Introduction
The larval settlement is one of the trickiest stages during the development of many benthic marine invertebrates due to the complex processes of interaction between biotic and abiotic factors operating at different spatial and temporal scales. Such a process interests the larval cycle of Holothuroidea species that is characterized by three main stages, a feeding planktonic auricularia, a non-feeding planktonic doliolaria, followed by the settlement into pentactula, which feeds on the substrate (Rakaj et al., 2018). Such a critical step is also reflected in the rearing of sea cucumbers, where the low settlement success and the low survival rate of post-settlers represent the bottleneck to the feasibility of commercial-scale production. Several cues have been used to stimulate the metamorphosis of sea cucumbers, including natural biofilms, the addition of seagrasses extract, and the conditioning of settlement plates with diatoms biofilms (Agudo 2006; Rakaj et al., 2018).
In order to improve the information on the role of algal biofilm in the induction of larval metamorphosis, we investigate the effect of different biofilms on the settlement success of the doliolaria of Holothuria tubulosa and we also provide information on the growth of juveniles reared for two years in a pilot-scale hatchery.
Materials and methods
The metamorphosis of doliolaria larvae of H. tubulosa was tested with three treatments: the diatom Amphora spp.; the green algae Ulvella lens and the Control (no biofilm). The experimental design considered multiple chamber plates (6 chambers each plate; 4 replicates each treatment) with a volume of 3 ml for each chamber. Before the trials, the plates were conditioned with the respective biofilms.
The larvae used in this study were bred in three 150-L tanks (1 larva ml-1) and were fed with the pabulum made of a mix of the microalgae Chaetoceros calcitrans, Isochrysis galbana, and Tetraselmis suecica. The larvae reached the doliolaria stage after 25 days post-fertilization. From this batch of larvae, 72 doliolaria were transferred individually in the trial chambers that were kept at 24°C with a photoperiod of 12h light and dark. The progress of the settlement was followed under a stereomicroscope, every five days, by observing the morphological changes from doliolaria to pentactula. When the second posterior podium appeared, the specimens were considered juveniles. The settlement success was calculated as the ratio of the number of settlers (pentactula and juveniles) to the number of initial larvae, expressed in percentage. The settlement performance was tested at the end of the experiment after 35 days. Prior to the one-way ANOVA (P < 0.05), Cochran’s C-test (P>0.05) was used to check the assumption of the homogeneity of variances. Post-hoc multiple comparisons were performed using the Tukey HSD test. The remaining doliolaria larvae of the batch described above (ca. 60000), were transferred in three 500-L tanks provided with Amphora spp. biofilms. For the following two months, larvae were fed with diatoms ad libitum. After this period, larvae were fed three times a week with spirulina (Arthrospira platensis) mixed with fine sand and a commercial feeding powder (at a feeding rate of 3% of juveniles’ weight). Every three months sea cucumbers were visually counted for survival rate determination and a subsample of 10 juveniles was randomly collected from each tank, measured (total length in mm), and weighed (total wet weight in g) for growth parameters determination.
Results
The settlement success at the end of the experiment was about 21%±12.5se with Amphora spp., and 54%±12.5se with U. lens, which was the only treatment significantly different from the Control. Moreover, the treatment U. lens showed the highest post-settler survival, with 25% of juveniles, while in the treatment Amphora spp. only 4% of the larvae reached the same stage. The percentage of swimming doliolaria decreased with the progression of the settlement, except in the Control plates, where at the end of the experiment, 62% of the doliolaria were still present.
The larvae transferred in the pilot-scale hatchery showed survival of about 1% after seven months post-fertilization, with the production of ca. 600 juveniles, of which, 50% survived after 15 months post-fertilization and showed a mean weight of 6.8g ±3.0sd, a mean length of 41mm± 10.4sd.
Discussion and conclusion
Sea cucumbers larvae bred in captivity are generally induced to settle on natural biofilms obtained by immersing the plates in diatoms cultures for a few days or adding an extract of Sargassum sp. or seagrass leaves (Agudo 2006, Rakaj et al., 2018). Our results showed that all the plates conditioned with algal biofilm allowed the settlement of the doliolaria and its metamorphosis into pentactula. The best settlement performance was achieved with U. lens, which allowed the metamorphosis of the 54% of the larvae into pentactula, confirming the high performance of this alga in fostering the settlement of echinoderms larvae (Hannon et al., 2017). It is particularly interesting to observe that larvae that survived in the Control plates were still in the doliolaria stage for the whole duration of the experiment. This fact indicates that the presence of some sort of cue is a necessary condition to proceed with the metamorphosis of H. tubulosa and that larvae are able to delay metamorphosis when no substrate is available. These results could be explained by the Desperate Larva Hypothesis, revised to be applicable to the non-feeding stage of the larval cycle (Botello and Krug, 2006). According to this hypothesis as the non-feeding stage continues, larvae became “desperate” to settle and the level of selectivity towards the substrata decreases. Indeed, the lower number of juveniles in the treatment with Amphora spp. (4%) can indicate that while the cue seems to be enough to induce the settlement, Amphora spp. alone, might not be able to ensure high survival of the post-settlers and their growth.
The results provide practical applications increasing the efficiency of the current rearing protocols for sea cucumbers. In fact, we reported the first record of growth-out of juveniles at a pilot-scale hatchery for H. tubulosa.
References
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