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).