**8. Morphological development of freshwater mussel**

Since the glochidia freshwater pearl mussel of *H.* (*L.*) *myersiana* was cultured in the artificial media that could develop to the adult (Kovitvadhi et al., 2006). Therefore, Kovitvadhi et al. (2007) could study for the morphological development of the juvenile through the adult *H.* (*L.*) *myersiana*. The mussels were collected in sequential developmental stages between 0 and 360 days old. Morphological development was observed by light microscope and SEM. SEM observations were prepared in fixative solution containing 10% neutral buffered formalin for 24 h and stored in 5% neutral buffered formalin for further process. The samples for SEM were thoroughly washed under running water for 30 min and then dehydrated in a graded series of ethanol and dried to critical point. Thereafter, they were mounted on SEM specimen stubs with conductive silver paint and coated with gold and observed with a Jeol Model JSM-5410LV scanning electron microscope operated at 25 KV. All samples before fixation, they were anesthetized in 2% chloral hydrate to observe the internal regions.

The morphological development of *H*. (*L*.) *myersiana* juveniles in culture (0-360 days old) is shown in Fig. 6. The early juvenile of *H*. (*L*.) *myersiana* at 0 days old after transformation has semi-oval, equivalve shells with an equilateral valve, presenting the same size and shape as the glochidium (Fig. 6A). Anterior shell growth was clearly seen in the first day of juvenile development (Fig. 6B), while posterior shell growth followed afterwards (Fig. 6C). The shells of 0-40-day-old juveniles were thin and transparent as seen under light microscope (Fig. 6D). The inner organs (i.e., stomach, intestine, gills, heart, foot, mantle, and cilia at the gills, mantle and foot) were clearly observed through the shell in this period (Fig. 6E). The shell, however, became thicker during the developmental process and covered all the inner organs (Figs. 6E-L). The first anterior and posterior wings appear in 50-day-old juveniles (Fig. 6E), with the posterior wing becoming dominant relative to the anterior from the 140-day-old stage (Fig. 6H). The mantle lobes of 0-50-day-old juveniles are joined dorsally and are free ventrally (Fig. 6F). The incurrent siphon and excurrent siphon appear after 50 days (Figs. 6G-6L). The complete adult morphology is apparent from 160 days old (Fig. 6I). Males and females reproductive organs are sexually mature about 270-360 days old mussel which depend on environment (Kovitvadhi et al., 2006; Srakaew et al., 2010) (Figs. 6K-6L).

A; Light microscopy of early juvenile (after transformation, 0-day-old) shell, note shell hinge (sh). B; SEM micrograph of 1-day-old juvenile, appearance in anterior region of new soft periostracum (pe), note glochidium shell (gs). C; Juvenile 10 days old, anterior (a) region appears before and grows more than the posterior (p), note growth line (gl). D; Light microscopy of development of shell, 40-day-old juvenile, note foot (f), gill (g), posterior adductor muscle (pa). E; Light microscopy of development of shell, 50-day-old juvenile, note anterior wing (aw); posterior wing (pw). F; SEM of ventral side of 50 day-old juvenile. G; Light microscopy of external morphology of 90-day-old juvenile, note excurrent siphon (es), incurrent siphon (is), umbo (u). H; Light microscopy of external morphology of 140-day-old juvenile, note rectum (r). I-L; External morphology of shell, 160, 180, 270 and 360 days old, respectively.

Fig. 6. Morphological development of 0–360-day juveniles of *Hyriopsis* (*Limnoscapha*) *myersiana*. (Kovitvadhi et al., 2006; 2007).
