INTRODUCTION
Pyropia is an economically important and widely cultivated seaweed in Asia, such as Korea, Japan, and China (He et al. 2021, Kim et al. 2022, Zuccarello et al. 2022, Zhong et al. 2023). The main cultivated Pyropia species are P. tenera, P. yezoensis, P. seriata, P. dentata, and P. haitanensis (Hwang et al. 2005, Luo et al. 2014, He et al. 2023, Kim et al. 2023). In Korea, four Pyropia species except P. haitanensis, are cultivated, but P. tenera is no longer farmed due to its low productivity and fertility (Hwang et al. 2010). As commercial production continues to grow, Pyropia is increasingly used not only as food but also in medicine and the chemical industry (Harada et al. 1997, Choi et al. 2016, Oh et al. 2018, Park et al. 2023, Wang et al. 2023).
In Korea, the Pyropia aquaculture industry has maintained its production level of 507,362 to 575,634 tons (wet weight) and economic value of US$332–400 million over the past three years (2021–2023). For the 2024 period (from September 2023 to May 2024), the production is similar to the previous year, but the economic value has increased by about twofold, reaching US$882 million. The Korean Pyropia industry grew rapidly, with exports increasing from US$100 million in 2010 to US$790 million in 2023 (Ministry of Oceans and Fisheries 2024), which was one of the reasons for the increase in unit price. The government aims to achieve US$1 billion in exports by 2027. To ensure the stability, sustainable growth, and increased exports of the Pyropia industry, aquaculture productivity must improve. Since Pyropia aquaculture relies on the seed industry (both free-living and shell-living conchocelis), improvements in seed production and management technology could lead to higher yield (Stekoll et al. 1999, Jo et al. 2013). However, seed production for Korean Pyropia species is inefficient due to the lack of standardized manuals. Some farmers still rely on the Japanese seed production technology for Pyropia yezoensis, which is not fully applicable to different species in Korea (Kawamura 2017) while others manage nursery productionbased on their own experience. For example, in P. dentata, the efficiency of artificial seed production using free-living conchocelis (FL-conchocelis), introduced to Korea in the 1970s, remains low. As a result, most farmers produce seeds using carpospores obtained from mature thalli (Kim et al. 2016). For the sustainable production and supply of Pyropia seeds, there is an urgent need for systematic and advanced cultivation and management technologies at the seed production stages, including FL-conchocelis, shell infiltration, and the growth and maturation of shell-living conchocelis (SL-conchocelis).
Although extensive research has been conducted on FL-conchocelis in the Pyropia seed industry (Notoya et al. 1993, Tang and Fei 1997, Kim 1999, 2000, Sahoo et al. 2006, Li et al. 2011, Redmond and Yarish 2011, López-Vivas et al. 2015, Jung et al. 2017), studies on SL-conchocelis are still very limited (Heo et al. 2021). Heo et al. (2021) reported that P. dentata had a lower shell infiltration efficiency compared to P. yezoensis and P. seriata under the same experimental conditions. They also suggested that further research is necessary to explore the physical factors that could affect the shell infiltration process of FL-conchocelis to improve the infiltration efficiency. Additionally, studies on these physical factors may be limited to a Japanese study that examined the number of holes perforated by FL-conchocelis (Kawamura 2017).
The objectives of the present study are to identify the optimal environmental conditions for shell infiltration in three major cultivated Pyropia species in Korea, and to determine the effects of physical factors, including quantity, fragment length, and cell length of FL-conchocelis on shell infiltration. This study will provide critical information for establishing optimal criteria for effective FL-conchocelis shell infiltration, thus contributing to the stable seed production of Pyropia species.
MATERIALS AND METHODS
Effects of temperature and light intensity on shell infiltration density
The present study was conducted using FL-conchocelis of three Pyropia species, P. yezoensis (SRI-R00171), P. seriata (SRI-R00251), and P. dentata (SRI-R00033) from National Fishery Bio-resources Bank of the Seaweed Research Institute at National Institute of Fisheries Science, Korea. Before the experiment began, FL-conchocelis of three Pyropia species have been cultivated within a temperature range of 20–25°C and at light intensity of 40 μmol photons m−2 s−1 and photoperiod of 14 : 10 h L : D (light : dark).
To determine the effects of temperature and light intensity on shell infiltration of FL-conchocelis, three pieces of oyster shell fragment (1–2 cm2) were arranged in a Petri dish (Ø 5 cm) with the prismatic layer facing upward. Then, 10 mL of the FL-conchocelis stock solution (conchocelis 0.1 g in 50 mL of seawater) was diluted to a 100-fold by adding autoclaved seawater and inoculated, as described in Heo et al. (2021). This experiment was conducted under a photoperiod of 14 : 10 h L : D with different combinations of two temperatures (20 and 25°C) and four light intensities (1, 10, 40, and 80 μmol photons m−2 s−1) (n = 3). Infiltrated conchocelis densities (No. of conchocelis cm−2) were calculated by counting under an optical microscope (BX51; Olympus, Tokyo, Japan) after 7 d of cultivation.
