4. DISCUSSION
This study investigated the relationship among three factors (larval
developmental stage, minute tubercle height, and position of the larvae
inside the host mussel) (Table 1; Figures 4, 5), and three main results
were found. Firstly, the minute tubercles were concentrated on EHR and
WLP of the Rhodeus bitterling larvae, being more developed in the
latter region than in the former. Second, during the formation, growth,
peak, and abrupt reduction stages, larval development occurred in the
interlamellar space of the demibranchs (U and M parts); larval migration
to the suprabranchial cavity (L part) occurred only during the reduction
stage, when the minute tubercles became shorter (Figures 5, 6). Thirdly,
when the larvae migrated to the suprabranchial cavity, morphological and
physiological changes regarding their locomotion ability were apparent;
in fact, individuals that migrated to the suprabranchial cavity clearly
developed faster than those that remained in the demibranches.
Two types of
minute
tubercles of R. pseudosericeus larvae were found: hemispheric and
vestigial-shaped. Among the Rhodeus bitterlings, R.
atremius , R. suigensis , R. ocellatus , and R. o.
smithi have only hemispheric minute tubercles, whereas R. uyekiiand R. pseudosericeus have both hemispheric and vestigial-shaped
tubercles (Suzuki & Hibiya, 1984a, 1984b; Suzuki & Jeon, 1988b; Suzuki
et al., 1985). The minute tubercles are unique features in bitterlings,
and as wing-like projections exist in Rhodeus bitterlings but not
in Acheilognathus and Tanakia , they were used as taxonomic
characteristics to differ among acheilognathinae genera (Kim, 1982,
1997). Suzuki & Hibiya (1984a, b) proposed the existence of three types
of yolk projections in bitterlings, and Rhodeus was considered to
have type-C projections, which was confirmed for R.
pseudosericeus in the present study. Suzuki & Jeon (1987) reported
that the type and morphology of these minute tubercles change over time
and from species to species. In the present study, R.
pseudosericeus larvae with two types of minute tubercle shapes similar
to those of R. uyekii larvae were found; however, R.
pseudosericeus larvae had a high concentration of hemispheric tubercles
only on the EHR and WLP, whereas R. uyekii larvae developed these
tubercles throughout most of their PR (Suzuki et al., 1985). The two
species are very similar not only in the shape of the wing-like
projection and the egg yolk during the development and disappearance
stages, but also in the morphology of the adult fish; however, there
were differences in the developmental area and height of the epidermis
(Kin et al., 2006). Moreover, R. pseudosericeus eggs are not
sticky and are laid in the interlamella space of the demibranches,
whereas those of R. uyekii are sticky and laid in egg masses in
the suprabranchial cavity (Kim et al., 2015). The reason for these
similarities and differences cannot be determined based on the results
of this study. Thus, in-depth studies on speciation based on ecological
characteristics and specific factors are necessary (Mayr, 1969; Arai et
al., 2001).
The hemispheric minute tubercles on the WLP were approximately twice as
large as those in the EHR, and the direction of the minute tubercles was
slightly inclined posteriorly, making it easy for them to fixate on the
gills but difficult to be removed, like a harpoon. The WLP was the
largest and most developed part of the entire larva, have also the
largest surface area. The minute tubercles on the WLP began to develop
shortly after hatching and began to shrink during the abrupt reduction
stage. The hatched larvae that entered through the mussel’s exhalant
siphon settled on the demibranches, growing in their interlamellar
space, during which time the larvae’s widest surface area is the WLP
(Song & Kwon, 1994). Mortality of bitterling larvae occurs by two main
factors: premature ejection by the mussel and death in the mussel gill
by asphyxiation or nutrient deficiency (Smith et al., 2000; Kitamura,
2005; Kawamura & Uehara, 2005). The minute tubercles are formed by
large unicellular epidermal cells and are presumed to be polysaccharidal
in nature; studies have shown that they perform an attachment function
that enables them to attach to vegetation and submerged objects (Laale,
1980). The minute tubercles occur only in larvae with no swimming
ability; when fins (and consequently, the larvae’s swimming ability)
start to develop, the minute tubercles are abruptly reduced (Table 1;
Figures 4, 5). The minute tubercles in Acheilognathus andTanakia bitterlings, which do not have a wing-like projection,
develop most intensively in the foremost part of the head, and the form
of the yolk projection is scaly or hilly, different from that ofRhodeus bitterlings (Fukuhara et al., 1982; Suzuki & Hibiya,
1985; Suzuki & Jeon, 1987, 1988a, 1988c, 1988d, 1989, 1990; Park et
al., 2008). The development of larger and sharper minute tubercles inAcheilognathus and Tanakia larvae compared to those ofRhodeus larvae (20–40 µm vs. 3–15 µm) is an adaptation strategy
that also prevents premature ejection and allows larvae to tightly fit
in the interlamellar space of the hosts’ demibranchs (Suzuki & Hibiya,
1985; Kitamura, 2006b). Further research will be required to compare
with migration inside mussels in Acheilognathus andTanakia larvae for investigating the role of minute tubercle as
their types.
