. 2015 and pipi (Paphies australis) (Hooker 1997) in New Zealand, macha (Mesodesma donacium) (Uriarte 2008, Ayerbe et al. 2017 in Chile and Peru, and the yellow clam Mesodesma mactroides (Reeve, 1854) in southern South America. M. mactroides is distributed from the coast of Rio de Janeiro, Brazil to Buenos Aires, Argentina (Rios 1994), standing out for its historical fishing value in these regions (Coscarón 1959, McLachlan 2018. Excessive extraction and massive mortalities drastically reduced their populations (Carvalho et al. 2013a, b, Santos et al. 2016), but the actual causes of mortalities are unknown. In the face of a threatening status, it is important to develop strategies to manage and increase their natural stocks (Gianelli et al. 2015).
The cultivation of yellow clam in the laboratory has been considered an option to foster repopulation programs and aquaculture activities, as it has already occurred with M. donacium in Peru (Ayerbe et al. 2017). However, the knowledge about the cultivation of M. mactroides, as many other tropical native species with commerce potential, is still lacking.
Developing culture methods for M. mactroides would be beneficial, as reduce the pressure on natural populations and would increase coastal productivity (Cáceres-Martínez & Vásquez-Yeomans 2008, López et al. 2008.
Previous studies on pathogen prevalence, anthropogenic influence, seasonality (Carvalho et al. 2013a, b, Santos et al. 2016, salinity tolerance, histopathology and immunology (Carvalho et al. 2015a(Carvalho et al. , b, 2016 have been carried out to comprehend the environmental behavior of M. mactroides. To cultivate a new species, it is important to understand its biological and ecological requirements and provide adequate simulation of environmental conditions in the laboratory for a better performance in subsequent cultivation systems (Urban 2000, Madrones-Ladja 2002, Huo et al. 2014. In this sense, little is known about the embryonic and larval development of M. mactroides in laboratory. Thus, our study describes the morphology and growth during the embryonic and larval development of the yellow clam M. mactroides under laboratory conditions, contributing to develop the technological package for this species.

Laboratory collection and conditioning procedures
Fifty adult specimens of M. mactroides were collected in April 2017 (autumn season), during a low tide in the tidal zone of Mar Grosso beach, in the municipality of São José do Norte, Rio Grande do Sul, Brazil (32°3'10"S 51°59'26"W). The animals were stored in 20-liter containment basins at room temperature and transported to the Laboratory of Marine Molluscs at the Universidade Federal de Santa Catarina, Brazil (LMM -UFSC).
The animals were acclimatized in 5-liter buckets containing sand (3 kg) and transferred to 40-liter tanks with a temperature of 18 °C, aeration and continuous flow of seawater (35 ppt). The diet of the broodstock was a microalgae mix composed of Chaetoceros muelleri, Isochrysis galbana and Rhodomonas salina (1:1:1) at the concentrations of 15 to 20 x 10 4 cellsmL -1 daily. The average total shell length and weight of the broodstock specimens selected for this experiment were 66 ± 3.82 mm and 27.15 ± 4.07 g, respectively.

Spawning induction
At the end of the third week of conditioning, spawning inductions were attempted thorough three different methodologies: two thermal inductions with a gradually raising temperature and an abrupt thermal shock, as well as a stripping technique.
The thermal induction with gradually increasing temperature method consisted of an acclimatization of 40 broodstock specimens in a tank (200 L) with continuous water flow and salinity of 35 ppt to a temperature gradually increasing from 18 to 26 °C during 9 hours. The second method of thermal induction was a shock method that consisted in exposing 40 broodstock specimens acclimated at 18 °C in a 2,500 L tank with mild aeration to an abrupt increase in the temperature (23.5 °C) for 15 hours (overnight). The tanks contained a mesh sieve of 18 µm at the water outlet to retain female gametes.
The spawning was performed using the stripping method, using eight specimens of M. mactroides evaluated under optical microscopy to certify the presence of viable gametes (Helm et al. 2004). The proportion of males and females used in this procedure was 1:1. Prior to the fertilization, the pooled eggs (10,000,000) were sieved and mixed in 15 L of seawater with sperm added (1:7).
The analysis of embryogenic and larval development was proceeded after fertilization. The zygotes obtained with the most efficient spawning method were selected for morphological evaluation.

