3. RESULTS
3.1. General features of the minute tubercles on R. pseudosericeus larvae
The larvae’s minute tubercles were observed in three sites: EHR, WLP, and PR; the different heights of the tubercles at each region are shown in Table 1 and Figures 4, 5. The shape and degree of development of the minute tubercles clearly differed among regions. Two types of tubercles were found: hemispheric and vestigial-shaped. The minute tubercles on the surface of EHR and WLP were of a well-developed hemispheric shape, whereas those on the PR had a shrunken and flattened vestigial shape. Immediately after hatching, the larvae had many minute tubercles on their surface, and the tubercles’ distribution and development changed with larval growth and development.
The hemispheric minute tubercles on the EHR and WLP were observed immediately after hatching. Their height gradually increased from day 1 after hatching, and the highest values were found on the WLP on day 7 [11.4 ± 2.0 µm (6.9–17.1)]. The height of the tubercles rapidly decreased approximately 60% from day 8 to day 10. The height of the tubercles continuously decreased from day 11 on, and on day 24, unidentifiable small protuberances were observed only around the eyes. No tubercles were observed on the epidermis of the larvae from day 27 on. The minute tubercles were mostly hemispheric but slightly inclined towards the posterior region; they were also denser and higher on the WLP than on the EHR. The minute tubercles on the posterior region were first observed on day 6, and they remained vestigial until day 11, when they became shrunken or flattened.
3.2. Larval migration inside the mussels
The changes in larval position in the mussels at each larval developmental stage are shown in Table 1 and Figure 4. The spawning ovipositor of R. pseudosericeus entered the gill demibranch (U and M parts) or suprabranchial cavity (L part) of the mussel through the exhalant siphon (Figures 1, 2), and the position of the larvae varied according to developmental stage. Changes in the position of the larvae per developmental stage were different before and after day 11 day, when larval migration from the gill demibranch to the suprabranchial cavity was first observed. Until day 10, the larvae were found in the interlamellar space of the demibranch (U and M parts, prevalence 100%, n = 110); from day 11 on, the larvae were more common in the suprabranchial cavity (L part; prevalence 92.4%, n = 134) than in the gill demibranch (U and M parts; 7.6%, n = 11). The larvae in the L part were faster than those found in the M and U parts at the same time. All the larvae found in the gill demibranchs and suprabranchial cavities had their heads facing the direction opposite to the exhalant and inhalant siphons.
3.3. Relationship between the height and position of the minute tubercles and the morphological and physiological characteristics of the larvae
The position of larvae inside the mussels and the larvae’s external morphological and physiological characteristics were closely related as larval development progressed (Table 1; Figures 4, 5). The changes in height of the minute tubercles were divided into six stages: formation, growth, peak, abrupt reduction, reduction, and disappearance.
3.3.1. Formation stage (from hatching to day 1 after hatching)
During this stage, the EHR and WLP of the larvae were already covered with hemispheric minute tubercles. On day 1, the heights of the tubercles on the EHR and WLP were 2.6 ± 0.5 µm (1.8–4.1) and 4.8 ± 1.7 µm (2.3–8.9), respectively; i.e., tubercles on the WLP were larger than those on the EHR. Tubercles on the PR of the larvae were not observed at the stage.
No larval migrations were detected immediately after hatching, but a pair of WLP, which were small and started to develop on the dorsal and ventral regions, were identified. The fin-fold of the caudal region was very small at this stage. Moreover, all larvae were found in the M part of the mussels.
3.3.2. Growth stage (day 2 to day 5 after hatching)
At this stage, the minute tubercles developed very rapidly, reaching approximately twice the size compared to the previous stage, and was abundant on the EHR and the WLP. Their heights on the EHR and WLP were 3.3 ± 1.0 µm (1.9–5.6) and 5.9 ± 1.4 µm (3.2–8.8) on day 2 and 4.2 ± 0.9 µm (2.7–7.1) and 7.5 ± 1.5 µm (4.5–12.0) on day 5, respectively. Vestigial minute tubercles began to appear on the posterior region, but were still very small.
On day 4, the larvae’s head developed slightly anterior to the egg yolk and the tubercles on the dorsal and ventral regions greatly developed. Between days 2 and 5, larvae were found only in the U and M parts of the mussels.