Determination of optimal quantity and fragment length of FL-conchocelis for infiltration
To determine the effect of conchocelis amount (wet weight, mg) on shell infiltration, FL-conchocelis were cut into small pieces, 200–400 μm in length, using a blender and then different amount of cut conchocelis (100, 150, 200, 250, and 300 mg wet weight) were sprayed onto oyster shells. This experiment also followed the infiltration methods of Heo et al. (2021). The samples were cultivated at a temperature of 20°C, a light intensity of 40 μmol photons m−2 s−1 and a photoperiod of 14 : 10 h L : D for 7 d (n = 3). The densities of SL-conchocelis (No. of conchocelis cm−2) were assessed using an optical microscope (as detailed above).
To investigate the optimal fragment length of FL-conchocelis for shell infiltration, five different lengths of conchocelis were tested. FL-conchocelis of each Pyropia species were blended at 8,000 rpm for various durations: 15, 25, 40, 60, and 80 s, respectively. After blending FL-conchocelis, average length of the conchocelis fragments for the three Pyropia species was 108.79 ± 1.81 μm (100 μm, 80 s), 209.13 ± 4.85 μm (200 μm, 60 s), 324.39 ± 3.62 μm (300 μm, 40 s), 411.48 ± 4.51 μm (400 μm, 25 s), and 519.74 ± 3.65 μm (500 μm, 15 s). This experiment also followed the infiltration methods of Heo et al. (2021). The samples (n = 3) were cultivated at the same conditions as described above, and the densities of SL-conchocelis were also determined following the above described method.
Observation of shell infiltration of FL-conchocelis in Pyropia yezoensis
Shell infiltration of FL-conchocelis was observed only in P. yezoensis. Fragments of conchocelis burrowing into the inner layers of oyster shells were observed using both an optical microscope and a Hitachi FE-SEM (field emission scanning electron microscope, S-4300; Hitachi, Tokyo, Japan) to examine the shell infiltration process of FL-conchocelis. The FE-SEM analysis required several pre-treatment steps: oyster shells having SL-conchocelis were fixed in 4% glutaraldehyde in a 0.6 M phosphate buffer at room temperature for 12 h. The fixed oyster shells were then washed in distilled water for 1 h, dehydrated in a graded ethanol series (10, 30, 50, 70, 90, and 100%, each for 10 min), and stored in 100% alcohol. The oyster shells fixed in 100% alcohol were then mounted on SEM stubs using carbon tape and sputter-coated with platinum before being examined under the FE-SEM (Jeon et al. 2018).
Statistical analysis
A two-way and three-way analysis of variance (ANOVA) were used to test the effects of environmental and physical factors on shell infiltration of FL-conchocelis for the three Pyropia species. When significant differences were detected between means (p < 0.05), Turkey’s honestly significant difference test was conducted (Sokal and Rohlf 1995). Prior to analysis, data homogeneity was tested using Cochran’s test of homogeneity of variance. Statistical analysis was conducted using SPSS version 20.0 (IBM Corp., Armonk, NY, USA).
RESULTS
Effects of temperature and light intensity on shell infiltration density
Shell infiltration densities of FL-conchocelis were significantly affected by species and light intensity (three-way ANOVA, p < 0.05) (Table 1). Combined effects of Pyropia species and light intensities were also significant in SL-conchocelis densities (p < 0.05), but the combined effect of species and temperatures or temperatures and light intensities were not significant. Each species showed variable infiltration densities, which varied according to the light intensities. At 20°C, P. yezoensis showed an increase in infiltration densities with increasing light intensity, with the highest density at 80 μmol photons m−2 s−1 (p < 0.05) while other two species exhibited no significant differences in different light intensities (Fig. 1A). At 25°C, P. yezoensis showed the highest infiltration density of 87.04 ± 13.12 conchocelis cm−2 at 40 μmol photons m−2 s−1, P. seriata 107.22 ± 12.92 conchocelis cm−2 at 80 μmol photons m−2 s−1, and P. dentata 48.72 ± 5.54 conchocelis cm−2 at 10 μmol photons m−2 s−1 (Fig. 1B). The conchocelis infiltration density showed significant differences between species (p < 0.001) and light intensity (p < 0.01), but the pattern of change according to light intensity was not consistent.