Embryonic development
After fecundation, the embryos were transferred to a 2,500-liter flat-bottomed tank at the density of 4,000 embryos L -1 , with seawater previously filtered and UV-sterilized, temperature of 25 ± 1 °C, salinity of 32 ppt and mild aeration. Embryos were monitored through their development and morphological analyses determining all embryonic phases lasted until they reached the D-veliger larva stage. Samples were taken at 5-minute intervals in the first hour after fertilization, 30-minute intervals for 8 hours and every 3 hours until completing 24 hours, using a 2 mL pipette and observed under a Sedgewick-Rafter chamber.

Larval development
The D-veliger larvae were transferred to a 4,000-liter flat-bottomed tank, with salinity of 32 ppt and mild aeration. Everyday, the water was completely renewed, the temperature was recorded before and after water exchanges, and the larvae were sieved with mesh sizes ranging from 35 μm at the beginning to 200 μm at the end of cultivation, with samples being collected for morphometric analysis. The larvae were initially fed with 1x10 4 cellsmL -1 of Chaetoceros calcitrans, Isochrysis galbana and Pavlova lutheri (ratio: 0.30: 0.35: 0.35x10 4 cellsmL -1 , respectively), increasing to 2x10 4 cellsmL -1 with the addition of Chaetoceros muelleri (ratio: 0.6: 0.7: 0.35: 0.35x10 4 cellsmL -1 , respectively). The larvae growth was monitored daily by measuring shell length and height of 10 to 20 specimens. After the previous observation verifying survival and motility, samples were fixed in 4% buffered formaldehyde for later analysis under optical microscopy. When all larvae reached the pediveliger stage and were retained at 200-μm meshes, they were ready to settle and the larviculture phase was considered to be completed

Morphometric analyses
Growth averages and standard deviation (± SD) (embryonic phase: diameter, larval phase: larval height and length) and morphology (cell divisions and structural development) were evaluated in living and preserved specimens under the x40 objective on a Carl Zeiss® microscope (Axio Imager A2) and Leica® DM500 (connected with the Leica LAZ EZ software) for embryonic and larval stages, respectively.

Statistics
Height and length of embryos and larvae were analyzed through Generalized Linear Model after the assumptions of normality and homoscedasticity were confirmed. Regression analyses were made to evidence patterns of the data as a function of time. The regression model was selected based on the determination factor (r 2 ). Growth was calculated as a function of the accumulated mean temperature values for each experimental day to evaluate the influence of temperature changes over time on the larval development (Degrees/Day). The analyses were performed using the software GraphPad Prism 6.

Spawning induction
From the three techniques used to obtain embryos, it was observed that both thermal induction with gradual increase of temperature and exposure to thermal shock were unsuccessful. However, the stripping technique showed a positive result for collecting gametes containing a visible germinal vesicle, which confirmed the fertilization.

Embryonic development
The different stages and morphological changes over time of M. mactroides embryos (n = 10 specimens) are shown in Table I and Figure 1. The newly fertilized oocytes had a spherical shape, with a mean diameter of 51.20 ± 6.64 μm (Figure 1a). Twenty minutes after fertilization, the first polar corpuscle was formed (Figure 1b). In the next 3 hours, rapid changes occurred, such as: the formation of the second polar corpuscle, first embryonic division, formation of the 2-cell embryo, and initial divisions until the multi-division phase (Figure 1c-h). Blastula and gastrula appeared within 4 and 5 hours after the fertilization, respectively (Figure 1i, j). Between 8-10 hours, the development of the first cilia was noticeable and the cells began the movements of slight rotation and swimming ( Figure 1k). The first trochophore larvae with active swimming emerged after 18 hours ( Figure  1l) with cilia growing over time, which allowed faster movements compared to the initial stages (Figure 1m, n). The first D-veliger larvae appeared at 24 hours ( Figure 1o).

