Combined effects of salinity and temperature on survival and growth during the early life cycle of the rock oyster Saccostrea cuccullata (Born, 1778)

Temperature and salinity are among the critical factors affecting the survival and growth of bivalve larvae. The combined effects of temperature and salinity on the embryonic and larval development of the rock oyster Saccostrea cuccullata (von Born, 1778) in culture conditions were investigated in a laboratory study on Inhaca Island, Mozambique. A factorial experimental design tested three temperatures (24, 30 and 34 ºC) and three salinities (30, 35 and 40 parts per thousand) over a seven-day period. Larval survival and growth (in height and length) were assessed by regular sampling by counting and measurement of larvae under an optical microscope equipped with a micrometric scale. Significantly higher larval survival was observed at the combination of 30 °C and 35 salinity. However, the mid-range temperature (30 ºC) and highest salinity (40) resulted in faster growth of the larvae. The lowest temperature (24 ºC) negatively affected growth regardless of salinity level and survival decreased linearly with increasing salinities. The present results will aid in the understanding of the environmental factors behind the natural recruitment of spat of S. cuccullata and contribute to the optimization of rearing protocols for the larval culture of this oyster species.


1999), Mozambique
and India (Tenjing, 2020). In Bangladesh, the decline of the rock oyster beds is leading to the collapse of breakwater reefs (Chowdhury, 2019). It is widely acknowledged that shellfish reefs provide relevant ecosystem services such as coastal protection, nursery grounds to enhance fisheries production, contributing to blue carbon capture, and pollutant removal and detoxification (Nagelkerken et al., 2015;Waltham et al., 2020).
S. cucculata is also an interesting species for aquaculture purposes due to its high commercial value, tolerance to extreme environmental conditions and fast growth. Most recent research and development efforts on S. cuccullata have focused on improving rock oyster natural populations through the development of improved substrates for spat collection and juvenile growth (Racuyal et al., 2016;Chowdhury, 2019).
However, the culture requirements for S. cuccullata during their early life stages are poorly known, which seems to be a major bottleneck for the development of the culture of this species. Major advances thus far on the larviculture of S. cuccullata were the production of embryos (Awati and Rai, 1931), and the description of the embryological development and suitable temperature and salinity conditions for the production of straight-hinge veliger larvae (Kalyanasundaram and Ramamoorthi, 1986). Joseph (1988a, 1988b) provided a general insight on the natural breeding cycle of S. cuccullata, including gonadal maturation and the description of the factors influencing the spawning cycle. Recently Thanormjit et al. (2020) characterized the gametes and early development of

S. cuccullata.
Temperature and salinity are major environmental factors that affect the survival and growth of bivalve molluscs during early life stages (Robert et al., 1988).
Considering the natural occurrence of S. cucculata in the intertidal zone, where environmental parameters are likely to reach extreme values, the current research tested a range of water salinity and temperature combinations to evaluate the most suitable conditions for larval culture of the rock oyster.

Breeding and experimental animals
Adult specimens of S. cuccullata were collected manually with a concave iron tool during low spring tide on the rocky shores of Ponta Torres (32° 57 ' S, 26° 4' E) on the island of Inhaca (Maputo, Mozambique).
During collection, water temperature (26.5 ± 1.0 °C) and salinity (35.5) were measured. The oysters were placed in a waterless plastic bucket and capped with Rachel mesh to minimize sunlight stress and brought to the Inhaca Marine Biology Station (EBMI).
At EBMI, the oyster shells were cleaned using a nylon brush and a knife, washed with distilled water and transferred to a 50 L aquarium with natural seawater at constant temperature (25 ± 2 °C) and salinity (40).
Aeration was continuous and illumination was based on natural light and photoperiod.
Ten oysters were randomly selected and their valves were opened through the sectioning of the adductor muscle (Legat et al., 2017;Santos et al., 2020;Thanormjit et al., 2020). The oysters were then washed with filtered (1 μm) and UV-treated seawater to remove microorganisms lodged in the inner layer of the valves. The sex of oysters was identified by gonad biopsy using an optical microscope (Olympus model CK40; magnification of 40 x).
The gonadal tissue was cut with a scalpel and the gametes were collected using a Pasteur pipette. They were then transferred to a 100 mL beaker (male gametes) and a 1 L container (female gametes) filled with filtered (1 μm) and UV-treated seawater (Absher et al., 2000).
The viability of the gametes was examined under the microscope, i.e., the sperm cells were mobile and the shape of the oocytes became spherical 20 min after being exposed to water. In vitro fertilization was performed adding 2.5 mL of the sperm solution in 1 L of oocyte suspension. Through microscopic observations, the necessary adjustments were made to establish a ratio of about 3 to 5 spermatozoa for each female gamete to avoid polyspermy. Of the 10 sacrificed adult oysters, only four females and two males were used to obtain the required number of gametes for use in the experiment. Each female contained on average 5 to 10 million eggs and males about 2 million sperm. For fertilization, gametes (spermatozoa and oocytes) were evaluated through microscopic examination according to Helm (2004): mature gametes were considered all ovules normally pear-shaped when removed for the first time and round-shaped in contact with sea water within 20 minutes; the sperm was considered mature when it was motile. Embryos of approximately 40-45 μm were transferred to a 10 L tank filled with 5 L of filtered (1 μm) and UV-treated seawater with moderate aeration at the density of 204 embryos mL -1 , estimated using an optical microscope by counting the embryos present in 1 ml. The temperature was set at 28 °C with the use of thermostats (Dophin 50W).
The embryos remained under these conditions until the appearance of D-larvae 24h after fertilization.
Since the time required to reach the D-larvae stage was previously unknown, the stages of embryonic and larval development were observed every five minutes.

