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.