Larval development
The larval growth of M. mactroides, including mean height and shell length as a function of time, is shown in Figure 2. The sequence of different stages of larval development with the average shell length are shown in Figure  3. The D-veliger stage is characterized by the presence of the first double transparent larval shell and the emergence of velum, which assists in swimming and feeding. Larvae had an initial mean height of 62.55 ± 2.20 μm and shell length of 79.69 ± 3.47 μm, showing a straight hinge and a semicircular shape over time as a characteristic of umbo formation. This semicircle conformation of shells remained until they reached the pediveliger stage, in which the umbo became more prominently pronounced (Figure 3a-f).
The early umbonate larvae were observed on the 8 th day with average shell height and length of 116.24 ± 1.29 μm and 142.59 ± 4.27 μm, respectively (Figure 3c). The larvae at this stage started to become rounded and the umbo was forming. The period between 10 th and 15 th day the shell height and length of larvae had enlarged from 140.03 ± 14.47 μm and 162.87 ± 17.15 μm to 182.47 ± 29.54 μm and 210.99 ± 39.35 μm ( Figure  3d, e). After 25 days pos fertilization, the larvae showed a functional velum and a foot, reached the pediveliger stage ( Figure 3f) with a mean shell height and length of 237.92 ± 19.86 μm and 281.11 ± 27.43 μm. At this stage the larva was able to crawl or swim. Throughout larval development, the spot that characterizes an eyed larva was not observed as a pattern for sand molluscs.
On the 27 th day, the larvae reached a mean height and shell length of 255.94 ± 14.22 μm and 295.14 ± 13.17 μm, respectively, being retained in the 200-μm nylon mesh. The velum was withdrawn and the activity of the foot (dense muscle tissue in the shape of an arrow) was intense, showing a crawling behavior in search for the sediment, which characterizes settlement

Temperature
The temperature of cultivation during the embryonic phase was maintained at 25 °C, while the average temperature during larviculture was 20.0 ± 1.8 °C, with minimum and maximum variations ranging from 16.5 to 24.5 °C ( Figure  2d). Although the experimental temperature was tried to set at 25 °C as an experimental constant, there were environmental variations during the experimental time that produced considerable temperature variations. Calculating the correlation coefficient between the specific growth rate (ln H f -ln H i /t) and the temperature, it was not possible to find a statistical interaction between the strong variables (r = 0.16 p > 0.06). evaluating the possibility of using temperature as a growth correction factor observed within the experiment, the degrees*hour for each experimental day were calculated, a regression analysis was performed that discarded the linear model (r 2 = 0.4 p > 0.05) as a descriptor of the behavior and a quadratic model was applied for the data, which presented the highest adjustment compared with other non-linear models (r 2 = 0.91 p < 0.05; Figure 2e), which rules out correction by degrees*hour as a correction factor and dismisses the range of temperatures presented in this experiment as a significant factor of variation (Stinner et al. 1974).

