Introduction
The fishery of the limpets Patella aspera and Patella candei (Patellogastropoda : Patellidae ) in Madeira surpasses 100 annual tons and concerns about overexploitation of the stocks led to different legislative protective measures
. Recently, the research on the limpets’ aquaculture achieved key methodological advances at hatchery level
, being highlighted the potential role of the crustose coralline algae ( acronym: CCA) to induce the limpet settlement
. This study took a step further to explore the seawater conditioned with CCA as settlement inducer for P. aspera and P. candei.
Material and Methods
The adult limpet s were captured during the breeding period (February to March 2022 ) and kept in captivity using an open system (20 ± 1 °C and 36 ± 1 psu) . The production and culture of larvae used gametes obtained by dissection following the procedures described by . The oocytes were matured artificially using a solution of NH4OH at pH 9 during 20–30 min. Fertilization used 105 sperm cells ml-1. Incubation and larval culture were done in glass beakers and plastic jars at 16 ± 1 °C. Incubation used 50 oocytes ml-1 and lasted 24h. Larval culture used 5 individuals ml-1 and lasted 48h.
The CCA were obtained from adult limpet shells of which the soft body was removed a nd the aperture was cleaned thoroughly following . Then, the shells were broken into pieces that were kept in small containers with filtered seawater treated with UV (acronym: FSS) during four days to obtain the conditioned seawater (acronym: CSW).
Two assays were done for each limpet species (P. aspera and P. candei) : 1) different CSW concentrations (ca. 17, 5, 2 and 0.7 mm2 CCA · ml-1 ) and 2) influence of frozen CSW (-24 °C) obtained from three different CCA communities (Pneophyllum sp., Neogoniolithon sp. 1 and Neogoniolithon sp. 2) . In P. candei , the influence of different treatments applied to the CSW was tested : raw and autoclaved kept at room temperature (0, 24 and 48h), raw kept in the fridge 2–4 °C (24 and 48h), raw frozen at -24 °C (48h) and f iltered (0 h). All the assays used FSS as negative control and CCA as positive control. Settle rs were identified by the loss of the velum (metamorphics ) and the teleoconch development (post-larvae ). The ratio of metamorphics and the ratio of settlers (metamorphics + post-larvae) were calculated.
Results and Discussion
T he ratios of metamorphics and settlers decreased with lower concentration of CSW in both P. aspera and P. candei , but the values were markedly lower in the former (Fig. 1A–B) . Regarding the frozen CSW, only Neogoniolithon sp. 2 induced the settlement in P. aspera , while all the frozen CSW treatments induced the settlement in P. candei . In P. candei , all the treatments applied to the CSW (raw kept at room or fridge temperature, autoclave and frozen) induced the settlement independently of the timing after obtaining the CSW (0 to 48h); being the single treatment without effect the filtered CSW ( glass microfiber 2.7 μm ) (Fig. 1C).
CSW promoted P. candei post larvae settlement in at a ratio positively related to its concentration with the potential to be preserved frozen. In P. aspera an efficient settlement response required the physical presence of CCA. Altogether, these results showed inter-specific differences in the sensitivity to the soluble cues released by the CCA, highlighting that limpet settlement requires to be studied at species level.