Determination of optimal quantity and fragment length of FL-conchocelis for infiltration
The quantity of FL-conchocelis significantly affected the conchocelis infiltration density (p < 0.001) (Table 2) with density increasing as the quantity increased (Fig. 2). The shell infiltration density according to the quantity of FL-conchocelis ranged from 44.81 to 146.15 conchocelis cm−2 in P. yezoensis, 51.40 to 190.49 conchocelis cm−2 in P. seriata, and 22.24 to 92.49 conchocelis cm−2 in P. dentata (Fig. 2). The conchocelis infiltration density was significantly lower in P. dentata compared to the other two Pyropia species (p < 0.001) (Fig. 2). Under the same culture conditions, P. seriata had the highest infiltration density, followed by P. yezoensis, and P. dentata. The results of Tukey HSD test presented significant differences in infiltration densities were observed among these groups: large (300 and 250 mg), medium (200 and 150 mg), and small (150 and 100 mg) (Table 2). Also, three Pyropia species showed a positive correlation between the conchocelis quantity and the shell infiltration density (p < 0.001) (Fig. 2).
Free-living conchocelis length also significantly affected shell infiltration densities (p < 0.001) (Table 3). Conchocelis infiltration densities were lower in P. dentata (22.02–49.67 conchocelis cm−2) than in P. yezoensis (55.40–131.91 conchocelis cm−2) and P. seriata (48.32–95.60 conchocelis cm−2) (p < 0.001) (Fig. 3). Shell infiltration densities were significantly affected by the combination of species and blending times (p < 0.001) and also between the combinations of species and conchocelis lengths (p < 0.01) (Table 3). The optimal conchocelis length to maximize infiltration density of FL-conchocelis was 300 μm with a blending time of 40 s for both P. yezoensis and P. seriata, and 300–400 μm (25–40 s) for P. dentata (Fig. 3). In all three species, FL-conchocelis length of 300 μm (with a blending time of 40 s) resulted in the highest conchocelis infiltration densities (Table 3).
Shell infiltration of FL-conchocelis in Pyropia yezoensis
P. yezoensis conchocelis fragments ranging from 200–400 μm in length were inoculated onto oyster shells to induce conchocelis shell infiltration (Fig. 4A & B). The conchocelis fragments penetrated the shell surface at the contact area and began to grow both inside and outside the shell (Fig. 4C–E). The microscopic FL-conchocelis that infiltrated the shells became visible to the naked eye as they developed into SL-conchocelis seeds (Fig. 4F). A smooth surface texture of the oyster shell (Fig. 4G) was observed through FE-SEM, with the shell infiltrating conchocelis growing on it (Fig. 4H & I). Under FE-SEM, the conchocelis were measured at 1.5–2.0 μm in diameter (Fig. 4I) and were observed infiltrating through pores (Ø 2–4 μm) formed on the shell surface (Fig. 4J & K).
DISCUSSION
The optimal conditions for shell infiltration of Pyropia FL-conchocelis slightly differed among the three Pyropia species: 20–25°C and 5–80 μmol photons m−2 s−1 for P. yezoensis, 20–30°C and 20–80 μmol photons m−2 s−1 for P. seriata, and 20–25°C and 20–80 μmol photons m−2 s−1 for P. dentata (Heo et al. 2021). However, the highest shell infiltration densities of FL-conchocelis were observed under temperatures of 20–25°C and light intensities of 40–80 μmol photons m−2 s−1 in all three species. In this study, a positive relationship between conchocelis infiltration density and light intensity was observed in the range of 1–80 μmol photons m−2 s−1 at 20°C, but no similar responses were found at 25°C. These results indicate that shell infiltration of FL-conchocelis is influenced by temperature, light intensity, and their interaction with variations among the three Pyropia species. Recently, Pyropia seeding technology has become critical, and many studies have been conducted to determine conchocelis shell infiltration conditions (Heo et al. 2021) and infiltration mechanisms (Wang et al. 2020).
The optimal growth temperature for each species of Pyropia conchocelis was confirmed to be 18–25°C for P. yezoensis (Ren et al. 1979, Wang et al. 2000, Zhou et al. 2007, Li et al. 2008), 15–20°C for P. seriata (Notoya et al. 1993, Kim and Notoya 2004), and 15–25°C for P. dentata (Notoya et al. 1993, Kim 2011, Kim et al. 2019), which were slightly lower than the optimal shell infiltration temperatures. In particular, in the case of P. seriata, the optimal growth temperature (15–20°C) and infiltration temperature (20–30°C) showed a difference of 5–10°C. This is related to the fact that conchocelis is active at higher temperatures because shell infiltration, growth, and maturation of conchocelis occur in summer (Blouin et al. 2011, Watanabe et al. 2014). Shell infiltration of FL-conchocelis for SL-conchocelis production in Korea is carried out late March to early April (10–13°C) which is different from the results of this study. This result suggests that the optimal infiltration temperature for each cultivated Pyropia species should be considered to increase shell infiltration efficiency.
