1. INTRODUCTION
Coevolution is a process that consists of reciprocal evolutionary
changes resulting from the interrelationship between a group of
organisms and associated populations and plays an important role in the
adaptation and speciation of almost all living organisms (Thompson,
1994). Prey-predator, host-parasite, and symbiont relationships are
typical examples of coevolution (Thompson, 2002; Liu et al., 2006).
Theoretical evidence of coevolution and adaptive traits have been
obtained mainly from studies on host-parasite interactions (e.g., avian
brood parasitism) (Thompson & Burdon, 1992; Takasu, 1998; Rothstein &
Robinson, 1998; Davies, 2000).
Oviparous fishes with parental care use different reproductive
strategies to select and prepare spawning sites to increase the number
and survival rate of larvae, for instance, by defending their eggs or
oxygenating the water around them (Smith & Wootton, 1995). Besides the
reproductive success of an individual, the choice of type and site of
spawning in species with parental care are also important factors that
influence larval survival (Smith et al., 2000; Mills & Reynolds, 2002;
Kitamura, 2005; Refsnider & Janzen, 2010). In contrast, species without
parental care are vulnerable to abiotic (e.g., low oxygen rates and
extreme temperatures) and biological factors (e.g., predators,
parasites, and competitors) during the larval stages (Smith & Wootton,
1995).
Bitterlings (Acheilognathinae) are small freshwater fish predominantly
distributed in Europe and Northeast Asia, and they have a unique
relationship with freshwater mussels (Bivalves: Unionidae) (Smith et
al., 2004; Damme et al., 2007). During the spawning season, the female
bitterlings elongate their ovipositors and spawn on the gills of mussels
through the mussels’ exhalant siphons. The male fish, who have nuptial
coloration and form territories around the mussels, release sperm that
enters the mussels’ inhalant siphons through their feeding and breathing
actions. The eggs are, therefore, fertilized in the gill cavity of
mussels where, depending on the temperature, they remain for 3–4 weeks
feeding on their own reserves until they become free-swimming larvae; at
this stage, the larvae leave the mussels and begin external feeding
(Aldridge, 1999; Smith et al., 2004).
Although bitterlings do not have parental care, they have very few eggs.
The host-parasite relationship between these fish and mussels presents
difficulties mainly during the moment of spawning and when a premature
ejection of larvae by mussels occurs; however, the period in which the
fish are most vulnerable (i.e., as eggs) is spent safely inside the
mussels, from where the larvae only exit after acquiring swimming
ability (Zale & Neves, 1982; Smith et al., 2004; Kitamura, 2008).
Host-parasite interaction and choice of oviposition site are critical
aspects of vertebrate ecology that have not been sufficiently studied
(Refsnider & Janzen, 2010). The relationship between bitterlings and
mussels is a notable example of coevolution between host and parasite
(Reynolds et al., 1997; Mills & Reynolds, 2003; Reichard et al. ,
2010; Rouchet et al. , 2017).
Recent studies have shown that the bitterling-mussel relationship is in
fact a type of host-parasite interaction (Reichard et al., 2001; Mills
& Reynolds, 2003; Spence & Smith, 2013). Bitterlings make
sophisticated oviposition decisions to prevent ejection and have several
unique physiological, behavioral, and morphological adaptations for
spawning on host mussels (Aldridge, 1997; Smith et al., 2004; Kitamura,
2006a, 2006c; Spence & Smith, 2013; Methling et al., 2018). The fish
larvae develop single-celled epidermal cells, called ‘minute tubercles’,
on their skin surface, which are known to play an important role in
preventing the larvae from being prematurely ejected from the gills
(Suzuki & Hibiya, 1984a, 1985; Suzuki et al., 1985; Suzuki & Jeon,
1987). Minute tubercles are common in all developmental stages of
bitterlings, even though the larval morphology is diverse. Previous
studies have reported that the minute tubercles are mainly developed in
the frontal part of the larvae and on the eyes of larvae, forming a
wing-like projection (Suzuki & Jeon, 1988a, 1988b, 1988c, 1988d, 1989,
1990; Kim et al., 2008; Park et al., 2008).
Many studies using in vitro insemination have briefly described the
development of the minute tubercles and the morphological
characteristics of larvae, and based on their results the minute
tubercles were assumed to prevent premature ejection of larvae from
their host mussels. However, no comprehensive studies correlating the
developmental stages of larvae with their morphological and
physiological characteristics, their migration inside the mussels, and
the development of the minute tubercles have been conducted. Therefore,
in the present study, my goal was to find direct evidence indicating
that the development of minute tubercles in bitterlings prevents
premature ejection. For this, I focused on the relationships among the
height of the minute tubercles, morphological and physiological
characteristics of the larvae during development, and the position of
the larvae in the mussels. Further I discussed the evolutional
advantages of the development of the minute tubercles and migration of
larvae inside mussel for better survival.