Introduction
Digestion in shellfish occurs extracellularly and intracellularly. The main site of intracellular digestion is the digestive cells located in the tubules of the digestive gland. During each digestion process, these cells go through four phases (resting, absorption, degradation, and reconstitution), and the diameter of the lumen of the digestive tubules also changes (Gavrilović, 2011). Determination of the digestive tubule index can indicate a lack of food or unfavorable (stressful) conditions in the culture environment in which filtration is reduced (Winstead, 1995; 1998). Given the rhythmic changes in the digestive cells, sampling should be carried out at shorter time intervals within a day to obtain relevant data. Considering that Ostrea edulis is often grown in areas near estuaries and/or underground fresh water sources, the aim of this research was to determine whether reduced salinity (hypoosmotic) affects food ingestion, i.e. the digestion process to optimize the gonad conditioning process in the hatchery.
Materials and Methods
Immediately before the start of the month-long experiment, two recirculating oyster conditioning systems were set up. Each system was stocked with 200 oysters from a farm in Mali Ston Bay. The basic parameters of the culture medium (oxygen saturation, temperature, salinity and pH) were kept constant and regularly monitored. Salinity in both systems was maintained at 35 psu during the first seven days of adaptation to the new breeding environment. After this period, in the first experimental group it was maintained at the same value, while the second experimental group was gradually acclimatized to 20 psu over the course of seven days. Feeding was carried out by giving an algae mixture in the amount of 3% of the dry mass of the soft oyster tissue. Four species of unicellular algae were used as feed: Isochrysis galbana, Pavlova lutheri, Tetraselmis suecica and Cylindrotheca closterium. The ratio of the listed algae species in the diet was 20%: 20% : 20% : 40%. The required volume of algae suspension was calculated according to Gavrilović et al. (2021). After one month of the experiment, 30 individuals from each system were sampled five times at one and a half hour intervals (total 150 per system). All oysters were opened and the soft tissue was fixed in Davidson fixative for routine histological processing. Hematoxylin-oezoin was used for staining. After microscopic examination, the digestive tubules index was calculated according to the method described by Winstead (1995). The mean value of the tubule index was calculated for each individual and then the mean value for each experimental group. ANOVA test was used to compare the values of tubule condition index at different sampling times of the same experimental group. After this test, the similarity of the mentioned groups was analyzed using MRT (Multiple range test). A statistical comparison of the values of the digestive tubule index between the group kept at 35 psu and 20 psu salinity was performed using the Chi square test.
Results and Discussion
The results of a microscopic examination of the digestive gland of both groups have shown that there is a rhythmical exchange of phases in the digestive tubules (Table 1), where in a single oyster the exchange was not synchronous in all of the digestive tubules, but they were in phases that were close to one another. Depending on the sampling interval all four phases of digestive tubules were found: resting, absorption, degradation, and reconstitution. Within the same experimental group ANOVA test has shown a significant statistical difference between individual sampling intervals (p<0,05), which confirms the existence of a rhythmical exchange in the digestive rhythm in the tubules. The results of a statistical comparison using a CHI SQUARE test showed that there was no significant statistical difference in the values measured between the two salinity groups (χ²<0.711, p<0,05). Winstead (1995; 1998) found that lower salinity affects intercellular digestion in Crassostrea virginica. Horedesky et. al (2019) reported optimal salinity for Crassostrea gasar in the range of 4 to 40 psu. From our results it can be concluded that O. edulis can gradually adapt to a 20 psu salinity, with no significant changes in the feeding rhythm, digestion rhythm and the digestive tubules index regarding the tested salinity value.
References
Gavrilović, A., 2010. Effect of plankton on the morphohistochemical and biochemical properties of the digestive system of the flat oyster, Ostrea edulis (Linnaeus, 1758) from the Bay of Mali Ston. PhD thesis, University of Zagreb.
Gavrilović, A., Jug-Dujaković, J., Ljubičić, A., Iveša, N., 2021. Dizajn i menadžment mrijestilišta školjkaša / Design and management of bivalve hatchery. University Juraj Dobrila of Pula. 130 pp.
Horodesky, A, Castilho-Westphal, G.G., Cozer, N., Gomes Rossi, N., Ostrensky, A., 2019. Effects of salinity on the survival and histology of oysters Crassostrea gasar (Adanson, 1757). Bioscience Journal, 35(2), 586-597.
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Winstead, J.T., 1995. Digestive tubule atrophy in eastern oysters, Crassostrea virginica (Gmelin, 1791), exposed to salinity and starvation stress. J. Shell. Res. 14 (1), 105-111.