Experimental design
Twenty-seven culture tanks each containing 5 L of filtered (1 μm) and UV-treated seawater were prepared about 24 hours after fertilization. Larvae were reared in the combinations of three temperatures (24, 30 and 34ºC) and three salinities (30, 35 and 40), each with three replicates. The D-larvae were directly transferred to those experimental treatments in three replicates per combined temperature x salinity. Approximately 25,000 D-larvae were transferred to each culture tank at an initial density of 5 larvae mL -1 . Water volume in each tank was maintained at 5 L. The salinities of 35 and 30 were obtained by diluting seawater (salinity 40) with distilled water. Moderate aeration was provided using air stones. Every 48 h, tank bottoms were siphoned (50 % water volume exchange) to ensure good water quality. To avoid the elimination of live larvae as well as to select the largest diameter larvae, 60 and 40 μm overlapping meshes were used (smaller mesh over the larger one). A density of 400 cells mL -1 of the microalgae Isochrysis galbana was provided daily to the D-larvae. A reduction of 50 % on the number of microalgae was provided in the days when no water was exchanged. Microalgal density was estimated daily with a Neubauer chamber. Growth was measured daily by sampling 30 larvae according to Hillerbrand et al. (1999). An optical microscope equipped with an ocular micrometric scale (Olympus CK40) was used to measure height (maximum distance between the dorsal and ventral regions) and length (maximum distance between the anterior and the posterior regions) (Hu et al., 1993). The mean (± SD) initial length and height of the larvae were 50.28 ± 0.30 μm and 51.30 ± 0.25 μm, respectively. Survival was estimated as the number of larvae alive at the end of the experiment in relation to the initial number.
Transparent larvae, which presumably indicated the absence of tissues, were considered dead (Ponis et al., 2003). The trial lasted seven days. The data obtained on length, height and survival of oyster larvae were initially tested for normality and homogeneity of variances by the Kolmogorov-Smirnov (distance) test and the Spearman's test, respectively. The data presented a normal distribution and homogeneous variances (p<0.05), therefore they were later submitted to parametric tests. A two-way analysis of variance (ANOVA) was used to verify the influence of temperature and salinity on larval growth and survival. If there were significant differences, the Tuckey test was applied for detailed comparison of the differences (Zar, 2010). At the end of the 7 day-long trial, means (± SD) of length ( Fig. 1) and height (Fig. 2) (Pinto, 1996). This confirms the findings of Kalyanasundaram and Ramamoorthi (1986) that S. cuccullata larvae tolerate a wide temperature and salinity variation though with different growth and survival trends.
Temperature and salinity are among the main environmental factors affecting the growth (Robert et al., 1988;His et al., 1989;Manoj Nair and Appukuttan, 2003) and survival of bivalve larvae (Yuan et al., 2016;Verween et al., 2007;Manoj Nair and Appukuttan, 2003;Robert et al., 1988). In this study, embryos and larvae reached all developmental stages defined by Kalyanasundaram and Ramamoorthi (1986) reported good larval development of S. cuccullata at salinities from 20 to 35. In contrast, Sudrajat (1990) determined an optimum salinity of 25 for embryonic development of S. cuccullata. In general, oyster larvae tolerate a wide salinity range between 25 and 40 (Lemos et al., 1994). However, this study showed that salinity affects the growth of S. cuccullata larvae, being the lowest at salinity 30. It may be possible that osmotic disturbances lead to energy allocations that ultimately impair other functions such as growth (Deaton, 2008).
Temperature is regarded as the most important environmental factor influencing bivalve culture (Robert et al., 1988;Helm et al., 2004). Temperature increments accelerate the growth of oyster larvae (Doround et al., 1999). The highest growth recorded here at 30 °C supports previous observations by Kalyanasundaram and Ramamoorthi (1986) that larval growth of S. cuccullata is higher at 30 °C. Yukihira et al. (2000) reported that the influence of temperature on the metabolism and physiological processes of oysters creates an optimal temperature range for each species providing a maximum rate of growth as well as survival. In fact, a similar trend was observed in the present study, where the maximum temperature tested (34 °C) resulted in reduced larval growth.
The greatest survival (73 %) obtained at the combination of salinity 35 x 34 °C, was higher than the one from the microalgae feeding experiments by Martínez-Fernández and Southgate (2007) in Pinctada margaritifera with 70.5 %. It was also greater than the 50 % larval survival obtained by Mafambissa (2009) for Crassostrea rhizophorae. Other oyster species such as Crassostrea gigas have shown higher larval survival rates of up to 87 % (Ponis et al., 2003).
The results from this study are in partial agreement with Nell and Holliday (1988) who reported higher larval survival of S. commercialis in salinities ranging between 23 and 39. However, the current results differ from those obtained by Coeroli et al. (1984) where they reported higher survival of Saccostrea echinata larvae at salinities ranging from 25 to 30 and temperatures from 25 to 29 °C. Heral and Deslous-Paoli (1990) demonstrated that C. gigas larvae tolerate higher salinities (from 45 to 50) with significant mortality above 50. These differences reflect the genetic variability among different species of oysters and their high adaptability to different environmental conditions.
The lowest larvae survival in the present study (26.4 %) was achieved at salinity 40, suggesting this is a threshold salinity level for S. cucculata larvae.
All treatments at 24 °C resulted in significantly lower survival rates. This seems to be the lower temperature limit tolerated by the larval stages of this species.
Another factor to consider is the abrupt submission of the larvae to low temperatures without any previous acclimation, which may have caused a thermal shock and contributed to a decrease in survival rates.
Opposite to what was previously reported by Kent et al. (1999), the fertilization method used here was very effective as high survival of larvae was observed in treatments where appropriate environmental