All species showed a similar pattern of shell infiltration responses to temperatures and light intensities, with P. dentata showing the lowest shell infiltration efficiency. In the subsequent experiment investigating the effects of quantity and fragment length of conchocelis on the shell infiltration density, the shell infiltration efficiency at 150 mg of P. yezoensis and P. seriata conchoclies was similar to that at 250 mg of P. dentata. This result suggests that P. dentata may require at least 1.5 times more cochocelis than the other two species to achieve the same level of shell infiltration efficiency. The blending time of conchocelis showed a different pattern in P. dentata compared to the other two species. P. yezoensis and P. seriata showed the maximum number of conchocelis infiltration at the conchocelis length of 300 μm (40 s blending time) while P. dentata showed peaks at 300–400 μm (25–40 s). Blending time affects the fragment lengths of conchocelis, resulting in different numbers of conchocelis cells. The cell length of FL-conchocelis from five strains of each of the three Pyropia species collected from National Fishery Bio-resources Bank of the Seaweed Research Institute at National Institute of Fisheries Science were measured (unpublished data). The conchocelis cell length was measured at 44.45 ± 2.96 μm (the average ± standard error, n = 150) in P. yezoensis, 46.45 ± 1.55 μm in P. seriata, and 68.44 ± 3.08 μm in P. dentata, indicating that cell length is longer in P. dentata than in the other two species (Figs 5 & 6). A conchocelis fragment length of the three Pyropia species was ca. 300 μm (blended for 40 s), which is the optimal conchocelis length (or blending time) for P. yezoensis and P. seriata. Five to seven conchocelis cells with a length of 45 μm were observed in P. yezoensis and P. seriata, while P. dentata showed 3–5 cells with a length of 65 μm. This result suggests that P. dentata may require less bending time (ca. 25 s) to produce 5–7 cells, the optimal cell number for infiltration, resulting in a total conchocelis length of ca. 400 μm. It is important to note that the blending time required to produce 5–7 cells may vary depending on the blending speed (rpm) and quantity of conchocelis.
This study confirmed the conchocelis shell infiltration of P. yezoensis using FE-SEM. We observed that the blended FL-conchocelis grew after infiltrating holes with diameters of 2–4 μm in the oyster shells, whereas Wang et al. (2020) reported holes with diameter of 6–10 μm in mollusk shell. Our experiment was specifically designed to examine the size of infiltration holes using oyster shells. However, Wang et al. (2020) did not provide detailed information about their experimental design and materials (e.g., types of shells used such as oyster or other mollusk shells). Consequently, a direct comparison between the findings of Wang et al. (2020) and this study is not possible. Nevertheless, the difference in infiltration hole diameter may be attributed to variations in shell materials (e.g., oyster, clam, or other mollusk shells), the growth stage of FL-conchocelis, shell properties, or pre-treatment methods for SEM analysis. The number of cells infiltrated increased depending on the area where the cells made contact with the oyster shell, and the holes were maintained throughout the conchocelis stage on the shell. Conchosporangia, the most important stage of Pyropia seed production, release conchospores after the formation of release tubes on the surface of the shell through growth and maturation (Kawamura 2017). This suggests a relationship between the perforation formed during FL-conchocelis shell infiltration and the subsequent formation of the conchosporangia, which requires further investigation.
In conclusion, we found that the optimal infiltration conditions of FL-conchocelis of all three Pyropia species were 20–25°C and 40 μmol photons m−2 s−1, which were similar to the optimal growth conditions of FL-conchocelis. However, under the same conditions, P. dentata had a lower number of conchocelis infiltrated than P. yezoensis and P. seriata. This appears to be due to the difference in FL-conchocelis fragments depending on the difference in cell length. Therefore, to increase the infiltration efficiency of P. dentata, the blending time may be shortened and the amount of conchocelis added may be increased by 1.5 times. The cell length of Pyropia FL-conchocelis can also vary depending on the culture methods (stationary vs. aeration culture) and strains (Redmond et al. 2014). Therefore, it is critical to measure the length of conchocelis cells before blending to optimize the infiltration rate. This study identified the key conditions to be considered for conchocelis infiltration by examining various environmental and physical factors. Future study should focus on verifying limiting conditions such as salinity, desiccation and temperature levels (2–3°C intervals) to enhance shell infiltration density and address the limitations of previous studies.