Mussels have one exhalant and one inhalant siphon. The bitterling’s
ovipositor enters a mussel’s exhalant siphon, and eggs are placed in the
suprabranchial cavity or interlamellar space of the demibranch (Wu,
1998). As the inhalant siphon is connected to the mantle cavity, when
the mussel’s shell opens, the larvae would become exposed to the
environment; therefore, bitterling spawning must occur in the exhalant
siphon to increase larval survival (Tankersley & Dimock, 1993a). The
interlamellar space of the demibranchs expands as the larvae grow and
becomes a limiting factor. The larvae that remained in the interlamellar
space for more than 11 days after hatching were found to have a slower
development than those that migrated to the suprabranchial cavity. By
migrating to the suprabranchial cavity, which is larger than the
interlamellar space, ventilation rates can be increased, thus increasing
oxygen supply and space (Davenport & Woolmington, 1982; Mills &
Reynolds, 2002). Song & Kwon (1994) reported that A. yamtsutaelarvae return to the U part as they gain physical abilities over the
developmental stages. A. signifier and R. sericeus larvae,
in contrast, remain in the interlamellar space only during the initial
developmental stages, and as their swimming ability increases, they
migrate to the suprabranchial cavity, in the direction opposite to the
exhalant siphon (Aldridge, 1997; Back & Song, 2005). A. rhombeuswas reported to initially remain in the suprabranchial cavity and then
migrate in the direction opposite to the exhalant siphon (Kim et al.,
2018). The bitterlings’ eggs inside the gills may compete with glochidia
for oxygen and space (Smith et al., 2001; Kitamura, 2005). The migration
of larvae from the interlamellar space of the demibranchs to the
suprabranchial cavity may reduce intraspecific competition and lower
larval mortality rate in the suprabranchial cavity by providing space
for growth and increased oxygen supply (Kitamura, 2006b; Spence &
Smith, 2013; Methling et al., 2018).
Many previous studies have reported that mussel gill structure and
conditions such as size, water flow speed, and dissolved oxygen content
vary among gill positions, sexes, and density of larvae (Tankersley &
Dimock, 1993a, b; Aldridge, 1999; Mills & Reynolds, 2002, 2003; Smith
et al., 2004; Kitamura, 2005, 2006a, b). No glochidia were found during
the present study, so the sex of the mussels was unknown. However,R. pseudosericeus larvae were mainly found in the two outer
demibranchs of the four gills. U. d. sinuolauts is known to brood
glochidia only in the outer demibranchs, but as the spawning season is
after May, no glochidia care was observed during this study. Aldridge
(1997) and Mill and Reynolds (2003) reported that the bitterlings mainly
use the inner demibranchs, which had more larvae than the outer
demibranchs, because of four reasons: active choice, space availability,
ovipositor accessibility, and ejection ability. Studies have reported
that A. rhombeus, A. cyanostigma , and R. o. kurumeus eggs
were found at a higher rate in inner demibranchs than in outer
demibranchs, suggesting that it is these species’ choice to avoid
competition for oxygen and space with glochidia of mussels that use the
outer demibranchs as brood pouches (Kitamura, 2006b, 2006c; Kim et al.,
2018). Tankersley (1992a) proposed that the total flow in gills during
brooding would be approximately 16% and 4% of those in non-gravid and
non-marsupial gills, respectively. Kitamura (2006c) reported that female
bitterlings may have been more constrained in their spawning inside the
inner demibranchs irrespective of mussel sex during group spawning.
Moreover, Mills and Reynolds (2003) reported that when mussels brood
larvae, bitterlings spawn in inner demibranchs, but that after the
mussels release their larvae, the widened outer demibranchs can be used
as spawning sites. Interestingly, when the spawning patterns of mussels
in March and April (i.e., before mussels brood the larvae) and in May
and June (after the brooding season) were analyzed, R.
pseudosericeus was found to have higher spawning rates in outer
demibranchs than in inner demibranchs (per. observation). For A.
signifier , twice as many larvae were identified in inner demibranchs
without brooding pouches compared to the outer demibranchs with brooding
pouchs (Kim et al., 2014). Further studies are necessary to elucidate
the selectivity of bitterlings regarding gill position and whether it is
related to gill structure or active selectivity of bitterlings
(Tankersley & Dimock, 1993b).
Bitterlings have a unique early life history. The bitterlings’ eggs can
be classified into four types: bulb-like, pear-shape, spindly, and
ovoid; moreover some eggs are sticky (Kim et al., 2006; Kim et al.,
2011). They lay a small number of eggs, develop unique tissue structures
called minute tubercles during the early stages of larval development,
have a very fast hatching time, and are unique in laying eggs in
mussels. However, this species, of which 60 types are known worldwide,
evolved due to various factors such as type of maturation, development,
spawning type, spawning position and larval migration in mussels, and
host selection (Smith et al., 2004; Nelson, 2006). In conclusion, the
present study, by examining the development of minute tubercles, the
migration of larvae inside mussels, and the physiological
characteristics of the larvae, provided direct and comprehensive
evidence that minute tubercles are developed to prevent the premature
ejection of larvae by their mussel hosts. Thus, this finding may enhance
our understanding of the evolutional advantages of the development of
the minute tubercles and migration of larvae inside mussel for better
survival. In this present study, however, the investigation was limited
to the determination of the main factors causing growth or reduction of
minute tubercles development and advantages of migration of larvae.
Therefore further physiological research will be required to determine
the role physiological factors.