A method for PCR-based identification of fish eggs attached to vegetation in Lake Biwa, Japan
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TECHNICAL NOTE
A method for PCR-based identification of fish eggs attached to vegetation in Lake Biwa, Japan K. Mabuchi1
Received: 7 February 2016 / Accepted: 6 April 2016 Ó Springer Science+Business Media Dordrecht 2016
Abstract Primers were developed for single-tube genotyping of four genera, Cyprinus, Carassius, Gnathopogon, and Ischikauia. Most of the six species in Lake Biwa belonging to these four genera (one is a native Japanese common carp) are endangered, and two of the six are important fisheries resources. However, many aspects of their reproductive ecology are unclear, because these species spawn similar looking eggs on similar substrates during the same time of year. The molecular method developed here enabled efficient and cost-effective genuslevel identification of such eggs, providing a breakthrough for developing effective conservation strategies for these species. Keywords mtDNA Cytochrome b Cyprinus Carassius Gnathopogon Ischikauia The native Japanese strain of common carp has been dramatically affected by invasion of conspecific domesticated strains from Eurasia. A survey of mitochondrial D-loop sequences from 11 locations in Japan (Mabuchi et al. 2008) revealed that half or more of the haplotypes in all 11 locations originated from domesticated Eurasian strains. However, an intensive mtDNA survey in the largest freshwater body in Japan, Lake Biwa, indicated that the deep off-shore water of the lake harbors a relatively pure native population of common carp (Mabuchi et al. 2010). One study using nuclear DNA markers (Mabuchi et al. 2012) confirmed that supposition (Mabuchi et al. in prep.), & K. Mabuchi [email protected] 1
Atmosphere and Ocean Research Institute, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8564, Japan
and the common carp population in the lake is now regarded as a ‘‘threatened local population’’ in the Japanese Red Data Book (Japan Ministry of the Environment 2014). It is necessary to determine the timing and location of spawning activities of endangered native and introduced Eurasian strains to properly manage the stock of the former strain. Common carp inhabiting the lake attach their eggs to submerged or floating vegetation in near-shore zones during May–July (Nakamura 1969). Surveys of these eggs can potentially provide detailed information on their spawning activities. A polymerase chain reaction (PCR)based method to distinguish native and introduced haplotypes has been reported (Mabuchi and Nishida 2006). Although a molecular survey of common carp eggs now seems possible, six non-common carp species belonging to the four genera Carassius, Gnathopogon, Hemigrammocypris and Ischikauia spawn similar eggs on similar substrates during the same time of year as those of common carp (Nakamura 1969). A simple application of the Mabuchi and Nishida’s (2006) method for these eggs resulted in misidentifications: for example, samples of Gnathopogon caerulescens were mistakenly identified as the introduced Eurasian strain of common crap (data no
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