DISCUSSION
The successful collection of mature gametes during the gonadal stage is the key factor to control spawning and reproduction in the laboratory (Reverol et al. 2004). In a previous study with M. mactroides in Argentina, two spawning events occurred in the spring and summer of one year (Herrmann et al. 2009). Accordingly, the broodstock in our study was collected during the autumn and similarly displayed mature condition for reproduction. This is an advantage for cultivating yellow clams activity. After metamorphosis was observed the development of gill arches and growth lines (Figure 3g). Statistical analyzes demonstrated positive larval development through biometric variables of height and length as a function of time (Figure 2). and rearing juveniles in laboratory, which is also observed by Hooker (1997) with Paphies australis. However, information on what triggers spawning in M. mactroides is still lacking. This is the first study regarding embryonic and larval development of M. mactroides in laboratory, adding essential information about the biology of Mesodesmatidae (Gadomski et al. 2015). Coscarón (1959) provided a partial illustration about the embryonic stage and young larvae specimens from planktonic samples of the environment, although the identification of species was uncertain. In addition, metamorphosis to D-veliger, in that case, did not reach its completion in laboratory. Indirect methods used by Coscarón (1959), such as abundance of the larvae in planktonic samples from the environment can lead to incorrect identification (Hooker 1997), since the identification of larvae by plankton samples is only precise when direct techniques are used, e.g. cultivation of the larvae in laboratory.
Coscarón (1959) observed that the size of mature oocytes ranged from 50 to 55 μm, similar to our results with M. mactroides (mean diameter ± SD of 51.20 ± 6.64; Figure 1a), smaller than other Mesodesmatidae species, which possess a diameter ranging from 60 to 73 μm (Gadomski et al. 2015). Apart from that, information concerning the chronology of events after fertilization, e.g. knowledge about the release time of the first polar corpuscle (Table I, Figure 1b), might be the basis for future studies with the yellow clam, including research related to triploid induction. We also observed the occurrence of metamorphosis into D-veliger stage in 24 hours, which was similar to the embryonic development of other species of Mesodesmatidae ranging from 22 to 45 hours, as described in Table II. The larval development period varied from 17 to 31 days among species from the Mesodesmatidae family and it lasted 27 days in our study (Table II). Morphologically, the larval development of M. mactroides (Figure 3) was similar to the larval description of P. ventricosa (Gadomski et al. 2015) and M. donacium (Ayerbe et al. 2017). This leads to the belief that such characteristics are representative of this family, e.g. the modifications of D-veliger into umbonated larvae, with round shape and an active velum, and the development of pediveliger larvae, with loss of the velum and presence of an initially retracted foot that becomes more active and functional after some time. However, the size of the total height and total length of the shells changed over time among P. ventricosa (Redfearn 1982, Gadomski et al. 2015, P. subtriangulata (Redfearn 1987) and P. australis (Hooker 1997) in the different stages of development (Table II).
The final shell length of pediveliger larvae (295.14 μm) was similar to P. ventricosa (270-300 μm) (Redfearn 1982) and larger than P. subtriangulata (230-260 μm) (Redfearn 1987) and P. australis (264.70 μm) (Hooker 1997) larvae (Table II). A similarity between M. mactroides and P. ventricosa was also observed in the slow development of pediveligers (27d and 22-31d respectively) compared to other representatives of this family (Table II). Gadomski et al. (2015) demonstrated that different temperatures directly affected the rate of embryonic and larval development of P. ventricosa, settling after 22 to 31 days. These authors reported that the rate of larval development depends on countless variables besides temperature, where phylogenetic differences may also contribute to slow or moderate rates of development, possibly assigning these traits to representatives of the Mesodesmatidae family. Mesodesmatidae species generally complete   their larval development in two to 3 weeks, like P. subtriangulata (17-18d), P. australis (18-22d); and M. donacium (23 d) according to Table II. As previously mentioned, temperature is a determining factor for larval development time in bivalve molluscs (Huo et al. 2017). In the present study, the larviculture was performed at room temperature, and it was not possible to gauge the extent to which this variable was decisive at the end of the experiment ( Figure  2), which had a duration of 27 days with live larvae, metamorphosed in pediveliger and ready for settlement. A preliminary descriptive study of the embryonic and larval development of Mesodesma donacium, with temperature controlled at 17 °C, lasted for 28 days with 100% mortality (Carstensen et al. 2006).
Salinity is another limiting factor for the embryonic and larval development of bivalves (Madrones-Ladja 2002) and fluctuations between 14 and 38 ppt affect environments where the yellow clam inhabits (Odebrecht et al. 2010). Although the juveniles and adults of this species are considered euryhaline, tolerating salinities ranging from 15 to 35 ppt (Carvalho et al. 2015b), it is uncertain if this could be applied with the embryonic and larval development in laboratory. In addition, salinity information was not mentioned in studies with other species of Mesodesmatidae. In this sense, we used a salinity of 32 ppt, which is close to the mean values found by Santos et al. (2016) in specific and seasonal analyses in places of occurrence of the yellow clam, which remained adequate until the end of the experiment.
These results provide an understanding of the embryonic and larval development of this species, serving as a basis for future research, e.g. effect of salinity and temperature on the survival and yield of embryos and larvae, as well as studies related to diets, in order to establish a complete technological package for this species.

CONCLUSION
This experiment showed that spawning using the stripping method is efficient for obtaining viable gametes. The distinct phases of embryonic and larval development present in this study demonstrated similar pattern to species from Mesodesmatidae. These findings ensure viability on production of M. mactroides juveniles after the period of 27 days of larval rearing at specific condition of temperature and salinity.