Spawning of <i>Carassius cuvieri</i> (Cyprinidae) in an artificially developed reed zonealong the Lake Biwa coast, outside of the reclaimed Hayasaki Lagoon, as revealed by DNA identification of field-collected eggs

<p>The Japanese white crucian carp, Carassius cuvieri, is a cyprinid fish end- emic to Lake Biwa, central Japan. Due to a drastic decline in the commercial catch, it is listed in the IUCN Red List as an endangered species. Its largest spawning ground was Hayasaki Lagoon, in the northeastern part of the lake. However, the lagoon was drained and converted into agricultural paddies in 1970, which was thought to have decreased the C. cuvieri population greatly. To promote the recovery of native cyprinid fish populations, including species such as Carassius buergeri grandoculis, Gnathopogon caerulescens, and C. cuvieri, the prefectural government developed artificial reed zones along the lake shore. However, it had not been confirmed whether C. cuvieri used these zones as spawning grounds. This study investigated the spawning of C. cuvieri through DNA identification of eggs collected in an artificial reed zone developed between 2002 and 2005, located outside the reclaimed Hayasaki Lagoon. The survey area consisted of Area-A, occupied by willow trees, located farthest offshore; Area-B, dominated by common reed (Phragmites australis); Area-C, an open-water area behind the reed area (partly floating herbaceous islands). These areas were searched for eggs attached to plants near the water surface between 13:00 and 13:40 on 11 May 2018 and the eggs were collected by hand. For each batch of egg samples, GPS data were recorded, and each egg was identified in the laboratory using DNA markers. The identification results were then mapped on a satellite image of the survey area on Google Earth, and the relationship between collection areas and spawning fish species was examined. While no eggs were collected in Area-A, 194 were collected from nine points in Area-B and 174 from seven points in Area-C. From these eggs, 80 and 56 were randomly selected for Areas-B and -C, respectively, and all were identified as belonging to Carassius by PCR using genus-specific primers. These eggs were then identified by PCR using species- specific primers. Of the 127 eggs, 79 (62.2%) were judged as C. cuvieri, and the remainder as either C. buergeri grandoculis or triploid Carassius species. By collecting area, 41.3 and 92.3% of the eggs from Areas-B and -C, respectively, were C. cuvieri; the proportion of C. cuvieri eggs in Area-C was significantly greater than that in Area-B. These results confirmed that C. cuvieri used the artificially developed reed zone studied here as a spawning ground, especially the open water area behind the reed zone.</p>