3.3.3. Peak stage (day 6 to day 7 after hatching)
The tubercles’ height was the highest at this stage (reaching approximately thrice the size compared to the formation stage), and their density on the EHR and WLP was very high. The heights of the tubercles on the EHR and WLP were 6.0 ± 1.3 µm (4.0–9.5) and 9.7 ± 1.8 µm (6.0–13.6) on day 6 and 6.6 ± 1.5 µm (4.2–11.0) and 11.4 ± 2.0 µm (6.9–17.1) on day 7—when they reached their peak heights—, respectively. Vestigial minute tubercles on the PR were first observed during this stage, but they were still very small and flat.
The larvae began to form eyes, and their heartbeat could be observed under their heads. Red blood circulation could be seen in front of the yolk, and the epidermis on the dorsal side began to shrink slightly, with the yolk lengthening backwards. No larval migration was observed, and the larvae were only found in U and M parts of the mussels.
3.3.4. Abrupt reduction stage (day 8 to day 10 after hatching)
At this stage, the minute tubercles on the the EHR and WLP became drastically smaller and shorter than in the previous stage. On day 8, the height of the tubercles on the EHR and WLP decreased to 4.7 ± 0.9 µm (2.8–7.0) and 9.0 ± 1.5 µm (5.2–12.9), respectively; and on day 10, it rapidly decreased to 2.4 ± 0.5 µm (1.7–3.8) and 4.3 ± 0.6 µm (3.1–5.7), respectively, reaching a height similar to that of the formation stage.
At this stage, the development of the lens in the larvae’s eyes was completed, their heart components were clearly differentiated, and the caudal fin began to develop. The tubercles on the dorsal region were significantly contracted and shortened. The larvae remained in the demibranchs, and no migration was observed in the suprabranchial cavity. The larvae were only found in the U and M parts of the mussels.
3.3.5. Reduction stage (day 11 to day 26 after hatching)
At this stage, the tubercles at all sites were smaller than in the previous stage. On day 11, the heights of tubercles on the EHR and WLP were 1.7 ± 0.3 µm (1.2–2.6) and 3.3 ± 0.4 µm (2.2–4.4), respectively. On day 24, some larvae without minute tubercles were found. On day 26, almost all minute tubercles had disappeared, only traces of them were left.
The pectoral and caudal fins of the larvae developed at this stage, and their eyes became clear and silver brown. Their heads, with complete upper and lower jaws, markedly developed. Their color darkened as the melanin pigment expanded, and their air bladders became complete, with two parts and a slightly larger front. The tubercles on the anterior side completely reduced, followed by the reduction of those on the dorsal side; some parts of the yolk remained. Most of the larvae were found in the suprabranchial cavity. From day 24 on, free-swimming individuals were found in the experimental tanks; the larvae that had remained inside the mussels also swam freely when removed from the mussels. A total of 2.3%, 6.2%, and 91.5% of the larvae were found in the U, M, and L part of the mussels, respectively.
3.3.6. Disappearance stage (day 27 to free-swimming larvae)
At this stage, only parts of minute tubercle were observed and only in some larvae. The pectoral, ventral, and caudal fins of the larvae were completely developed, the mouth and anus were open, and the yolk sac was completely absorbed. Larvae were found only in the L part of the mussels.
3.4. Host mussel utilization by R. pseudosericeus larvae
No significant differences in shell length were found between the mussels that had larvae (53.66 ± 6.65 mm; range, 38.42–68.82; n = 85) and those that did not (54.05 ± 7.20 mm; 40.10–69.96; n = 65; two sample t-test; t148 = 0.341, P = 0.734). The number of larvae inside mussels was 3.01 ± 2.27 (range, 1–13; n = 127). The number of larvae in the left outer, left inner, right inner, and right outer gills of the mussels was 1.89 ± 1.17 (1–5; n = 56), 1.30 ± 0.67 (1–3; n = 10), 1.00 ± 0 (1; n = 5), and 2.36 ± 1.92 (1–10; n = 56), respectively (Figure 6A); no significant difference among the four demibranchs was found (Kruskal-Wallis H test, P = 0.148; Figure 6A).
A total of 49, 30, 6, and 0 larvae were found in one, two, three, and four parts of the mussels’ gills. The frequency of appearance of larvae in the left outer, left inner, right inner, and right outer gills of the mussels was 44.09% (n = 56), 7.87% (n = 10), 3.94% (n = 5), and 44.09% (n = 56) among each of the four demibranchs (Figure 6B). The larvae were significantly more frequent in the two outer demibranchs than in the inner demibranchs (Kruskal-Wallis H test, P< 0.001; Figure 6B).